摘要:

GERTRUDE E . PERLMANN 77 THE GLOBULAR-FIBROUS PROTEIN TRANSFORMATION BY E. BARBU AND M. JOLY Service de Chimie-Physique et Service de Chimie Biologique, Institut Pasteur, Paris Received 7th May, 1952 Some globular proteins can be transformed into fibrous proteins by moderate heating, salt effect or high pressure. It has been studied by electron microscopy, viscosity, electrophoresis, ultracentrifuge, paper electro- phoresis, fractional precipitation and streaming birefringence. The problem is to know whether this transformation is due to any unfolding of the polypeptide chains or to the end-to-end aggregation of protein molecules that remain globular. A great number of experimental facts support the second assumption. The denaturation of protein molecules is an activation process.The molecules thus activated aggregate when they collide. The kinetics of this process has been studied both experimentally and theoretically. This transformation is partially reversible. The transformation of globular proteins into fibrous is generally performed under very drastic conditions,l-4 e.g. at pH 10-1 1, in the presence of urea or deter- gents, by violent extruding and precipitation. The analogy of corresponding X-ray diagrams with those of stretched keratin prompted the authors to interpret the globular-fibrous (g-f) protein transformation as an unfolding of polypeptide chains. Previous research has shown that less powerful treatment of protein solutions can cause the formation of thread particles by end-to-end aggregation of globular molecules. This occurs, for instance, in the first stages of the sol-gel transformation of gelatin,s* 6 in the thermal denaturation of horse 7-9 or human 10 serum albumin and of egg albumin,ll-13 in insulin gelation 14-16 or in fibrinogen association in- duced by thrombin.17 We have recently studied the effects of different physical and chemical factors on the process of linear aggregation in the thermal denaturation of horse serum alb~min.7~ 8 The purpose of the present paper is to give some new experimental and theoretical information on the g-f transformation and to extend this investi- gation to other proteins and denaturing agents.78 GLOBULAR-FIBROUS PROTEIN TRANSFORMATION EXPERIMENTAL MATERIALS.-The horse serum albumin and the hen egg albumin were prepared by salting-out with (N&)2so4 and by successive crystallization according to Sorensen.18 The conservation of our sample of Cohn's crystalline bovine serum albumin (no. 59, control 128-1 65) was tested by electrophoresis.The horse y-pseudo-globulin was pre- cipitated three times by 33 % saturated (NH&S04, dialyzed against H20 and centrifuged to eliminate the euglobulins. Rabbit actin was prepared according to Straub 19 and dissolved in twice distilled water cooled in absence of CO2. METHODS.-(a) Streaming birefringence.-The capacity of the apparatus (with moving inner drum) used for the streaming birefringence measurements was 5 cm3 ; the cylinders were 7 cm high with a gap of 0.025 cm ; the velocity gradient could be varied between 80 sec-1 and 6,000 sec-1.The extinction angle X was determined with an accuracy of f 0.1" by the differential method of Frey-Wyssling and Weber.20 For elongated particles the apparent lengths Z(g) for each value of the velocity gradient g were determined from the X (g) values using the tables of Scheraga, Edsall and Gadd.21 The size I f of the most frequent particles and the polydispersity P of the solutions were derived using the semi-empirical relationships given by one of us.% 23 The accuracy was 5 % for I f and 10 % for P. The precise significance of I f and P was given in a preceding paper.22 We shall use later the rate of polydispersity T which is, by definition, the quotient P/If. For globular particles the computation of precise relationships giving the most fre- quent diameter df and the polydispersity is now in progress ; here we shall give only the mean diameter d obtained empirically by extrapolating the d(Z/g) curve against Z/g to 0 (the validity of this method was checked by electron microscopy).From the experimental values of the streaming birefringence A = n, - no, we can deduce for very dissymmetrical monodisperse particles the order of magnitude of the intrinsic particle anisotropy A = n12 - n22, 11: and 112 being the principal refractive indexes of the particles. We have A N A/cf(X), f(X) being a function of the extinction angle, and c the volume concentration of the particles. This quotient is independent of c and g and so bas real significance. For polydisperse systems A/cf(X) is not independent of g and of the solution composition ; we can only determine apparent values A(g) of the anisotropy, of which we shall give the mean value A-for velocity gradients ranging from 80 sec-1 to 5,500 sec-1.In a mixture of small globular and large elongated particles, only the threadlike particles give streaming birefringence, and so, by comparing the values of 2 during a g-f transformation, we can estimate the rate of transformation. By this means we find, for instance, with horse serum albumin solutions (c = 2.4 %, pH = 4.15, acetate buffer M/50) heated for 10 min at 65" C and 80" C, a ratio of the transformation rates equal to 60 % (in good agreement with the value 61 % given by precipitation in 16 % Na2S04). If the particles are not very elongated, A/cf(X) + n12 - n22 and the determination of f ( X ) requires a knowledge of the axial ratio p of the particles.But for any system we can arbitrarily define, by means of the relationships valid for very elongated particles, an equivalent length of the most frequent particles [b], an equivalent rate of polydispersity [;z] and an equivalent mean anisotropy [a; these parameters have no direct physical significance, but they are very useful in the study of globular or quasi-globular aggrega- tion 24 and of photoelastic effect or dynamic turbidity.25 For instance, if we know If and A for a given protein under such conditions that the fibrous state of the particles is com- plete, and if for the same protein under different conditions the apparent values [ I f ] > r f and [A] < 2, we can conclude that the second type of particle is much less dissymmetrical, and from these values it is possible by successive approximations to obtain the order of magnitude of the axial ratio p of the quasi-globular aggregates. (b) Other teclzniques.-For the viscometry measurements we used a Baume capillary viscometer, the constant K of which was 4945 x 10-5, The electrophoresis was carried out in a Moore apparatus following the method of Tiselius, and the paper electrophoresis was performed according to the technique of Durrum as used by Macheboeuf and Rebeyrotte.26 The ultracentrifuge was of the Phywe type, running at 36,000 or 42,000 rev/min, with an optical design by Wielgosz.27 The precipitation at the isoelectric point was studied by N titration of the super- natant obtained by centrifugation, 3 h after putting the protein in a 14 % or 16 % Na2S04 -E.BARBU AND M. JOLY 79 solution at pH4.75. The solutions were compressed at high pressure in the apparatus built by Basset using the technique developed by this author.28 EVIDENCE OF AGGREGAnoN.-From the data of streaming birefringence we have shown in preceding papers7, 8 that the heating of dilute serum albumin solutions gives rise to filaments, the length of which can attain several thousand A, and that the properties of these filamentous particles (e.g. rheological behaviour and tensile strength) cannot be explained by the unfolding of initially folded polypeptide chains, but can be easily under- stood using the concept of end-to-end aggregates. On the contrary, large globular aggregates grow by adding tri- or tetravalent ions to the protein solution.We shall show, too, that the high pressures gave rise in the solu- tions to quasi-spherical aggregates with a diameter of several hundred A. For characterizing these different types of aggregation we examined some solutions of 2.5 % horse serum albumin at pH 7.3, without treatment, after heating for 10 min at 65" C and 80" C, and after compression for 1 h at 37" C under 10,OOO Kg/cm2. (a) Streaming birefringence.-The solutions before treatment do not show streaming birefringence. For the treated solutions we found : heated at 65" C, L) = 700& T = 1.7, = 2-4 X 10-3 ; heatedat 8O"C, b= SOOA, n = 1, A= 3 x 10-3; The quotient of the values of 2 at 65" C and 80" C was 0.8 in good agreement with the ratio 0.85 given by the solubility test.(6) Zsoelectric precipitution.-3 h after mixing with 14 % Na2S04 (PH 4.75) we found as percentage of soluble serum albumin : 100 % for the untreated solution, 17 % for the solution heated at 65" C, 2 % for that heated at 80" C and 18 % for the compressed solu- tion. All these results agree with the values of the mean intrinsic anisotropy. (c) Viscosity.-The measurements were carried out at 22°C in a phosphate buffer pH 7-3 (p = 0.15) with a protein concentration of 0.94 % for the solution heated at 80" C and 0.63 "/, for the other samples. The approximate values of the intrinsic viscosity were 12.9 for the solution heated at 65" C, 16.7 for that heated at 80" C and 11.6 for the compressed solution. The corresponding values of the axial ratios (following Guth's relationship 29, 30) are at a first approximation 13 and 15 for the solutions heated at 65" C and 80" C, 12 for the compressed solution.The results concerning the heated samples fit well with those of streaming birefringence and lead to a width of about 50A for the rod-shaped particles in satisfactory agreement with the approximate diameter of the serum albumin molecules.31 But the value for the compressed solution is hardly compatible with a suspension of rigid and compact spheres. ( d ) Electrophoresis.-The detailed electrophoresis study of the heated or compressed serum albumin solutions will be published in a forthcoming paper.32 We summarize here the results concerning the solutions we investigated. The electrophoresis was carried out after dilution of the protein to 1 % in phosphate buffer (PH 7.5 ; p = 0-15 NaCI) and dialysis against the same buffer.Fig. 1 shows the diagrams (ascending boundary) after 3 h electrophoresis (V = 110 V, i = 18 mA) of the untreated and heated solutions. The electrophoretic patterns are the same for the samples compressed or heated at 65" C . We found for the mobilities the following approximate magnitudes : untreated solution 6.2 (one fraction only) ; solution heated at 80" C, 5.8 (one fraction only) ; solution heated at 65" C, 6.5 (16 %) and 5.5 (84 %) ; solution compressed at 10,000 atm, 6.5 (15 %) and 5.5 (85 %). There is a good agreement of the transformation rates derived from electrophoresis with those obtained from the solubility experiments. The small difference between the mobility of the native protein and that of the heated or compressed, proves that the ratio charge/ frictional coefficient differ very little for the single protein molecules and for the linear or globular aggregates.We particularly verified that this ratio was, for very elongated rod-shaped particles, practically independent of the length of the particles. (e) Puper-electrophoresis.--'Fhe experiments were carried out with 2.5 % solutions. Fig. 2 shows the diagrams obtained by 6 h paper-electrophoresis. We note the great mobility and homogeneity of the untreated sample. The mobility of the particles heated at 80" C was much lower and the corresponding spot spread considerably. The solutions heated at 65" C or compressed at 10,000 kg/cm2 show two spots: one strong with a compressed at 10,000 kg/cm2, 2 =; 350 A.80 GLOBULAR-FIBROUS PROTEIN TRANSFORMATION mobility lower than that of the untreated solution and one weak with a mobility of the same order as that of the native protein.The discrepancy between the mobilities of un- treated and heated protein was much larger in paper-electrophoresis than in ordinary electrophoresis. The mobility of the main fraction of the solution heated at 65" C was a little lower than that of the corresponding fraction of the compressed solution. These differences between ordinary and paper-electrophoresis are related to the fact that a very dissymmetrical particle moves less easily in paper than a globular one does even though the ratio charge/frictional coefficient in water is the same for both the particles.(f) Ultracentrifuge.-The ultracentrifuge studies were carried out with solutions of 1 % of horse serum albumin, in phosphate buffer (pH 7.3; p = 0.15). Fig. 3 shows the sedimentation patterns after 1 h run at 42,000 revlmin for the untreated and at 36,000 revlmin for the compressed or heated samples. The mean values of the sedimentation constants were : for the slow fraction of all the samples (only this one for the untreated solution) s = 4.6 x 10-13 (in good agreement with the literature values) ; for the rapid fractions, 15 x 10-13 with the compressed solution, 20 x 10-13 with the solution heated at 65" C and 29 x 10-13 with that 9f 80" C. From the general relationship of the ultracentrifuge M = -____ ( p solvent density, V specific volume and D diffusion constant of the particles) we can derive the size of the aggregates according to the assumptions made on their shape. For instance, for linear aggregates made of only one row of serum albumin molecules, the length L of the particles is related to s by L N 25 exp (1.6 x 1012 s) A, and for linear aggregates made of two adjacent rows of molecules L N 25 exp (1.2 x 1012 s)A; for a globular but rigid and compact aggregate of serum albumin molecules, the diameter d is given by d N 1-14 108 6 A .By applying these equations to the preceding values of s and by assuming the particle to be a single row of spherical molecules of diameter 50 A, one finds L = 600 A for the solution heated at 65" C ; this is in good agreement with the value derived from streaming birefringence data ( I f = 700 A).For the solution heated at 80" C , the values found by ultracentrifuge and by streaming birefringence are the same (L = I f = SOOA), if we as- sume the particles to be composed of two adjacent rows of protein molecules. This as- sumption is consistent with the mean width of the particles as deduced from electron micrographs and agrees with the variation of the mean intrinsic anisotropy A with the temperature of heating. But for the globular aggregates, the sedimentation measure- ments gives d = 140 A, whereas streaming birefringence and electron microscopy lead to d= 350 A. This means (like the results of viscometry) that the globular aggregates are not rigid and compact globules and that the solvent can partly flow through them.(8) Electron microscopy.-Fig. 4 shows photographs of untreated, heated and com- pressed solutions. The two types of aggregation, filamentous and globular, are evident. One can always object that this effect is produced by some artefact in electron micro- scopy and that such an interpretation of the photographs should not necessarily carry weight. One knows also how uncertain is the discussion of viscometric results in the yet imperfect state of the theory of colloid viscosity. The precise explanation of the electrophoresis or paper electrophoresis patterns and of the sedimentation diagrams is frequently difficult. We have emphasized in preceding papers 22,23, 33 the caution necessary in interpreting streaming birefringence data.But when there is rather good agreement between the results that we obtain by applying such varied techniques, in which the fundamental assumptions are different, it seems sound to draw the following conclusion. Protein denaturation is frequently accompanied (according to the experimental conditions) by linear or globular aggregation of the molecules remaining individually globular. This nevertheless does not exclude partial modification of their shape. Particularly we can legitimately say that the globular-fibrous protein transformation, in all the cases we studied, proceeds by end-to-end aggregation of the globular molecules, and in no circumstance by complete unfolding of the chains. In the following we examine the details of this transformation for different proteins and for various treatments of the solutions.HORSE SERUM ALBUMIN.-The linear aggregation by heating of the horse serum albumin molecules was systematically studied,', 8 and we recall here only the main features of the results. If we consider the variation of the length rf of the most frequent particles during the thermal treatment, for a given temperature, pH, ionic strength and protein concentration, RTs D(I - Vp)'A B C A B C FIG. I . FIG. 3. FIG. 1 .-Electrophoretic diagrams (ascending boundary). t = 3 h ; Y = l l O V ; i = 1 8 m A ; c = 1 % of serum albumin ; phosphate buffer pH 7.5 ( p 7 0.1 5 ) . A, untreated solution. B, sample heated for 10 rnin at 80' C C , sample heated for 10 min at 65" C . FIG. 3 .-UI tracentrifuge patterns.t = 1 h ; c = 1 04; pH 7.3 ( p = 0.15). A, untreated sample B, sample compressed at 10,000 atm C, sample heated for 10 min at 80" C. FIG. 2.-Diagrams of paper electrophoresis. t = 6 h ; c = 2.5 %; pH 7.5 ( p = 0.15). A, solution heated for 10 min at 80 C B, solution heated for 10 min at 65' C C , solution compressed at 10,000 kg,icm2 D, untreated solution. [To face page 80FIG. 4.--Electron micrographs. A, untreated solution pH 7.3 B, solution at 2.5 %, pH 7.3, heated for 10 niin at 80 C and then diluted to 0.01 7; C, solution at 4 %, pH 7.3, compressed for 1 h at 37" C under 8000 kg/cm2 and then diluted to 1 % D, solution at 2.5 %, pH 4.3, heated for 10 inin at 80' C in acetate buRer and then diluted to 0-01 7:. [See puge 8 1E . BARBU AND M .JOLY 81 we observe that it attains after a time 0 a value Lf which then remains practically constant, often for a very long time (see table (1)). Lf depends upon concentration and pH ; 0 decreases when the temperature and the ionic strength increase. After this period of stationary length, If increases again more or less regularly and, according to the values of pH, concentration and ionic strength, coagulation or gelation of the solution can occur. The variation with the time t of the rate of polydispersity 77 depends greatly on the temperature of heating. The increase of the mean intrinsic anisotropy 2 during the first period of the particle growth (and, for low temperatures, at the beginning of the flat part of the If against t curve) corresponds to the increasing number of the particles large enough to promote streaming birefringence. The decrease of A along the flat part of the I f against t curve (or only at its end, for low temperatures) and during the second period of aggregate growth, seems to correspond to the increase of the particle width due to side-by-side aggregation of several rows of molecules, which is in agreement with the information from electron microscopy (see fig.4, D) and ultracentrifugation. One conceives for very elongated linear aggregates, that the polarizability in the length direction is inde- pendent of the number of the end-to-end aggregated molecules, while the polarizability in the width direction changes greatly when there are several side-by-side aggregated rows of molecules instead of one. If the solutions are heated in absence of a neutral electrolyte, 0 is significantly larger, but the flat part of the curve is often very short and sometimes even completely hidden by the second step of growth or by the effect of very strong interactions between the particles (see table 2).TABLE GROWTH OF THE PARTICLES OF SERUM ALBUMIN c = 2.4 % ; pH = 4-15 ; M/50 acetate buffer T("C) t (min) 1.0 57 I f ( & I A x 103 - 65 I,- - 2 x 103 - 80 r f - 90 I f - 2 x 103 - n - rr I 77 - 77 - 100 I f 650 rr 1.5 ;i x 103 - 5 - - - - - - 1100 0.1 1850 1.5 2.4 2600 0.95 - 10 - - - 950 - 4-2 2100 0.25 2200 1.2 3.5 2750 1.1 3.1 20 1000 - 1.5 1050 2.0 4.3 2450 0.55 2200 2.1 3.3 2800 1.6 - 40 I150 0.4 1.4 1100 0.9 4-4 2500 0.75 2450 2-8 - 2850 2.4 - 80 1250 0.5 1.9 1600 0.13 7.0 2400 1.7 2650 3-1 - 2800 3.0 - TABLE 2.-GROWTH OF THE PARTICLES OF SERUM ALBUMIN c = 2.9 % ; pH = 4.4 ; without buffer T("C) t (min) 2 2.5 5 6 10 20 35 - 800 - 1050 1250 - 2400 77 - 0.5 - 1.8 1.4 - 0.7 100 If 1750 - - - 2450 2400 - n 0.7 - - - 0.8 2.7 - 80 I f ( @ 160 1200 0.5 3.0 21 50 tom1 5.4 2450 3.0 3250 3-8 - 3050 4.7 - 40 - - 3350 0.55 We have described in detail the effect of salts in preceding papers.7.8 The variation of I f , n and 2 with the temperature of heating (all other conditions being the same) seems irregular if this is determined after a given time of treatment (see table 1) ; but the vari- ation of Lfis regular as is seen in table 3,82 GLOBULAR-FIBROUS PROTEIN TRANSFORMATION The dilution after heating does not affect the size of the particles. On the contrary, Ef varies considerably with the protein concentration for a given time of heating (all other conditions being the same), the law of variation not being simple, chiefly at the low concentrations, as we see from table 4 ; but Lf increases with q c a s follows from the theory of the end-to-end aggregation (see below).TABLE 3.-vARIATION OF Lf WITH T FOR SERUM ALBUMIN c = 2-4 % ; pH = 4.15 ; M/50 acetate buffer T ("C) 50 57 65 80 90 100 Lf (A) < 300 1200 1600 2400 2600 2800 TABLE 4.---VARIATION OF If WITH C OF SERUM ALBUMIN T = 100°C ; t = 20 min ; pH = 4.15 ; M/50 acetate buffer c (%I 0.3 0.6 1.2 2.4 r, (A) 3 50 1600 2000 2750 The variation of the aggregation as a function of the pH was systematically studied.7, 8 Generally speaking, the size and mainly the rate of polydispersity of the aggregates in- crease with proximity to the isoelectric point.If the pH of the solution is very near the isoelectric point, the aggregation is no longer linear but random, the solution becoming turbid and precipitation frequently occurs. The aggregates built up by heating are not stable indefinitely and the size of the particles spontaneously decreases ; when the aggregate dissociation starts, n decreases rapidly (see table 5). If the spontaneous change of an initially slightly aggregated solution occurs at moderate temperature, some particles dissociate while others aggregate and the system becomes more homogeneous, the particles being larger but less numerous (see table 6). TABLE 5.-sPONTANEOUS VARIATION OF AND 7~ FOR SERUM ALBUMIN c = 2.5 %; pH = 7 ; t = 10 min; T = 80" C ; without buffer after : Oh 15 h 40 h I f (A) 2600 2450 1000 71 1-35 1.0 - The results for compressed solutions will be reported in a forthcoming paper.24 Gener- ally high pressures lead to the formation of globular aggregates, the mean diameter of which varies between a few hundred and a few thousand A.Frequently the compressed solutions show photoelastic effect characterized by low values of the extinction angle which are nearly independent of the velocity gradient, and this is similar to the phenomenon observed with partly hydrolyzed alkaline solutions.34 TABLE 6.-sPONTANEOUS EVOLUTION AT MODERATE TEMPERATURE c=2*4%; pH=7; t = 10min; T = 6 0 " C ; withoutbuffer at 37" C during : Oh 1 h 5 h 71 8.7 1.1 0-1 2 x 104 5.7 12.0 8.5 I f (A) 250 700 1200 It was interesting to find the effect of successive heating and compression.Gener- ally, when a solution is compressed after heating the linear aggregation diminishes, as is seen in table 7. In some cases the decrease of the linear aggregation is accompanied by a globular aggregation as is shown in table 8. Conversely, by heating a previously compressed solution the globular aggregation diminishes and is replaced by a linear aggregation, but to a degree less important than by direct heating, as one can see in table 9. It is recalled that the horse serum albumin solutions frequently show photoelastic effect or dynamic t~rbidity.7.8~25E. BARBU AND M . JOLY 83 TABLE 7.-EFFECT OF PRESSURE A m R HEATING 2.4 % serum albumin heated for 10 rnin at To C and then for 1 h at 37" C under pressure p P H T ("C) P (atm> I/(& 7l 2 x 104 7-0 60 1 - - 1000 7.1 80 1 - - 1000 7.3 65 1 7.7 60 1 - I 1000 - 65 1 - - 1000 - - 1000 < 700 750 400 250 850 350 600 150 750 600 1.1 2-3 5.8 10.0 - - 2.3 16.0 1.8 3.0 12.0 4.8 8.3 1.0 24-0 4.7 6.3 3.3 4-5 - TABLE EFFECT OF PRESSURE AFTER HEATING 2.4 % serum albumin heated for 10 rnin at To C and then for 1 h at 37" C under pressure p (in M/50 acetate buffer for the sample at pH 4.4) PH T ("C) JZ ;? x 103 2 (A) - 2.5 - 600 - 750 - 750 1.7 - - 350 0.9 - - 450 BOVINE SERUM ALBUMIN.-The aggregation ability of the bovine serum albumin is much weaker than that of horse albumin as shown in table 10.Dynamic turbidity appears frequently and greatly increases the polydispersity.Near the isoelectric point the side- by-side aggregatior: grows, inducing a net decrease of 3 (e.g. for a 2.4 % solution at pH 4.4, heated I0 min at 80" C, r f = 900& n = 5.3, 2 = 4.2 X 10-4). The addition of electrolytes during the thermal treatment greatly increases the aggregation (e.g. at pH 4-2 in acetate buffer M/50, with a 2.4 % solution after 10 min heating at 80" C, 6.9, the heated solutions of bovine serum albumin show photoelastic effect. Thus, a solution at 2-4 % and pH 6.9 heated 10 rnin at 80" C does not show streaming birefringence if M/IO NaCI is added, which proves that (in the limits of the sensitivity of the method) it does not con- tain aggregates larger than 300A. On the contrary, when salt is not added for the measurement, the same solution shows streaming birefringence (see table 1 l), from which we deduce the equivalent values 25 [ I f ] = 4800 A, [n] = 6.7 and [A] = 2.8 x 10-5.= 2700 A, 'ii : 4.2, A = 3.9 x 10-3). For the pH values, < 3.9 or TABLE EFFECT OF HEATING AFTER COMPRESSION 2.4 % serum albumin at pH 7.3 compressed for 1 h at 37" C underp atm or heated for 10 rnin at 65" C P T V C ) +(A) A x 103 d(A) 8 50 l h 8000 10 min - 65 8 50 2.4 - 65 900 0.35 - - - - - - - aooo - r l h I+ 10min - Globular aggregation is induced by high pressure with greater difficulty than is found with horse serum albumin. Also aggregation by compression is more difficult than aggregation by heating. For instance, a solution of 1 % bovine serum albumin at pH 7-15 in phosphate buffer 0.= 0.15) does not produce aggregates after 1 h at 37" C under a pressure of 8000 kg/cm2 while the same solution heated 10 rnin at 80" C gives linear aggregates ( r f = 400 A, 3 = 2.6 s 10-2).84 GLOBULAR-FIBROUS PROTEIN TRANSFORMATlON TABLE 1 O.-HEAT AGGREGATION OF BOVINE SERUM ALBUMIN c = 2.4 ; t = 10 rnin ; T = 80" C ; without buffer PH lf& 2 x 103 4.2 850 0.8 4.3 6.3 1600 1.6 3.5 6.6 650 7.9 1.8 TABLE 1 1 .-PHOTOELASTIC EFFECT OF BOVINE SERUM ALBUMIN SOLUTIONS g (sec)-1 328 638 1195 2585 5500 X O 13.9 13.6 13.3 12.7 12.2 (ne-llO) x 107 1.0 1.2 1.2 1.6 1.8 iz ACTIN FROM RABBIT MUSCLE.-In its globular form (G-actin), the freshly prepared actin does not show streaming birefringence. It in fact comprises small globular par- ticles 35,36 with a molecular weight 19 of about 70,000.By storage at low temperature (near 0" C) linear aggregation (with high polydispersity) appears spontaneously in the actin solutions. For instance, a 0.017 % solution at pH 6.6 after a 2-days' storage contains aggregates in which If = 3950A, T = 6-6 and 2 = 9 x 10-3 ; a 0.034 % solu- tion at pH 6.6 gives after 10 days - 6150 A, T = 8.2 and A = 3.6 x 10-3. This spon- taneous aggregation is diminished by heating (e.g. a 0.068 % solution at pH 6.95, heated 10 rnin at 100" C after 3 days' storage, gives If= 2950A, T = 6.1 and ;? = 4.5 x 10-3). Likewise the aggregates are partly destroyed by high pressure (e.g. the same solution as above compressed 1 h at 37" C under 8000 atm gives lf.=2200 A, T-6 and ;?=56x 10-3).The modes of transformation G-actin --f F-actin have been studied by many authors 33-42 and the formation, probably in two steps,40,43 of very long filaments seems now well established by electron microscopy, and is in agreement with our streaming birefringence data. Thus a freshly prepared solution at 0.017 % of G-actin at pH 6.85 gives on addi- tion of M/10 KCl (after 30 min at room temperature) very polydisperse linear aggregates of F-actin (b= 113OOA, T = 1 1 , 2 = 4.7 x 10-2). With the same solution one finds by viscometry an axial ratio equal to 64, leading to a mean diameter of 170A (electron micrographs give 100-200 A). The decrease of the rate of the transformation G-actin --f F-actin after storage in an ice-box was reported by Straub.44 We found that the number, as well as the length, of the aggregates diminishes ; e.g.for a 0.017 % solution at pH 6.3 after 4 days' storage the aggregates obtained by adding M/10 KCl (after 24 h at room temperature) correspond to If= 9250b1, T = 8.9 and 2 = 8.1 x 10-3). The effect of KC1 concentration is summarized in table 12. It was interesting to discover the effect of heat or pressure on the G-actin -+ F-actin transformation induced by KC1. Generally both factors reduce the length of the aggregates; e.g. the most TABLE 12.-THE G-ACTIN -+ F-ACTIN TRANSFORMATION c = 0.034 % actin stored for 8 days ; pH 6.5 ; 24 h of contact with KCl - [KCU 'f (A) 7c A X 103 M/100 8750 8.7 3.3 M/10 9250 8-9 8.6 M/2 3950 6.5 5-0 frequent length I f = 9250 A of the aggregates obtained by adding M/10 KCl to a 0.034 % solution at pH 6.5 after 4 days' storage is reduced to 5800 8, by 10 min heating at 65" C, to 2050A by 10 min heating at 100" C and to 4050 8, by compressing for 1 h at 37" C under 8000 atm.The effect is similar if KCl is added after heating or compression ; e.g. the addition of M/lO KC1 (24 h of contact) to 0.034 % solutions of G-actin at pH 6.8 (after 10 days' storage) untreated, heated for 10 rnin at 60" C and compressed for 1 h at 37" C under 8000 atm, gives rise (at practically the same rate of transformation) to elongated aggregates of Factin, the most frequent lengths of which are respectively 8900 A, 7000 8, and 5800A. HEN EGG ALBUMIN.-The heat denaturation of egg albumin has been exhaustively studied by Foster and his co-workers,'L 12 who have shown the importance of the heat aggregation at different pH values and the existence of a minimum particle length (about 300 8, for c -- 0.39 %, t -- 15 min, T = 100" C) near pH 2-2.5.These authors suggest an unfolding of the egg albumin molecules to explain the behaviour of the solutions.E . BARBU AND M. JOLY 8 5 Our experimental conditions are a little different from their conditions : our egg albumin was prepared by their method but was neither lyophilized nor diluted with glycerol. We have only studied solutions which have been heated for 10 min at 80' C and at pH values between 2 and 4. The behaviour of the egg albumin differs largely from that of serum albumin. A 2.4 % solution (heated for 10 min at 80" C) of serum albumin was clear even at pH 4.5 (0.3 below the isoelectric point), while the egg albumin solution was very turbid from pH 4 (0.5 below the isoelectric point) and shows dynamic turbidity.The serum albumin did not give rise to aggregates detectable by streaming birefringence by heating below pH 3.5, while the egg albumin was greatly aggregated at very low pH value. 2 was always much weaker for egg albumin than for serum albumin; its maximum was at pH 3.95 (2 = 1.1 x 10-3) for particles of I f = 2100 8, and n 1 1.8. At pH 4 the par- ticles were a little longer ( I f 2 2500 A) but a little less dissymmetrical (2 = 9 x 10-4). Between pH 3.8 and 2, the relatively low values of the birefringence n,-no associated with the low values of the extinction angle X suggest the behaviour of large rather sym- metrical particles, the size of which reaches a minimum at pH 2-7.Table 13 gives a few values obtained by the extrapolation method.24 TABLE 13 .-HEAT AGGREGATION OF EGG ALBUMIN c = 1.6 %; f = 10min; T = 80°C PH 3.8 3-7 3.5 3.2 2.8 2.5 2.0 d (4 1050 750 700 650 600 600 800 - [A] x 104 0.9 2.8 0.8 1.7 2.1 2.0 1.4 By adding NaCl to solutions at pH 2.5 after heating, the particle size did not change as long as c[NaCl] < M/20. For M/10 NaCl the size decreased slightly (li = 550A) while the dissymmetry increased ([A] = 4.9 x 10-4 instead of 2 x 10-4). In M/6 NaCl large globular aggregates grow (d N 850A, [A] = 1.3 x 10-4); in M/5 NaCl the solu- tions became extremely turbid, and later the precipitation occurred. The dilution of these solutions gave rise to a large increase in the size of the aggregates : e.g.d was 850 8, instead of 600 8, by dilution to 0-8 % in H20 and nwas 900 8, instead of 550 8, by dilution to 0.8 % in M/10 NaCl, but without any striking change in [A]. The particles obtained by heating a more dilute solution (c = 0.4 %) at pH 2.5, with or without the addition of M/20 NaCl after the heat treatment, were practically of the same length as those produced in a 1.6 % solution, but were much more dissymmetrical. Therefore the increase of concentration greatly promotes the side-by-side aggregation. At pH 10 a solution at 1.6 % egg albumin showed without heating a very marked photoelastic effect (&] = 5350& [n] = 5.5, [A] = 9 x 10-5) of the same type as shown by alkaline serum albumin solutions.34 HORSE y-PSEUDO-GLOBULIN.-The behaviour of the globulins is very different from that of the albumins.We have only reported in the present paper results from rela- tively concentrated solutions on the alkaline side of the isoelectric point. One obtains exceptional and unreproducible solutions which do not contain aggregates and do not show streaming birefringence. Generally the concentrated globulin solutions contain, without any treatment, large globular aggregates of small equivalent anisotropy and probably of very weak compact- ness. With the addition of electrolytes (NaC1) the size of these aggregates decreased slightly at low salt concentrations, but increased at high concentrations as it can be seen on table 14. The dilution of the solutions gives rise to a striking increase of the relative number of aggregates, as shown by the variation of [ I ] ; e.g.for a 1.1 % solution at pH 6.1 in M/10 NaC1, [A] = 3.2 x 10-4. The main effect of heat is to augment the dissymmetry of the particles and, in fact, to tiansform the globular into elongated aggregates. Thus, for 3.3 % solutions at pH 8.3 heated for 10 min, the shape and the size of the particles remain constant until 50" C with 2 = 850 A and [A] = 6.6 x 10-5 ; then the aggregates lengthen progressively and at SO" C (always for 10 min heating) one observes elongated particles with I ) = 1900 A, T = 5.9 and 3 = 2.5 x 10-4.86 GLOBULAR-FIBROUS PROTEIN TRANSFORMATION TABLE 14.-THE EFFECT OF ELECTROLYTES ON THE SPONTANEOUS AGGREGATION OF THE y-PSEUDO-GLOBULIN c = 3.3 % PH [NaCl] 2 (A) [s] X 105 6.1 0 900 6.8 - M/5 700 8.2 - M12.5 900 5-7 7.9 0 800 6.4 - M/5 900 4.3 The solutions heated above 80" C show dynamic turbidity; the flow induces the formation of large aggregates of low dissymmetry, of which the diameter is about 1000 A.Likewise a 3.3 % solution at pH 7-9 containing initially globular aggregates with 2 = 800 A and [z] = 6-4 x 10-5, shows after 10 min heating at 80" C elongated particles with & = 1450A, T = 5.5 and ;? = 2-5 x 10-4. By adding a small amount of electrolyte after heating, the length of the most frequent particles decreases slightly but their number increases (e.g. for the preceding solution, on adding M/10 NaC1, &varies from 1450A to 1350A and 3 from 2.5 x 10-4 to 3.9 x 10-4). On the contrary, in the presence of a large amount of electrolyte the solutions become turbid and sometimes precipitate (e.g.in M/5 NaC! the preceding solution is completely opaque). THEORETICAL TREATMENT OF THE AGGREGATION PROCESS The first theory of the aggregation of colloidal particles was proposed by Smoluchowslu 45 for the globular aggregation of spherical particles, the process being considered as a diffusion process. By assuming the probability E for two particles to remain aggregated after collision to be independent of the particle size, and by setting nk the number per cm3 of particles comprising R molecules, Rk and Dk being the action radius and the diffusion constant of the particles, the variation of nk as a function of time is given by i = l with h defined by 2 ini = no, no being the initial number of molecules per cm3.Smoluchowski assumes in a first approximation (Q + Dk)(Ri + Rk) = 2 DR, D and R being the diffusion constant and the diameter of the initial molecules. By setting i- 1 p = 47rDRE and i - A ni = N* i - 1 the total number of particles per cm3 at the time t , we find and ylk rises to a maximum at the timeE . BARBU AND M . JOLY 87 With numerous assumptions Kleczkowski 10 showed that Smohchowski’s theory fits for the aggregation (studied by salt precipitation) of diluted (0.25 - 1 %) solutions of human serum albumin (PH 6-8, phosphate buffer M/15) heated for 10 min at 83” C. La Mer46 extended the collision theory to charged particles and ions, and Collins,47, 48 by a more rigorous treatment, has shown that the calculation of Smoluchowski is valid only for low values of E and for small enough particles.Donnet and Sadron 49 have checked experimentally the validity of the Stokes relationship and of the equations of Brownian motion and diffusion for particles as small as 100 A, which justifies the application of the diffusion theory to the ag- gregation process. But in spite of these improvements, the theory of Smoluchowski does not suffice to explain all the experimental features of the protein aggregation. Particularly this theory cannot explain the spontaneous dissociation of the aggre- gates, the limited values of the particle sizes, the relative homogeneity of the solu- tions and particularly the linear aggregation in the globular-fibrous protein trans- formation.By plotting the equipotential curves of the interaction between one charged particle and one aggregate made of two such particles, Rees 50 has shown that the potential barrier to be surmounted by the single particle in order to collide with the aggregate falls to a minimum at the ends of the double particle. Therefore, in explaining the linear aggregation, we shall follow a treatment analogous to the polymerization kinetics developed by Tobolsky.51 With the same notations as above and by setting Aim equal to the addition fre- quency of the particles containing I and m molecules, and Bq to the dissociation frequency of the particles made of q protein molecules, we can write He found k < 7 and E N 10-8. The dissociation of a linear aggregate corresponds to the breaking of the temporary bonds between two elementary joined particles; one can therefore put as a first approximation Bq = B, independent of the particle size.By generalization of Frenkel’s relationship 52 we can write N being Avogadro’s number, A4 the molecular weight of the protein, k Boltzmann’s constant, and W the mean value of the interaction energy between one molecule and all its neighbours in the same aggregate. We have now to calculate Aim. Consider two rod-shaped particles AB and CD (fig. 5), whose centres are 0 and P. For linear aggregation of these particles it is necessary that : (i) B and C collide, i.e. they diffuse towards each other and they surmount the intervening potential barrier; (ii) the angle AB-CD is smaller than a given angle a ; (iii) the protein molecules in B and C are activated at the time of collision so that the two particles form one single aggregate.If AFis the free energy of this activation, corresponding probably to the freeing of one or several functional groups initially coupled inside the molecule, the prob- ability that the molecules in B and C will be activated at collision time is wa = exp (- AF/kT). We shall call this activation “ predenaturation ”. If U is the height of the potential barrier between the particles, only kinetic energies greater than U allow the particles to collide. Following Rees,sO U is a minimum at the ends of the particles and, according to the bead structure of the rodlike particles, we can suppose that this minimum value is independent of the88 GLOBULAR-FIBROUS PROTEIN TRANSFORMATION particle length.By assuming the validity of the Maxwell-Boltzmann law for the whole of the aggregates, the probability for a particle to surmount the potential barrier of height U is Wb = 1 - - (kT)-”’[ exp (- z/kT) 42 dz = F(U). 2 U d; 0 Table 15 gives a few values of this function. TABLE 15.-vALUES OF Wb = F(U) UIkT wb UIkT *b UIkT wb 0.0 1.0 1 0.57275 5 0.0 195 0.01 0.99925 2 0.2625 7 0.003 15 0.1 0097765 3 0.1 125 10 0~00022 0.5 0.80135 4 0.04655 20 0*000004 Without the potential barrier, the collision frequency of particles having diffusion constants Di and Dm and an action radius RIm is (Dl + Dm)Rlm. For a rod-shaped particle made of 1 spherical molecules of diameter R aggregated end-to-end, the mean value of DI is - 21 (7 being the solvent viscosity).As the efficient 3 q l R ’ A I FIG. 5.-End-to-end aggregation of two rod-shaped particles. collisions are only by the ends, and as the angle a is assumed to be small, and therefore kT(1 + m) (- In 21 + !!!?), 6n7 1 m (01 + Dm)RIm which we can replace in practice by kT(1 + m)2 27~7Im ’ Among all the particles which have diffused towards 0, only those having their centres at the portion of the spherical surface PP’ (see fig. 5) can collide by their end with B, and the corresponding probability is f = a2/(1 + m)2. Thus we can write For greater simplification, according to the dissymmetry of the potential energy around each particle, we can introduce an effect of mutual orientation of theE . BARBU AND M. JOLY 89 particles in contact, this effect being stronger the longer the particles.It is therefore not necessary to keep cc constant and it seems sufficient for a linear aggregation that cc is small as m and 1 are small. So we can, for instance, set ct2 = (ml, 5 being a numerical factor about 10-2. At least, independent of I and m, and the general equation for end-to-end aggregation is For globular aggregation, we can assume with Smoluchowski that in a first approxi- mation and thus i.e. the same relationship as above, but with 4 = 4/3. It is only by a rather rough approximation we can assume B to be independent of the size of a globular aggre- gate and use the same equation for the globular and the linear aggregation. By setting the integration of the general equation leads 51 to nk = noPk-l (1 - p)2 1 with p = - noAt 2 A + (A2 - l)* coth - (A2 - 1)i If we consider the average number x of protein molecules per particle, 1 knk 1 1 - -- 1 x=- 5 nk 1 - p p ) 1 we can in a first approximation substitute it for the molecule number of each particle of the most frequent size and write x == l f / R for the linear and x = __ for the globular aggregates. We find then 4 2 ~ 3 B 2B , B2 ~ Q A 2B B2 + as (a T n$A2 --)* coth - 2 (--- + n$A2 -)*t B 2B B2 )* coth + -)'t ' x = ____ noA 2B B2 n$A2 and as the aggregation is measureable only for B/noA < 1, we have to consider in mactice90 GLOBULAR-FIBROUS PROTEIN TRANSFORMATION in good agreement with the experimental If against t curves, and the value reached at infinite time x, N dg, related to Lf by xoo -= LfIR.than in5--is given by t, = By substituting A and B for x, in their expression as a function of W, U, AF, molecular weight M and concentration c of the protein, one finds (R being the gas constant and k Boltzmann's constant) : - The time ts at which the ratio x/x, rises to a given value s-sufficiently larger J' noAB arctan s. and x, = w d / c exp (W - AF/2kT)F* ( U ) (x) RT f , with w N 0.46 and T N 5.8 for the linear aggregation and w N 5.2 and T N 0.49 for the globular aggregation. Therefore RTctsF(U) arctan s exp (AF/kT) = #--- Y M x, and with C$ N 0.38 and $ N 0.078 for the linear aggregation, and 4 N 0.4 and 4 N 0.9 for the globular aggregation. By applying these results to the experimental data obtained with a solution of 2.4 % horse serum albumin at pH 4.15 heated in M/50 acetate buffer at 65" C or 80" C (see table l), we find W N 16 kT and AF < 10kT.DISCUSSION AND CONCLUSION.-we think that a more systematic study by this method of the cohesion energy W of the aggregates will give a lot of information on the nature of the bonds in the large protein particles. We see that the free energy of the activation, which we called predenaturation, is considerably smaller (3 or 4 times) than that found in the literature 53-56 for the free energy of the protein denaturation. We may inquire if in the ordinary denaturation experiments some secondary process (such as the aggregation) does not increase the apparent free energy. Indeed, if instead of writing that the probability for a particle to collide and to be aggregated as A = a F( U) exp (- AF/kT) (a being a constant), we consider a free energy of activation AF' for the whole process of aggregation by setting A = exp (- AF'lkT), we find, in the same case as before, AF' of the order of 40 kT, in good enough agreement with the values generally given for the free energy of de- naturation.Thus the real protein denaturation (an intramolecular phenomenon) is perhaps only the activation process which we have described above as pre- denaturation, and all the other features generally included in the word denaturation are only secondary effects, involved in all the experiments on the denaturation, but related to the presence of other molecules. The chemical aspect of the protein denaturation was extensively studied,5g9 59 but the physical mechanism of this process was less investigated.In agreement with many authors 53-55,57 we have suggested 7 9 8 that the protein denaturation proceeds by the freeing of groups initially coupled inside the molecule and this allows the formation (in particular places of each molecule) of weak intermolecular bonds like Van der WaaIs forces, hydrogen or salt bridges. This freeing of functional groups certainly corresponds to a profound perturbation of the molecularE . BARBU AND M. JOLY 91 structure but does not necessarily involve considerable unfolding of polypeptide chains. Several authors suggest even for moderate denaturation, extensive unfolding but, it would seem, without very convincing evidence. The existence of particles practically all of the same size does not involve, as suggested by Foster,lz that each comprises only one kind of molecule ; we have seen from the above calculations that the aggregation of molecules does not lead to a random size distribution and that the heated solutions can show relatively low polydispersity.In addition, one has to be cautious in the interpretation of the streaming birefringence data for very acid or very alkaline protein solutions, because they show photoelastic effect depending upon the structure of the solution as a whole instead of upon the particle size. We have noted elsewhere 7,s that many features are in contradiction with a large unfolding of the chains, e.g. variation of Z j and with the pH, tensile strength of the elongated particles, partly reversible behaviour of the denaturation (and even in certain cases, conservation of the immunological properties).Moreoever if the rod-shaped particles were made of one single unfolded chain we could expect an intrinsic anisotropy considerably greater than that observed. If, instead of having a relatively rigid structure, the molecules of denatured protein were randomly folded chains, even for pH values rather far from the iso- electric point (but without hydrolysis), its mean configuration would be quasi- globular .6*-63 To explain the high values of the viscosity and Maxwell effect, it would be neces- sary to consider them as due to the flow ; but to reach the observed particle length, the corresponding velocity gradient should rise to values considerably higher than used in the measurements.64 On the contrary, one frequently finds by examination of the X against g and A against g curves that the particles obtained by moderate heating of dilute solutions or by compression after heating, show a dissymmetry increasing with the velocity gradient and probably comprise more or less folded end-to-end aggregates, which the flow progressively unrolls.It is therefore possible when fibres are ob- tained by extruding and precipitation of concentrated protein solutions at high temperature in alkaline medium or with detergents,l-4 that stretching, unfolding and side-by-side union of such aggregates occur and these comprise end-to-end aggregated globular molecules, instead of the unfolding of the chains of each protein molecule.X-ray diagrams of keratin would then be due to the general orientation of the molecules and not to the unfolding of their polypeptide chains. One has probably the same frame in the natural protein fibres as is observed in electron micrographs .65-68 The linear aggregation of the globular proteins seems to play an important part in the formation of the protein natural or artificial fibres. It is still difficult to say whether the aggregation process (concomitant or not with the denaturation) occurs in one or two steps ; it seems to be a two-step mechanism for certain concentrated proteins.16-403 43 To emphasize the biological importance of the globular-fibrous protein trans- formation is redundant. As a general conclusion perhaps we may quote a sentence of Szent-Gyorgyi : 44 ‘‘ It may be asked whether the globular form is not the basic form of protein and whether all fibrous proteins are not built up of globular par- ticles.” Our sincere thanks are due to Prof.Schapira, who provided us wth the actin sample, to Miss Croissant who carried out the electron microscopy work, to Mr. Wielgosz for his aid with the ultracentrifuge, to Mr. Basset, who helped us greatly with the high pressure experiments, and to Mr. Bjormholm and Mr. Rebeyrotte for their experimental assistance in the viscometric and electrophoretic measurements.92 GLOBULAR-FIBROUS PROTEIN TRANSFORMATION Palmer and Galvin, J . Amer. Chem. SOC., 1943, 65, 21 87. Senti, Eddy and Nutting, J . Amer. Chem. Soc., 1943, 65, 2473. 2 Putnani and Neurath, J. Biol. Chern., 1943, 150, 263.4 Senti, Copley and Nutting, J. Physic. Chem., 1945, 49, 192. 5 Joly, Bull. SOC. Chim. biol., 1948, 30, 398 ; 1949, 31, 105. 6 Bourgoin and Joly, J . Chim. Phys. (in press). 7 Joly and Barbu, Bull. Soc. Chim. biof., 1949, 31, 1642 ; 1950, 32, 116, 908. 8 Joly and Barbu, J. Chim. Phys., 1951, 48, 636. 9 Joly, Kolloid-Z., 1949, 115, 83. 10 Kleczkowski, Biochem. J., 1949, 44, 573. 11 Foster and Samsa, Science, 1950, 112,473. 12 Foster and Samsa, J . Amer. Chem. Soc., 1951, 73, 3187, 3190, 5388. 13 Jaggi and Waugh, Fed. Proc., 1950, 9, 66. 14 Waugh, Amer. J. Physiof., 1941, 133, 484. 15 Waugh, J. Amer. Chem. Soc., 1946, 68, 247. 16 Farrant and Mercer, Biochim. Biophys. Acta, 1952, 8, 355. 17 Waugh and Livingstone, J. Physic. Chem., 1951, 55, 1206.18 Sorensen and Hoyrup, C. Lab. Carlsberg, 1915-17, 12, 12. 19 Straub, Inst. Med. Chem. Univ. Szeged, 1942, 2, 3 ; 1943, 3, 23. 20 Frey-Wyssling and Weber, Helv. chim. Acta, 1941, 24, 278. 21 Scheraga, Edsall and Gadd, J. Chem. Physics, 1951, 19, 1101. Z2 Joly, Trans. Faraday Sac., 1952, 48, 279. 23 Joly, J. Chim. Phys., 1951, 48, 536. 24 Barbu, Basset and Joly, to be published. 25 Joly and Barbu, to be published. 26 Macheboeuf, Rebeyrotte and Brunerie, Bull. Soc. Chim. biol., 1951,33, 1953. 27 Wielgosz, to be published. 28Basset and Macheboeuf, Compt. rend., 1932, 195, 1931 ; 1933, 196, 1540; 1933, 197, 796 ; 1935, 200, 1072. 29 Guth, Kolloid-Z., 1936, 74, 147 ; 1936, 75, 5. 30 Guth, Gold and Simha, Kolloid-Z., 1936, 74, 266. 31 Dervichian, Fournet and Guinier, Bull. Soc. Chim. biof., 1949, 31, 101. 32 Barbu, Macheboeuf and Rebeyrotte, to be published. 33 Joly, J. Physique, 1951, 12, 900. 34 Barbu and Joly, Bull. Soc. Chim. biol., 1950, 32, 123. 35 Jakus and Hall, J. Biol. Chem., 1947, 167, 705. 36 Astbury, Perry, Reed and Spark, Biochem. Biophys. Acta, 1947, 1, 379. 37 Feuer, Molnar, Pettko and Straub, Hung. Acta Physiol., 1948, 1, 150. 38 Hall, Jakus and Schmitt, Biol. Bull., 1946, 90, 32. 39 Snellman and Erdos, Biochim. Biophys. Acta, 1948, 2, 660. 40 Rozsa, Szent-Gyorgyi and Wyckoff, Biochim. Biophys. Acta, 1949, 3, 561. 41 Dubuisson, 1st Int. Congr. Biochem. (Cambridge, 1949), p. 132. 42 Dubuisson, Biochim. Biophys. Acta, 1950, 5, 426. 43 Szent-Gyorgyi, Faraday Soc. Discussions, 1951 , 11, 199. 44 Szent-Gyorgyi, Acta Physiol. Scand., 1945, 9, suppl. 25, 33. 45 Smoluchowski, Z. physik. Chenz., 1918, 92, 129. 46 Umberger and La Mer, J. Amer. Chem. Soc., 1945, 67, 1099. 47 Collins and Kimball, J. Colloid Sci., 1949, 4, 425. 48 Collins, J. Colloid Sci., 1950, 5, 499. 49 Donnet and Sadron, Compt. rend., 1952, 234, 69. 50 Rees, J. Physic. Chem., 1951, 55, 1340. 51 Blatz and Tobolsky, J. Physic. Chem., 1945, 49, 77. 52 Frenkel, Kinetic Theory of Liquids, Oxford, 1946, p. 2. 53 Eyring and Stearn, Chem. Rev., 1939, 24,253. 54 Bull, Adv. Enzym., 1941, 1, 1. 55 Stearn, Adv. Enzym., 1949, 9, 25. 56 Sizer, Adv. Enzym., 1943, 3, 35. 57 Mirsky and Pauling, Proc. Nut. Acad. Sci., 1936, 22, 439. 58 Neurath, Greenstein, Putnam and Erikson, Chem. Rev., 1944, 34, 157. 59Anson, Adv. Protein Chem., 1945, 2, 361. 60 Sadron, J. Chim. Physique, 1946, 43, 142 and 1947,44, 22. 61 Guinand, Boyer, Kawenoki, Dobry and Tonnelat, Compt. rend., 1949,229, 143.E. BARBU AND M. JOLY 62 Kuhn, Bull. SOC. Chim. Belg., 1948, 57, 421. 63 Hermans, Bull. SOC. Chim. Belg., 1948, 57, 164. 64 Frenkel, Acta Physicochim., 1944, 19, 51. 65 Astbury, Brit. J. Derm. Syph., 1950, 62, 1. 66 Noda and Wyckoff, Biochem. Biophys. Acta, 1951, 7, 494. 67 Pratt and Wyckoff, Biochem. Biophys. Acta, 1950, 5, 166. 68 Weibull, Faraday Soc. Discussions, 1951, 11, 195. 93

ISSN:0366-9033    

DOI:10.1039/DF9531300077

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

E. BARBU AND M. JOLY 93 GENERAL DISCUSSION Prof. H . Neurath (University of Washington, Seattle) said : There is a question in my mind whether Dr. Doty and Myers have not subjected their experimental data to a more rigorous interpretation than is warranted by the material under investigation. Previous light scattering measurements from our laboratory 1 and from Dr. Doty’s 2 have demonstrated that different preparations of crystalline zinc-insulin may differ significantly from one another in their scattering properties These variations are not merely due to differences in species from which the com- mercial preparations were prepared, nor due to different commercial sources but they persist even if different lots of crystalline beef insulin obtained from the same commercial source are compared to one another.Even measurements of the same lot in the hands of two groups of investigators (in Seattle and Cambridge, re- spectively) have not yielded entirely concordant results. While it would take us far afield to speculate about the causes for these discrepancies, the important point which follows from the present consideration is that the thermodynamic constants calculated from the measurements by Doty and Myers need not, and quite probably do not, have absolute significance. Although the question of the minimum molecular weight of insulin has not specifically been raised by the authors, I should like to comment on it, since it has been subject of a controversy to which I should like to contribute the results of more recent ultracentrifugal experiments by Dr.Frank Tietze and myself. In these measurements, the experimental conditions described by Fredericq and Neurath 3 for the dissociation into a 6,000 molecular weight unit were observed, i.e. phosphate buffer, pH 2.6, ionic strength 0.1, but measurements at 0-2 ionic strength were also made. The present measurements extend over a wider con- centration range than those of Fredericq and Neurath and approximately 20 experimental points were determined under each set of conditions to define the dependence of sedimentation constants on insulin concentration. The results, shown on the slide, and to be published in detail elsewhere, when extrapolated to zero protein concentration yield a sedimentation constant of s20 = 1-75 S which is compatible with a molecular weight of 12,000 rather than 6,000.While these findings remove the last discrepancy from the reported values for the minimum molecular-kinetic unit of insulin as obtained by physicochemical methods, the value of 6,000, obtained by Harfenist and Craig 4 by countercurrent distribution studies on partially substituted dinitrophenyl insulin cannot be ignored. Indeed, a simmering thought remains in my mind that in the final solution of this problem the tool of the organic chemist may yet prove to be sharper than that of the physical chemist . Referring to the paper by Dr. Barbu and Joly, I should like to caution against undue generalizations concerning the mechanism of denaturation processes. Proteins differ within wide limits in their susceptibility to denaturation and various denaturation agents may have quite different effects on the structure of 1 Tietze and Neurath, J.Biol. Chem., 1952, 194, 1 . 2 Doty, J . Amer. Chem. SOC., 1952, 74, 2065. 3 Fredericq and Neurath, J. Arner. Chem. SOC., 1950, 72, 2684. 4 Harfenist and Craig, J. Amer. Chem. Soc., 1952, 12, 308.94 GENERAL DISCUSSION a given protein. The effect of heat depends on its intensity (time, temperature) as does that of urea or guanidine hydrochloride (time, concentration) and it appears that in general, the latter agents are more effective than the former in inducing unfolding of polypeptide chains. Dr. A. Wassermann (University College, London) said: The suggested explana- tion of the small entropy change accompanying the dissociation of the insulin dimer refers to effects which play a role both in simple electrolytic dissociations and in the equilibria of uncharged species in inert organic solvents.An extreme case of this latter kind is the thermal dissociation of dianthracene into mono- anthracene, which is accompanied by an entropy decrease (unpublished calcula- tions, based on Weigert’s measurements). In the reaction discussed by Doty, and in other dissociation processes of the type a + b + c, the possibility has to be taken into account, moreover, that the vibrational entropy of a is considerably larger than the sum of the vibrational entropies of b and c. This effect will be particularly important if a is a complex species in which vibrational or torsional modes of very low frequency occur.Dr. P. Doty (Harvard University) (communicated) : Our contribution to this discussion aims to support the view that in acid solutions of insulin a rapid dynamic equilibrium exists among the 12,000 units and multiples thereof and that the equilibrium constants involving monomers, dimers, and trimers can be determined from light scattering measurements. This conflicts with the osmotic pressure measurements of Gutfreund which do not reveal any simple equilibrium relations and with the sedimentation measurements which do not appear to permit as high an average molecular weight as we obtain. Consequently we have, with the help of Prof. Masaji Kubo, extended our measurements to test the self- consistency of our conclusions over the greatest range possible. Indeed, part of our argument rests upon the fact that under numerous conditions where the monomer-dimer equilibrium is dominant, the light scattering measurements can be fitted over as much as a fifty-fold range of concentration with a single equilib- rium constant.This observation, together with the fact that the values of the equilibrium constants vary with conditions in an intelligible manner, is the best refutation to the suggestion that random amounts of impurities invalidate our conclusions. Inasmuch as Gutfreund had found that dissociation to the monomer was essentially complete in citrate solutions, we have carried out measurements in sodium -+ citric acid buffer of ionic strength 0.1 and pH 2.6. Dissociation was found to be only slightly greater than for phosphate (Kzl = 6 x 10-4).Another question of interest is the maximum number of monomers that can combine into a polymer. The decreased solubility with increasing pH may be due to the formation of very large polymers. Or, on the other hand, a certain polymer size may be required to act as the nucleus for phase separation. Ex- amination of saturated solutions above pH 4 shows the maximum molecular weight to lie between trimer and tetramer, indicating that the tetramer is the highest polymer formed. The effect of ionic strength was examined in phosphate buffer at pH 1.9 by varying the ionic strength from 0-05 to 0.2. The value of K21 fell continuously from 10 to 4-8 x 10-4, showing the expected effect of increasing dissociation with ionic strength. At ionic strength of 0.05 the Ke/R90 plot showed for the first time a positive slope (at concentrations greater than 0.5 %).This is to be anticipated as the ionic strength decreases and its occurrence sets a limit to the ionic strength range in which we can assume that the value of B in eqn. (1) is negligible. It should be emphasized that the absence of a positive slope at 0.1 ionic strength is the best direct evidence that B is essentially zero in the experiments reported here. With respect to the lack of reproducibility of results mentioned by Prof. Neurath, we have examined the effect of filtration of insulin solutions throughGENERAL DISCUSSION 95 sintered-glass filters and find that this increases the dissociation somewhat, al- though not enough to account for all the difference between his results and ours on the same insulin sample. We have encountered no irreproducibility in our own work even though measurements have been made by different persons over a period of two years.Only slightly different results have been obtained with a sample of Boots' insulin. Prof. F. Haurowitz (Indiana University, U.S.A.) said : Entropy increases have been observed also when proteins combine with dyes (Klotz) or when antigen pro- teins combine with antibodies (Haurowitz and Sowinski). In both these cases the interpretation was that bound water molecules are released. In the antigen antibody combination it could be shown that the entropy increase did not depend on the ionization of the combining groups. Has Dr. Doty any reason to attribute the entropy increase to the dehydration of ionic groups? Dr.L. Nanninga (Leiden) said: From the agreement of the equivalent weight 800 from the lowest P content with M = 24000 for DFP-trypsine, it follows that there is 1 DFP to 1 trypsine mole/mole. It follows that trypsine has one active group per molecule. This agrees with the results of Hartley and Kilby 1 on chymo- trypsine. Prof. W. T. Astbury (Lee& University) said: With regard to the paper of Barbu and Joly, it has not been claimed, from our own X-ray studies at least, that the denaturation of corpuscular proteins leads always to complete unfolding of the polypeptide chains, but rather that there is a disorganization tending more and more in that direction according to the severity of treatment. Almost always, though, some kind of a P-diagram is obtained, especially after stretching the de- natured product, and it is very hard to believe that, in general, this can arise from a regular end-to-end aggregation of the original corpuscular units.Such an inference would involve the assumption that most corpuscular protein molecules are similarly constructed from long regular folds of chains in the /3-configuration- for which there is no evidence-and that moreover these folds, after certain heat treatments, tend naturally to fit together so as to form a linear aggregate with the ,&chains pointing accurately along the axis of the aggregate. I do not say that a process of this kind cannot happen; indeed, evidence is accumulating that it does happen in the actual biogenesis of protein fibres, and we have now the very suggestive in vifro models provided by the reversible formation of fibrils from corpuscular insulin, tropomyosin, actin, and perhaps also trypsinogen ; but the experiments described by the authors seem far removed from biogenesis, and I feel that they offer no systematic interpretation of the X-ray findings on protein denaturation in general, including both fibre and film formation, which I believe is based primarily on disorganization followed by chain-unfolding as far as the intramolecular linkages and experimental conditions allow, culminating finally in aggregation into ,8-'uundles.Dr. E. Barbu (Paris) (communicated): Since our paper was written observa- tions have been made on the behaviour of horse y-pseudoglobulin solutions in the acid range, pH 1.5-4.2.We find low values for the equivalent mean aniso- tropy [A] associated with large values of the equivalent length of the most frequent particles [If], what corresponds to large globular aggregates. The mean diameter d of the particles shows a minimum between pH 2 and 3 associated with a maximum By heating the solution of horse y-pseudoglobulin at pH 3.2, d decreases and [A] increases ; by adding salt d becomes larger. The table on page 96 summarizes the results obtained 10 min after acidification of the solutions. Dr. W. E. F. Naismith (Z.C.Z. Ltd., Dumfiies) said: Dr. Barbu and Joly have presented a considerable amount of evidence to show that the globular-fibrous transformation brought about by various treatments of proteins is due to 1 Flartley and Kilby, Biochem.J . , 1952, 50, 672. Is it possible to deduce the nature of this active centre in trypsine? of [ Z ] .96 GENERAL DISCUSSION TABLE 1 .-SPONTANEOUS AGGREGATION OF THE HORSE 7-PSEUDOGLOBULIN IN THE ACID RANGE OF pH c = 3.3 % remarks PH [If1 [%I [;i 1 ri 4.2 3-2 3.2 2.8 2 8 2-3 1.9 1.5 5900 5650 2900 3800 5450 2750 5900 5650 6.4 7.6 6.15 5.9 5-75 6.1 5 6.75 5.6 4-55 x 10-5 5-8 x 10-5 1-01 x 10-4 7 x 10-5 5.1 x 10-5 6-75 x 10-5 4.2 x 10-5 4-8 x 10-5 1000 950 750 775 900 750 97s 950 after 10 min at 80" C in the presence of M/3 NaCl end-to-end aggregation of globular protein molecules rather than unfolding of the polypeptide chain of a single molecule. We are very interested in the mechanism of the globular-fibrous transformation in connection with the spinning of fibre from alkaline solutions of groundnut protein. It has been found that treatment with alkali at relatively high ionic strength causes a considerable increase in the viscosity increment indicating some elongation in the shape of the molecule.The molecular weight of the protein, however, is drastically reduced. These facts can be simply explained on the basis of the Astbury 1 hypothesis that the chains unwind and are then broken up, but could not be so easily explained on the basis of the theory of Dr. Barbu and Joly. Prof. F. Haurowitz (Indiana University, U.S.A.) said: Are we justified to generalize the conclusions of Dr. Barbu and Joly, and to assume that all types of denaturation are due to similar changes from the globular to the fibrous state? In some cases, e.g.in alkali denaturation, true unfolding may play a role. Mr. A. J. Hyde (Bradford Tech. Colf.) (communicated): In Bradford Mr. Manogue and myself have been investigating a globular -+ fibrous change in a different protein from that of Dr. Joly-in fact the whole system was of a different type. The protein was silk fibroin in aqueous solution. Fibroin can be dissolved in water using either cupri-ethylene diamine hydroxide solutions 2 or strong lithium bromide solutions 3 as has been described previously. A 1 % fibroin solution gels spontaneously in about a month giving a thixotropic gel. We have observed the structural elements of this gel in the electron microscope. The particles are rod-like and are approximately lOOA wide and high and have a most frequent length of about 1700A.Addition of almost anything (except alkali and heat) accelerates the gelation (or in some cases precipitation). The protein present in the original solutions could be detected with the electron microscope, but the structure was not resolved. The addition of soap solutions forms rather less elongated rods, and ammonium sulphate forms dense large globules (2000 8, diam.). In solution, the fibroin is in a folded form, probably the a-form of the Courtauld's school 4 with the ribbon of this structure randomly coiled : this makes it a different type of system from Dr. Joly's where there is specific secondary folding in such molecules as serum albumin. A further difference is the complete irreversibility of the transformation, The transformation to the gel form involves a transforma- tion from an a-chain to a @-chain, as has been shown by X-ray diffraction methods ; but clearly there is more to it than this.1 Astbury, Dickenson and Bailey, Biochem. J., 1935, 29, 2351. 2 Coleman and Howitt, Proc. Roy. Soc. A , 1947, 190, 143. 3 Ambrose, Bamford, Elliott and Hanby, Nature, 1951, 167, 264. 4 Elliott and Toms, Nature, 1952, 169.GENERAL DISCUSSION 97 Whilst we were doing this work, Mercer 1 published a picture of similar aggre- gates from the sac of the silkworm and has since informed us privately that he tried to obtain them artificially-but we are unaware of his conditions and reason for failure. The structure and formation of the fibrils could be explained either by uncoiling and aggregation of the polypeptide chains or end-to-end aggregation of globules-however, the latter would require the existence of specifically folded, parallel-chained, roughly isodimensional units as for normal globular proteins.Work is at present in progress on the immediate pre-gelation state and it is hoped that full results will be published shortly. Dr. M. Joly (Paris) said: I would like to emphasize several experimental results, the comparison of which shows that the elongated particles of denatured serum albumin are built up of nearly globular aggregated molecules and not of one completely unfolded polypeptide chain : (i) The particles obtained during the denaturation are frequently much longer than the total length of a polypeptide chain with the same molecular weight as serum albumin.(ii) All other conditions being the same, the length of the particles increases greatly with the protein concentration during the denaturation, a property which is not explainable by chain unfolding. (iii) The length of the particles obtained by heat denaturation increases when the pH values approach the isoelectric point ; unfolded chain would behave in a contrary way. (iv) The length of the particles obtained by heating the protein with salt is greater than without salt, in contradiction to an unfolding process. (v) The length of the particles does not depend on the velocity gradient, con- tradictory to the unfolding of a coil. (vi) The energy required for breaking one particle of denatured serum albumin is considerably lower than the energy of a covalent link. (vii) After denaturation, the particle length decreases spontaneously at room temperature.(vii) Moderate heat denaturation is partly reversible. In reply to Prof. Astbury, the techniques we use do not allow us to judge if the denaturation induces in each globular molecule the ct + /3 transformation of the polypeptide pattern in absence of a general unfolding of the chain. More- over it does not seem from the literature that such structural determination has been made by X-ray study in the denaturation of serum albumin by moderate heating in dilute solution and without stretching the particles. In reply to Prof. Neurath, Naismith and Haurowitz, the process described in our paper concerns serum albumin, egg albumin and y-pseudoglobulin, and is related to the denaturation by compression or moderate heating without a de- naturing agent like urea. This process is perhaps valid also in other cases, but there is not any experimental reason to affirm that is the unique process of de- naturation. In the alkaline denaturation of serum albumin, we have shown 2 that, as long as the hydrolysis does not induce the breaking of the molecules, the unfolding of the chain into a filament does not occur. Without mechanical treatment or precipitation, we observe only the swelling of remaining globular particles (con- sequently the alkaline solutions do not show orientation birefringence), with very strong interactions between the particles inducing high viscosity, photoelasticity of the solutions and, for high protein concentration, gelation according to a process similar to that described by Kirkwood.3 By adding salt, the interactions diminish, the viscosity decreases and the photoelastic effect disappears. 1 Mercer, Nature, 1951, 168, 792. 2Barbu and Joly, Bull. SOC. Chirn. Biol., 1950, 32, 123. 3 Kirkwood, Colloque international m r les changements de Phase (Paris, 195 1) (in press).

ISSN:0366-9033    

DOI:10.1039/DF9531300093

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

III. HIGH MOLECULAR WEIGHT SYSTEMS THE PHYSICQCHEMICAL EXAMINATION OF THE CONARACHIN FRACTION OF THE GROUNDNUT GLOBULINS (ARACHZS HYPUGAEA) BY P. JOHNSON AND W. E. F. NAISMITH* Department of Colloid Science, The University, Cambridge Received 17th April, 1952 Preliminary results are reported on novel association-dissociation reactions occurring in the conarachin fraction of the groundnut globulins. It has been shown by ultra- centrifugation and light scattering that the dissociated state, corresponding to a molecular weight of ca. 190,000, is favoured by high ionic strength and high pH. With lowered ionic strength at constant pH, or lowered pH at constant ionic strength, the degree of association increases, at least four new species, with molecular weights rising as high as 2 x 106, appearing.No indication of the occurrence of these changes was obtained from electrophoresis, so that electrophoretic mobility seems to be independent of degree of association. Light-scattering measurements demonstrated that equilibrium was attained, after change in ionic strength, within 1 min. The globulins of the groundnut (Arachis hypugaea) were first investigated by Ritthausen 1 who extracted the proteins from the oil-free groundnut meal with aqueous sodium chloride and weakly basic solutions, and precipitated them by acidification. He considered the solids so obtained to be identical. Johns and Jones 2 showed that the globulins extracted with aqueous sodium chloride could be separated into two fractions, which they named arachin and conarachin, by an ammonium sulphate fractionation of the saline extract.Jones and Horn 3 later showed that arachin could be obtained from a 10 % sodium chloride extract by dilution or dilution to permanent cloudiness followed by saturation by carbon dioxide, or by 40 % saturation with ammonium sulphate. Conarachin, which was the more soluble fraction, could not be separated by dilution but could be prepared from the filtrate of the arachin precipitation by dialysis against water or complete saturation with ammonium sulphate. Irving, Fontaine and Warner,4 who carried out an electrophoretic investigation of the groundnut proteins in ammonia buffer (ionic strength I = 0.1, pH = 9.26), showed that at least 3 and probably 4 protein components were present, and that arachin and conarachin each consisted of at least 2 of these components.Because the major components of arachin and conarachin had the same electrophoretic mobilities they were regarded by these authors as being identical. Johnson 5 showed, from an ultracentrifugal examination of the proteins, that conarachin, prepared by dialysis of the supernatant liquid after precipitation of the arachin with ammonium sulphate, gave in the ultracentrifuge an ill-defined sedi- mentation diagram for which the sedimentation constant was approximately 10.927. However, the insolubility of the material and its polydisperse character indicated that during dialysis the protein had become altered and partly denatured. Conarachin, prepared by saturation with ammonium sulphate gave small quantities of slower sedimenting materials of s values 4.8 and 6.2s.Johnson, however, did * Present address-I.C.I. Ltd., Nobel Division, Dumfries Factory, Drungans, Dumfries. 98P. JOHNSON AND W. E. F . NAISMITH 99 not definitely apply the name " conarachjn " to any of the above sedimenting species. The present work is concerned with the physicochemical investigation of con- arachin, ultracentrifuge, light scattering, and electrophoretic techniques being employed. It is shown that conarachin contains at least 2 different molecular species ; one of these is very susceptible in its state of aggregation to differences in external environment. In this connection and in its solubility behaviour conara- chin is significantly different from arachin though in other properties surprising similarities are revealed.The various environmental factors which influence the state of aggregation of the conarachin proteins have been investigated and are discussed together with other association-dissociation systems. EXPERIMENTAL APPARATUS.-The ultracentrifugal measurements were made using a Phywe air-driven instrument equipped with a Philpot 6 diagonal Schlieren optical system. In view of con- siderable temperature rise during sedimentation velocity determinations, rotor tempera- tures were recorded throughout and a correction factor evaluated to reduce sedimentation constants to the viscosity and density of water at 20" C . For peaks of reasonable area and resolution, such S& values (given in Svedberg units) are accurate to f 2-3%. Since, in this work, the different components observed are in the main the different aggregated states of a single protein, the relative areas quoted give a useful indication of relative concentrations. In view of other approximations, no correction for dilution in the sectoral ultracentrifuge cell has been applied to area measurements.Peaks are numbered 1, 2, 3, . . . beginning with the most rapid. It should be noted that in the single sedimentation diagrams reproduced here, it is not always possible, owing to the large range of s values, to include all observed components. Diagrams are chosen at a stage of sedimentation which gives resolution of the maximum number of components. The apparatus and methods of Goring and Johnson 7 were used for the measurements of light scattered at 90" to the incident beam and over a GO" arc around this direction.The temperature was 18 f 2" C. The solutions were prepared dust free by ultrafiltration through nitrocellulose membranes as described by Goring and Johnson.8 Using this technique, dissymmetry values as low as 1.04 were reproducibly obtained. Electrophoretic measurements were made in a Tiselius electrophoresis apparatus manufactured by L.K.B. Produkter, Fabriksaktiebolag, Stockholm, and provided with the Svensson modification of the diagonal Schlieren optical system. Changes of ionic strength were routinely performed by dialysis of the protein solution in a slowly rotating Cellophane bag against a large volume of the required buffer solution. Osmotic equilibrium was achieved in ca.1 h and 3 or 4 changes of solvent were always carried out. MATERwLs.-Buffer salts were A.R. or of equivalent purity. The groundnut meal from blanched Spanish Bunch nuts, defatted by extraction with petrol, was supplied by T.C.I. Ltd. (Nobel Division). PREPARATION OF PROTEINS.-The groundnut meal was extracted at room temperature for 4 to 5 h with a 40 % saturated solution of ammonium sulphate. According to previous work3 this should leave the arachin and extract only the conarachin. The latter was pre- cipitated from the extract which had been filtered through paper pulp, by saturating to 85 % with solid ammonium sulphate. No more protein was precipitated by further addition of ammonium sulphate and tests for protein by trichloracetic acid and biuret were negative.The precipitate after redissolving in 40 % saturated ammonium sulphate and reprecipitating at 85 % saturation (40/85 fraction) was stored under 85 % saturated ammonium sulphate. In most of the experiments described, the fractions precipitated over the ranges 40-65 % (40/65) and 65-85 % saturation with ammonium sulphate (65/85) were used. According to the above statements, conarachin is precipitated between 40 and 85 "/, saturation with ammonium sulphate. However, Jones and Horn 3 defined conarachin as the fraction precipitating from 10 % NaCl solution on adding ammonium sulphate to between 40 and 85 % saturation. It has, however, been confirmed that the presence of 10 % NaCl does not appreciably affect the fractionation limits so that the two definitions of conarachin are equivalent.100 GROUNDNUT GLOBULINS Jones and Horn3 state that after the arachin was removed at 40 % saturation with ammonium sulphate from a 10 % aqueous NaCl extract of the meal, there was no further precipitation on the addition of ammonium sulphate until a fraction separated at between 70 % and 80 % saturation.We found that a fraction precipitated (from either a 10 % saline extract after removal of the arachin, or from a 40 % saturated ammonium sulphate extract) at between 40 % and 65 % saturation with ammonium sulphate, and another fraction between 65 % and 85 % saturation. The fractionation, however, was by no means sharp, as will later be seen. Arachin was prepared by precipitation from a 10 % saline extract of the groundnut meal by 40 % saturation with ammonium sulphate and purified by several similar repre- cipitations.drying to constant weight at 120" C for 4 h, conarachin was found by the micro Kjeldahl method to contain 18.2 % nitrogen (cf. 18.3 % quoted by previous workers 3). Using the value 18.2 % the results of fig. 1 were obtained by interferometer measurements and DETERMINATION OF PROTEIN CONCENTRATION AND PARTIAL SPECIFIC VOLUME.-After FIG. 1.-a n (from interferometer) against c for conarachin. were used for all concentration determinations. The partial specific volume V was calculated from the equation,g 1 - - w dm (1 - V p ) = - - m dw' where p = density of the solution for which the solute weight fraction is w, and m = mass of a given volume of the solution. m was determined with a pyknometer for a range of protein concentrations.From fig. 2 in which m is plotted against w, the value ij = 0.72 0.004 was obtained. Absence of curvature indicates the constancy of this value over the concentration range covered. It is of interest to note that the more insoluble globulin from the groundnut, arachin, also has the value 6 = 0-72 f 0-005.5 the different conarachin preparations in phosphate buffer, I = 0.5 and pH 7-5. Three IN PHOSPHATE BUFFER (0.032 M Na2HP04 . 12H20, 0.003 M KH2P04, 0.04 M NaCI) AT ULTRACENTRIFUGE RESULTS : FRACTIONATION.-Tabk 1 summarizes the results on TABLE 1 .-SEDIMENTATION AND DERIVED DATA FOR THE DIFFERENT CONARACHIN FRACTIONS I = 0.5, pH = 7.5 ; PROTEIN CONC. = 0.8 g/100 ml wt.-average mol. wt. peak 1 peak 2 peak 3 --I___ --__--I_ __ _._.__ S s area .? a:7a (calc.) area fraction 0' % 0 A 40185 15 16 8.7 44 2 40 150,000 B 40/65 14 20 - I 2 80 90,000 C 65/85 18 4 8.7 78 2 18 175,000P . JOHNSON AND W . E . F. NAISMITH 101 main components occur in the 40185 fraction, mainly two occurring in the other fractions. The slow component of s w 2s occurs largely in the 40/65 fraction whilst the prominent peak of s = 8.7 is largely precipitated in the 65/85 fraction. Most of the work here discussed is concerned with the latter material. Although the s2 component appears to be inert compared with s8.7 (see later) it is desirable to remove it completely from the fraction but no simple method has yet been devised. On lowering the ionic strength of the 40/85 fraction to I = 0.1, pH = 7.7, a profound change occurred in the sedimentation pattern, the following sio values being observed : (1) 30, (2) 20, (3) 12.5 and (4) ca.2, with the respective percentage peak areas (1) 11 (2) 37 (3) 19 and (4) 33. Apparently the s2 component remains unaltered but the other components disappear completely, new ones appearing. Thus definite evidence of asso- ciation at low ionic strengths was obtained. On adding an equal volume of arachin solution, also at I = 0.1, pH = 7-7, of equal total protein concentration, the resulting sedimentation pattern was in good agreement with that expected from the addition of the separate patterns, arachin appearing as a separate component with sio = 14.2 (cf. sio = 14.6 for arachin alone at Z = O.l).lO Thus distinguishability from conarachin was proved as well as the lack of appreciable interaction effects between arachin and con- arachin components.In a similar experiment with the 65/85 fraction at I = 0.5, pH = 7.5, sedimentation patterns were again apparently additive (fig. 3), arachin appearing as a new w x / 0 2 FIG. 2.-m (mass of a given volume of solution of conarachin for which the solute weight fraction is w) against w. component with s;o = 13.5 to be compared with sio = 13-3s for sedimentation alone at this ionic strength.7 Thus arachin and conarachin are separate and distinguishable sys tems. Since the 65/85 fraction contains a high proportion of the labile s;., component with much smaller amounts of s2 and s15 than the other fractions, further work was concen- trated on it. The ~ 1 5 and 32 components have not, as yet, received any further direct study.Since diffusion data are not available for the different components, molecular weight calculation depends on some type of assumption concerning frictional ratios (flfo). As many seed globulins approximate to ellipsoids of revolution of relatively small asymmetry, fife has been assumed throughout to be 1.3 which allows for a contribution from hydration as well as from asymmetry (e.g. 30 % hydration, axial ratio 4). Table 2 contains observed sedimentation constants and molecular weights calculated thus, both sets of values being suitably rounded. Using these figures and relative proportions as indicated by peak areas, weight average molecular weights were calculated for the fractions and are included in table 1 and elsewhere. * A species of sedimentation constant 8.7 Svedberg units is for convenience termed the sg.7 component.102 GROUNDNUT GLOBULINS 65/85 FRACTION : EFFECT OF VARIATION OF IONIC STRENGTH.-At Constant pH (7.6 & 0.2) and protein concentration (0.8 g/100 ml) the ionic strength of a solution of the 65/85 fraction was varied by stages from 0.5 to 0-025 by dialysis.Sedimentation diagrams and constants are shown in table 3 and calculated weight-average molecular weights and relative areas of the various components in table 4. Since it was not possible to use the same preparation for all experiments, some variability in peak areas is to be expected from fractionation differences. Thus the areas of ~ 1 2 . 5 and s20 components at I = 0.1 TABLE 2.4BSERVED SEDIMENTATION CONSTANTS AND MOLECULAR WEIGHTS CALCU- LATED ASSUMING f/fo = 1 *3.0 820 2 9 12.5 14 20 30 50 (Svedberg units) mol. wt. 19,000 190,000 290,000 380,000 560,000 1 - 1 x 106 2.2 X 106 TABLE 3.-sEDIMENTATION DIAGRAMS AND CONSTANTS * OF THE 65/85 CONARACHJN FRACTION AT pH = 7-6 ( f 0.2) AND PROTEIN CONCENTRATION = 0.8 g/100 ml buffer composition (M) NazHPO KH2P04 NaCl I sedimentation constants 8.7 2 - A 0032 0.003 0.40 0-50 18, B 0-032 0.003 0.25 0.35 20, 9.3 2 C 0016 04015 0.15 0-20 30, 19, 12.6, 2 - - D 0.016 0.0015 0.05 0.10 32 21, 12-5, 2 . _ E 0.008 0.0008 - 0.025 33 20, 12.8, 2 - * any component occurring > 30 % (by area) underlined. TABLE 4.-wEIGHT-AVERAGE MOLECULAR WEIGHTS AND RELATIVE AREAS (%) FOR THE DIFFERENT SEDIMENTING COMPONENTS OF THE 65/85 FRACTION AT pH = 7.6 (4: 0.2) AND PROTEIN CONCENTRATION = 0.8 g/100 ml 330 s20 s12.5 s9 sz wt.- average mol.wt. (calc.) 0.50 - 4 - 78 18 175,000 0.35 - 4 - 84 12 185,000 0.20 4 43 40 - 13 405,000 0-10 4 60 23 - 13 450,000 0.025 3 54 31 - 12 430,000 were somewhat variable even under identical conditions as far as could be judged. How- ever, tables 3 and 4 show clearly the disappearance of S8.7, with lowered ionic strength, the appearance or growth of ~12.5, s20 and to a smaller extent 830, and the relative constancy of ~ 2 . At the lowest ionic strength, sedimentation constants and patterns may be subject to charge effects ; the slight indication of smaller degree of association at I = 0-025 than at I = 0-1 is not therefore necessarily significant.At higher ionic strength than 0.5, no further change in pattern was observed. Weight-average molecular weight values indicate clearly the occurrence of increased association with lowered ionic strength. Replacement of NaCl by Na2S04 at I = 0.5 did not introduce any sedimentation changes ; nor did the addition to a similar solution of CaCl2 to M/1000 or HgC12 to 5 x 10-4 M (i.e. 1 atom of Hg/2 moles of protein) give any sign of change in degree of association (cf. Hughes 11). Since dialysis occurs over several hours, in order to estimate the speed of the processes involved, experiments were performed in which the ionic strength was raised from 0.1 to 0.5 by addition of solid NaCl and lowered from 0.5 to 0.1 by diluting a concentratedA B C FIG.3.-Sedimentation dia- grams of: ( a ) arachin, (b) 65/ 85 conxachin, (c) mixture of (a) and (b) ; phosphate buffx I=0*5, pH=7.5 ; total protein conc. * 0.8 g i l 0 0 ml. A A B B C D C E TABLE 1. TABLE 5 . [To fuce page 102 TABLE 3.A B C D E C TABLE 10. F G TABLE 6. [To face page 103P . JOHNSON AND W . E . F . NAISMITH 103 protein solution with water. In both cases, ultraceiitrifuge runs were imniediately per- formed, sedimentation patterns obtained being characteristic of the final ionic strength with no further changes occurring. Thus the changes occurring were complete within the first hour (see later). Whilst the effects of extreme change in ionic strength seemed completely reversible (i.e. 0.1 --'t 0-5 or vice versa) it was observed that if the intermediate ionic strength 0.2 was approached from higher and lower values, some differences in the sedimentation patterns persisted even after prolonged dialysis.This result is not understood and may indicate the importance of some factor as yet overlooked. EFFECT OF PROTEIN coNcmTRATIoN.-In experiments reported above the protein con- centration was kept constant since it was conceivable that variations in it would affect the equilibrium position. No observable change in the proportions of the sedimenting components was observed at I = 0.5, pH = 7.5, on varying the protein concentration from 0.4 to 2.5 g/100 ml, but at I = 0.1, pH = 7.7, it was found that increase in protein concentration tended to favour greater association (table 5). The constancy of the area of the s2 component is again noticeable, whilst increased total protein concentration causes a transfer from the ~ 1 2 .5 area to that of s20. A simiIar conclusion is reached from the increase in weight-average molecular weight with protein concentration. VARIOUS CONCENTRATIONS IN PHOSPHATE BUFFER : (Na2HP04 . 12H20,0.016 M ; KH2P04, TMLE 5.-sEDIMENTATION AND DERIVED DATA FOR THE 65/85 CONARACHIN FRACTION AT 0.0015 M ; NaCl, 0.05 M) I = 0.1, pH = 7.7 wt .-average mol. wt. peak 1 peak 2 peak 3 peak 4 .__I__ ~ _ _ _ _ protein concentra- tion area area area area (calc.) A 2-10 - * ca. 2 22 74 11-5 12 2 12 475,000 (g/100ml) (yo) (%> (Yo) (70) B 0.85 36 4 23 60 12-4 23 2 13 450,000 C 0.43 33 4 20 56 12.3 28 2 12 440,000 * not measurable because of ill-defined peak.EFFECT OF pH vARIA-rIoN.-Solutions of 65/85 conarachin at constant protein concen- tration (0.8 g/100 ml) and ionic strength (0-l), were prepared at various pH values by dialysis from I = 0.1, pH =I 7.7, and examined in the ultracentrifuge. Summarized data is presented in tables 6 and 7. At pH values below 4 irreversible changes occurred since on dialyzing back to pH7.7, considerable precipitation of protein took place and the soluble protein now gave an altered sedimentation pattern. On the other hand, re- dialysis to I = 0.1, pH = 7.7 after exposure to pH's above 6 gave the characteristic pattern for these conditions. With increasing pH above 6, the constancy of the s2 area was again shown, and there was a gradual transfer from the more rapid to the slower sediment- ing components which was confirmed by weight-average molecular weight calculations. At I = 0.1, pH = 10.3, the sedimentation pattern was very similar to that at Z = 0.5, pH = 7.5, and similar weight-average molecular weights were observed.Thus high pH and high ionic strength affect the degree of association of the protein very similarly. The main peak at pH9.4 was noticeably broader than those obtained at other pH values and the s& value, 14.7, seemed to fall between those of the two well-defined species ~ 1 2 . 5 and s20. Whilst these facts have not been further investigated, it seems possible that some rapid reversible type of reaction (e.g. 2~12.5 + s20) is involved. At I = 0.5, pH variation within the range 9-4-59 had little or no effect on the ultra- centrifugal pattern.Only when the pH was reduced to 4.7 (which is approximately the isoelectric point of conarachin at I = 0.5) was considerable aggregation observed, the pattern in table 6 being obtained. At I = 0-35, lowering of the pH from 7.6 to 6.1 resulted in only slight association. Thus apparently the solvent action of the aqueous medium at I = 0-5 is so good that only the discharge of the protein at or near the isoelectric point affects the state of association. On the other hand, at lower ionic strengths the weaker solvent action is shown by the higher state of association at a given pH, and by the effect on association of small pH changes. EFFECT OF TIME.-NO change in the ultracentrifugal pattern of a solution of the 65/85 conarachin in phosphate buffer at I = 0.5, pH = 7.5 was observed with time even after104 GROUNDNUT GLOBULINS 3-4 weeks.At I - 0.1, pH =: 7.7, little change was observed over a period of two weeks but thereafter gradual dissociation occurred until after 3-4 weeks a pattern similar to that at I = 0.5 was observed. This result, which was confirmed, was most surprising, and is difficult to reconcile with the definite reversible changes in association which occur on changing the ionic strength. Its occurrence with old and often obviously infected protein solutions suggests the effects of microbiological attack. TABLE 6.-sEDIMENTATION DIAGRAMS AND CONSTANTS * OF THE 65/85 CONARACHIN FRACTION AT DIFFERENT pH VALUES I = 0.1, protein conc. = 0.8 g/100 ml PH _______-____ ~ -__ -- buffer composition (M) sedimentation constants - -_ gl ycine 0.09 NaOH 0.0 1 A NaCl 0.0098 10.3 21, gly cine 0.094 NaOH 0.0056 B NaCl 0.014 9.4 9.4 2 14.7 2 - Na2HP04 .12H20 0.01 6 C KH2P04 0.0015 7.7 32, 21, 12.5 2 - NaCl 0.05 Na2HP04. 12H20 0.0086 NaCl 0.05 D KH2P04 0.024 6.3 50, 32, - CH3COONa 0.05 NaCl 0.05 29, 17, - E CH3COOH 0.23 3-8 glycine 0.01 6 HCI 0.004 F NaCl 0.096 2.9 I = 10-5, protein conc. = 0.74 g/100 ml 12.3 2 2 2 2 CH3COONa 0.1 NaCl 0.4 0.0048 4.7 46, 27, - G CH3COOH 11.7 2 * any component occurring > 30 % (by area) underlined. DIFFERENT SEDIMENTING COMPONENTS OF THE 65/85 FRACTION AT DIFFERENT pH VALUES I = 0.1, protein conc. = 0.8 g/100 ml TABLE 7.-wEIGHT-AVERAGE MOLECULAR WEIGHTS AND RELATIVE AREAS (%) FOR THE s2 wt.-average mol. PH s50 s30 S20 s l 4 s12.5 $9 wt.(calc.) 10.3 - - 5 - - 83 12 190,000 9.4 - - - 87 - - 13 330,000 7.7 - 4 60 - 23 - 13 450,000 6.3 5 70 - - 13 - 12 900,000 3.8 - 3 42 - - 39 16 345,000 2.9 - - - 30 - - 70 130,000 I = 0.5, protein conc. = 0 . 7 4 g/100 ml 4.7 8 58 - - 21 - 13 880,000P. JOHNSON AND W . E . F . NAISMITH 65/85 and 40/65 fractions of conarachin in phosphate buffer, Z = 0.5, pH = 7-5, has given weight-average molecular weights of 190,000 and 70,000 respectively to be compared with 175,000 and 90,000 from ultracentrifuge calculations. In view of the approximations made and the fact that light scattering and ultracentrifuge work was carried out on different protein preparations, the agreement in molecular weight values is as good as could be expected. C/T against c (c = concentration in g/cm3) for the 65/85 fraction in phosphate + NaCl buffers at pH 7.6 0-2 over a range of ionic strengths.Each plot gives a horizontal 105 LIGHT SCATTERING EXAMINATION.-EXam~atiOn by light Scattering Of SOlUtiOnS Of the 65/85 FRACTION : EFFECT OF VARIATION IN IONIC STRENGTH.-Fig. 4 Contains plots Of i c x 10' (p +') 2 2 4 6 8 I 1 /o FIG. 4 . 4 7 against c for the 65/85 conarachin fraction at pH 7.6 ( + 0.2) and different ionic strengths. 0 I = 0.5. 0 I = 0.35. A Z = 0.2. I = 0.1. straight line and the intercepts on the C/T axis were used to calculate the weight-average molecular weights of table 8, column 2. Satisfactory agreement with calculated values from ultracentrifuge data is obtained. It was further shown that changes produced were readily and quantitatively reversible at the extremes of the ionic strength range, but at TABLE 8 .-CHANGE IN WEIGHT-AVERAGE MOLECULAR WEIGHT OF THE 65/85 FRACTION WITH IONIC STRENGTH AT pH = 7.6 (& 0.2) weight-average molecular weight - calculated from ultracentrifugal data Z light scattering 0.50 190,000 175,000 0.35 210,000 185,OOO 0.20 395,000 405,000 0.10 550,000 450,000 I = 0-2 the molecular weight value again depended to a small extent upon whether the solution had been previously at a higher or lower ionic strength.To estimate the rate at which association or dissociation occurred, rapid changes in ionic strength were caused by addition of water or concentrated sodium chloride solutions (carefully filtered) to the protein solutions. Light scattering measurements taken within 1 min of such additions corresponded with the final jonic strengths and no further change was observed. D106 GROUNDNUT GLOBULINS EFFECT OF pH VARIATION.-Fig.5 contains plots of C/T against c for the 65/85 fraction in buffers at I = 0.1 and various pH values, horizontal straight lines again being obtained. Table 9 contains derived weight-average molecular weights compared with calculated ultracentrifugal values. Reasonable agreement is again obtained, and as previously suggested on the basis of ultracentrifuge patterns, the weight-average molecular weight at I = 0.1, pH = 10.3, agrees well with that for I = 0.5, pH = 7-5. Within the pH range covered, the changes observed were readily reversible. It was shown by ultracentrifuge examination that increased protein concentration at I = 0.1, pH = 7.7, caused greater association and higher weight-average molecular weights, and this effect might be expected to modify the horizontal C / T against c plots expected for a moderately charged protein in solution at appreciable salt concentration.However, the concentration effects observed occurred largely at above 1 % protein con- centration, whilst light scattering data was obtained at lower values where any curvature FIG. 5.+/7 against c for the 65/85 conarachin fraction at I = 0.1 and different pH values. 0 pH = 10-3. A pH = 9.4. pH = 7.7. El pH = 6-3. due to altered degree of association would be very small. No such curvature is detectable in the light scattering results reported here. TABLE g.-CHANGE IN WEIGHT-AVERAGE MOLECULAR WEIGHT OF THE 65/85 FRACTION WITH pH AT IONIC STRENGTH = 0.1 _____ weight-average molecular weight calculated from ultracentrifugal data PH light scattering 10.34 206,000 190,000 9.43 290,000 330,000 7-65 548,000 450,000 6.29 1,040,000 900,000 ELECTROPHORETIC ExAMmATIoN.-Only a brief outline of electrophoretic results is given here, a more detailed report being planned.19 Electrophoretic diagrams at 20" C of the 40/85,40/65 and 65/85 conarachin fractions in phosphate buffer at I = 0.1, pH = 7.7 are shown in table 10 with mobility and peak area data calculated from the descending patterns.From the mobilities it seems likely that the same 2 components occur in all fractions though in differing amounts. Comparing the 65/85 electrophoretic pattern with that from sedimentation at I = 0.1, pH = 7.7, considerable differences are revealed but there is some similarity with theP .JOHNSON AND W. E. F . NAISMITH 107 sedimentation pattern at I = 0.5, the major electrophoretic component occurring in proportions similar to the sum of the 2 faster sedimenting components. Since the latter appear to be merely different stages of association of the same chemical entity, it seems legitimate to group them together and the conclusion emerges that such association is TABLE 1 ELECTROPHORETIC AND DERIVED DATA FOR DIFFERENT CONARACHIN FRACTIONS IN PHOSPHATE BUFFER (I = 0.1, pH = 7.7) AT 20" C; PROTEIN CONC. = 0.8 (& 0.1) g/100 ml major component minor component -- fraction 7; mobility* area (Yo mobility * A 40185 10.9 79 5-1 21 B 40165 10.7 72 5.2 28 C 65/85 10.8 92 5.2 8 * units : cmz volt-1 sec-1 x l o 5 not detected by electrophoresis.Similarly on varying the pH between 6.5 and 9 and the ionic strength between 0.05 and 0.2 no change in the relative areas of the electrophoreti- cally different components occurred. Thus apparently the very different components observed by the ultracentrifuge have either identical electrophoretic mobilities or are so similar in this respect that no separation occurs in electrophoresis. DISCUSSION Although an ideal separation of protein constituents was not obtained, the 65/85 fraction contained only ca. 10 % of s2 component and ultracentrifuge ex- periments (especially tables 4, 5, 7) demonstrate clearly the inertness of this com- ponent in the reactions being studied.A comparison of sedimentation diagrams for the 40/65 fraction at I = 0.1 and 0-5 confims this point further. The reactions involve only the S8.7 component in the most highly dispersed form of the fraction from which it has been shown that more rapidly sedimenting species only form under a variety of other conditions. From the discrete nature of the new sedi- menting peaks and the constancy of their sedimentation constants over an ap- preciable range of conditions, the phenomena seem, more in the nature of well- defined processes than irregular colloidal aggregation or coagulation. For this reason the terms " association " and " dissociation " have been used throughout this paper in preference to " aggregation " and " disaggregation ".However, while the regular nature of the association reactions is to be stressed, it should also be men- tioned that at low ionic strengths and pH values near 6 where association is con- siderable, solubility is low and the association processes may merge into irregular coagulation. Assuming regular association, the reactions occurring may be summarized in the form : 178.7 . S8.7 + n12.5 . ~ 1 2 . 5 + n20 . s20 + n30 . ~ 3 0 , etc., (1) where ns represents the number of molecules ss of sedimentation constant s which take place in the chain of reactions. In the absence of diffusion or other suitable data to make possible the calculation of unambiguous molecular weights, the n values cannot be fixed. Certain probable features may, however, be mentioned.Large changes in frictional ratio by the di- or trimerization of ellipsoidal units of small axial ratio would not be expected so that the relative magnitudes of the molecular weights in table 2 are probably not greatly in error. Thus under con- ditions where association was considerable, molecular weights are in the ratios 1/2/4/8, the lowest value corresponding with ~12.5. The absence of sg under such conditions is perhaps surprising but may be significant in that the molecular weights108 GROUNDNUT GLOBULINS of sg and ~12.5 seem to be in the ratio 2/3. Possibly the onset of appreciable as- sociation requires a preliminary packing of a basic unit (of molecular weight ca. 100,000) into three’s (~12.5) rather than two’s (sg). Association reactions according to eqn.(1) would then remove the trimer as it was formed so that s9 would eventually disappear completely. The effectiveness of high ionic strength or pH in giving maximum dispersion and the rapidity of the dispersion or association processes suggests that electro- static forces are largely responsible for the binding of the smaller units. At the highest pH values studied (10-3), all basic groups except the guanidinium will be almost completely uncharged and the excess negative charge apparently suffices to disperse the protein completely by repulsion of the sg units. As the pH is lowered, +amino groups of lysine become charged positively (the net negative charge now assuming a lower value) and at pH 9.4 and I = 0.1 when this process must be largely complete, it will be recalled (table 6) than an anomalously broad sedimenting boundary was observed.It seems possible that at this stage there is an approximate balance between the electrostatic repulsion of higher pH values and the attraction of lower pH values which takes control when the net negative charge is further reduced. Since at higher ionic strengths such attraction is not effective, it must in part at least be electrostatic. A correct disposition of charged groups on the unit structures wouId make possible such an attraction, but it may also involve Van der Waal’s forces and hydrogen bonds. At lower pH values the attractive forces become more predominant, and the equilibrium moves towards the most rapidly sedimenting species whose boundaries are now of more normal sharpness.Since it has been shown by light scattering that equilibrium is rapidly attained, association and dissociation must occur continuously during ultracentri- fugation, but providing both processes are relatively rapid no noticeable effect on boundary contours would be expected. Nor would observed s values be affected unless the equilibrium position were changed appreciably. The special conditions at pH 9.4, I = 0.1, are probably responsible for the modified contours and sedi- mentation constant observed. To check the above picture it is planned to compare with electrophoretic, analytical and titration data. It is of interest to compare the main features of conarachin with those of other dissociating systems. Dissociation at high pH values seems to occur very generally, being reported for the haemocyanins,l2.13 insulin,l4 thyroglobulin 15 and for arachin,lo the less soluble and quite separate globulin from the groundnut.As to the effect of addition of electrolytes, dissociation or association may occur ac- cording to the protein studied. Dissociation occurs with Helix pomatia haemo- cyanin, whilst association is promoted with haemocyanin from Paludina vivipara.12 The latter effect is probably more general since it has been reported also for arachin,lo insulin,l3 and thyroglobulin.~s As yet no pH effect on reaction velocity, as was observed with arachin, has been detected, but further investigation of this question is required. Nor have any specific ion effects, comparable with those observed for Helix pomatia haemocyanin, been observed.A comparison of sedimentation and electrophoresis diagrams for the 65/85 fraction at I = 0.1 and pH values below 10 reveals striking differences which can only mean that electrophoretic behaviour is largely independent of degree of association. It was shown similarly 16 that the dissociation product of arachin was electrophoretically very similar to the undissociated molecule. Svedberg 17 observed that components of a dissociation would not be distinguishable electro- phoretically but, on the basis of electrophoretic theory, this is not readily explicable as has been pointed out elsewhere.18 The advisability of applying more than one method to the examination of protein systems is clearly indicated. The assumption of identity on the basis of similar electrophoretic mobility at a given pH, as was made by Irving, Fontaine, and Warner,4 is particularly dangerous. The similarity of arachin and conarachin electrophoretically has been confirmed in this work,lg but clear differences in other properties leave no doubt that the proteins are different.P . JOHNSON AND W. E . P . NAISMITH 109 We are grateful to Miss Susan Preger for technical assistance with the ultra- centrifuge and to Messrs. I.C.I. Ltd. (Nobel Division) for seconding one of us (W. E. F. N.) for one year to work on this problem and also for supplies of oil-free ground-nut meal. 1 Ritthausen, Arch. Ges. Physiol., 1880, 21, 81. 2 Johns and Jones, J. Biol. Chem., 1916, 28, 77. 3 Jones and Horn, J. Agric. Res., 1930, 40, 673. 4 Irving, Fontaine and Warner, Arch. Biochem., 1945, 7, 475. 5 Johnson, Trans. Faraday Soc., 1946,42,28. 6 Philpot, Nature, 1938, 141, 283. 7 Goring and Johnson, Trans. Faraday SOC., 1952,48, 367. 8 Goring and Johnson, J. Chem. Soc., 1952, 33. 9 Svedberg and Pedersen, The Ultracentrifuge (Oxford, 1940), p. 58. 10 Johnson and Shooter, Biochim. Biophys. Acta, 1950, 5, 361. 11 Hughes, J. Amer. Cliem. Soc., 1947, 69, 1836. 12 Brohult, J. Physic. Chem., 1947, 51, 206. 13 Pedersen, Cold Spring Harbor Symposia, 1950, 14, 140. 14 Gutfreund, Biochem. J., 1948, 42, 554. 15 Lundgren, Nature, 1939, 143, 896. 16 Johnson, Shooter and Rideal, Biochetn. Biophys. Acta, 1950, 5, 376. 17 Svedberg, Chem. Rev., 1937, 20, 81. 18 Alexander and Johnson, Colloid Science (Oxford, 1949), p. 332. 19 Johnson and Naismith, to be published.

ISSN:0366-9033    

DOI:10.1039/DF9531300098

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

P . JOHNSON AND W. E . P . NAISMITH 109 THE EFFECTS OF CERTAIN IONS AND NEUTRAL MOLECULES ON THE CONVERSION OF FIBRINOGEN TO FIBRIN BY SIDNEY SHULMAN School of Medicine, The University of Buffalo, Buffalo, New York Received 8th May, 1952 Certain reagents interfere strongly with the clotting of fibrinogen by thrombin. A large number of compounds have been studied under the same conditions of pH, ionic strength, and protein concentrations, and about 40 of these reagents were found to delay clotting for at least 24 h ; this has been termed inhibition. A like number of substances were found which, even at high concentrations, caused much less clotting delay ; this has been termed retardation. Inhibition is reversible and does not involve destruction of either protein. It can be achieved even if the reagent is added after the thrombin, at any time up to the moment of actual clotting.Correlations are made between the molec- ular structures of the reagents and their inhibitory effectiveness. Certain general struc- tural features may be required, though specific structural details are also significant. The clotting of fibrinogen by thrombin is markedly affected by the presence of certain reagents. Except for the very few substances that have been found to accelerate the reaction, these reagents may be classified into two main groups- retafders and inhibitors. Members of both groups will increase the time required for the first appearance of a clot after mixing the two protein solutions, and de- crease the final opacity of the resulting clot. The clotting time increase and the opacity decrease are progressively augmented with increasing concentration of reagent .Is 2 The retarders, even a t very high concentrations, increase the clotting time by a t most a factor of 10 or 20, compared to similar systems containing no reagent, while the more effective compounds, even at moderate concentrations,110 CONVERSION OF FIBRINOGEN TO FIBRIN delay clotting for at least 24 h, corresponding to a factor of about 120.This action has been defined as inhibition,3 since a 24-h delay in clotting generally means that the mixture will remain fluid indefinitely. This has generally been studied at pH 6.2 and ionic strength 0.45; the effects of altering these variables will be discussed below. Inhibition has been found to be a reversible phenomenon as shown by the fact that apparently normal clotting will result from dialysis of such mixtures against salt solution.Further evidence of the innocuous nature of such a reagent is pro- vided by mixing it with either of the proteins for 24 h. On removal of the reagent by dialysis, the protein solution remains clear and fluid and clots in a normal manner on mixing with the other protein. Some of the reagents have been ob- served to cause denaturation of fibrinogen, as evidenced by turbidity increase and formation of a fragile opague coagulum, but this occurs only at concentrations considerably higher than those required for inhibition. It was also found that inhibition can be produced by adding the reagent solu- tion after mixing the two protein solutions, provided the addition precedes the actual gelation.3 This has been termed post-thrombin inhibition, and is of im- portance in showing that the inhibitor must be influencing fibrinogen rather than thrombin, since the enzyme must have completed its action by the time of clotting, and any interference with thrombin should not then have any effect on the sub- sequent fibrinogen polymerization.The inhibitors can be conveniently classified into five groups, viz. neutral alcohols, neutral amides, dipolar ions, cations, and anions. Some representative examples 3 are listed in table 1, along with the minimum inhibiting concentration of each. Some reagents of similar structure that are only retarders 3 are shown in table 2. The inhibitors bromide and iodide were first studied by Edsall and Lever,4 who employed different conditions of protein concentrations and ionic strength, and a different criterion of clotting delay, so that comparison with the inhibitors as defined above is somewhat difficult. But they did find that guanidine and thiocyanate interfere markedly with clotting, as did also urea, iodide, and acetyltryptophanate.Under their conditions, bromide was not very effective. In a recent study,5 sodium bromide and iodide were examined in the same manner as in the earlier inhibitor investigation, with the results indicated in table 1. TABLE 1 .-INHIBITORS OF FIBRINOGEN CLOTTING Fibrinogen, 5.0 g/l. ; thrombin, 1-0 unit/ml ; pH 6-20 ; ionic strength, 0.45 neutral alcohols cations minimum inbibiting (molell.) reagent concentration reagent heptamethylene glycol 0.24-0.28 guanidine hexamethylene glycol 0-3 1-0-33 histamine*b pentamethylene glycol 0-43-0.48 imidazole tetramethylene glycol 0-90-1.01 n-butylamine *c tetrahydrofurfuryl alcohol 0.38-0.50 lithium bromide *d 1 : 4-cyclohexanediol 0.45-0'52 anions 1 : 2-cyclohexanediol 0.21 -0.43 minimum inhibiting concentration (mole/l.) 0.12-0.1 6 0.1 4-0.20 0.31-0.36 0.36-0.72 0.22-0'34 neui urea methylurea semicarbazide formamide sodium ascorbate *e 0.44-0.45 0.36-0.42 sodium tetrathionate 0.1 2-0.14 0.42-0.56 sodium thiocyanate 0.24-0'32 0'26-0.34 sodium sulphite *f 0.19-0.38 1.01-1.35 sodium bromide *g 0.34-0.45 sodium iodide *JJ 0.22-0.34 sodium benzenesulphonate 0.33-0-36 'ral amides neutral dipolar ion cys teine*a 0'34-0.42 * at a total ionic strength of : a, 0.45-0.56 ; b , 0 3 1-0.53 ; c, 0-63-0.8 1 ; d, 0.56-0-62 ; e, 0-89-0.90 ; f, 0.98-1.51 ; g, 0-62-0.68 ; h, 0-56-0-62.SIDNEY SHULMAN 111 The effectiveness of an inhibitor (excepting the inorganic ions) is markedly influenced by ionic strength.6 The minimum inhibiting concentration becomes smaller with an increase in ionic strength.This increased effectiveness can be accounted for in terms of an enhanced binding of inhibitor by fibrinogen, and in general, a decreased solubility of the inhibitor. It is essentially a salting-out effect. Before attempting to explain the action of the inhibitors, one may consider the influence of pH and ionic strength on clotting.7.4 At constant pH, say 6.2, in- creasing concentration of sodium chloride causes an increasing clotting time, but inhibition cannot be obtained by this means.At the same time the nature of the final clot shifts from opaque to transparent. Both of the criteria demonstrate a TABLE 2.-RETARDERS OF FIBRINOGEN CLOTTING Fibrinogen, 5.0 g/l. ; thrombin, 1.0 unit/ml ; pH 6.20 ; ionic strength, 0.45. reagent maximum relative concentration clotting time tested (mole/l.) increase t neutral alcohols trimethylene glycol 0.80 2.8 1 : 3-butanediol 0-43 11.8 glycerol 2.16 2.6 mannitol 0.44 1.8 ethanol 1 -09 2.0 ethylene glycol 3.14 4.3 cyclohexanol $ 0.19 11.2 biuret $ glycine taurine histidine *a lysine *b methylamine neutral amide 0.14 25.4 neutral dipolar ions 0.72 0-22 1.27 1.3 cat ions 0.41 1.5 0-76 21.8 0.36 2.7 anions sodium thiosulphate *c 0.35 5.8 sodium m-benzenedisulphonate *d 0.32 3.7 sodium isopropylsulphonate *e 0.57 5.1 sodium chloride*f 1.72 56.0 * at a total ionic strength of: a, 0.50; b, 0.83; c, 1.11 ; d, 0.77; e, 1-02; f, 1.72.$ concentration limited by reagent solubility. a factor of 5 corresponds to a clotting time of about 1 h. reduced tendency toward aggregation, and can be explained by the decreased activity coefficient of a single fibrinogen molecule relative to that of an aggregate caused by increasing salt concentration, as is also demonstrated by the effect of salt addition on the solubility of the protein. The effect of pH is a little more complicated. The isoelectric point of fibrinogen is about pH 5 and the protein has invariably been studied at pH values alkaline to this.At the lower values of pH, the clot is opaque, while at higher values it is transparent. This is illustrated in fig. 1. The actual pH of transition depends on the ionic strength. No com- pletely satisfactory explanation of this complicated behaviour has yet been offered, and we must presumably wait until there is some understanding of the distribution of ionizable groups on the fibrinogen molecule.112 CONVERSION OF FIBKINOGEN TO F l B R I N The effect of pH on clotting may now be considered. It has been found that a clotting time minimum occurs in the vicinity of pH 7, depending somewhat on FIG. 1 .-Clot opacity plotted against pH for 5.0 g/l. fibrinogen and 1.0 unit/ml thrombin. The figures denote values of ionic strength I 0.8 0.6 0.4 0.2 0 5 6 7 8 9 10 PH the ionic strength, and that clotting is progressively delayed at higher and lower values of pH, until inhibition is obtained at pH 5.3 and at pH 10.This is illus- 3 .O 2 .o In 3 c .- E + 0 .- c FIG. 2.-Logarithm of clotting g 1.0 time plotted against pH for 5.0 g/l. fibrinogen and 1.0 unit/ml thrombin. The figures denote values of ionic strength. - 0 5 6 7 8 9 10 PH trated in fig. 2. To account for this behaviour, two mechanisms were postulated.7 The clotting delay caused at high pH can be explained in terms of the increasing electrostatic repulsions between the more highly charged fibrinogen units. At lowSIDNEY SHULMAN 113 pH one can assume that some ionizable groups in one, or both, of the proteins is involved in the reaction.Considering the pH region in question, the only likely group is the irnidazole ring of histidine. According to this hypothesis, the neutral form of imidazole is reactive, while the addition of a proton produces an inactive component. Inhibition by acid was studied by Laki and co-workers, who were the first to establish the presence of a definite intermediate stage in the clotting of fibrinogen.gY9 By neutralizing acid-inhibited mixtures of fibrinogen and thrombin after various time intervals, it was shown that some transformation was occurring in the acid environment even though no clotting took place, for the neutralized mixture clotted faster, the longer it had first stood at low pH. The action of inhibitors other than acid or base can be interpreted in terms of the reagent being bound to protein, producing an equilibrium between free and 70 60 u) 5 0 L 1 0 r .c 40 E Q) .- c g 30 * 20 .- c c 0 - 10 0 10 20 30 4 0 5 0 Concentration of hexamethylene glycol in g./l.FIG. 3.Xlotting time plotted against concentration of hexamethylene glycol for 5.0 g/l. fibrinogen, 1.0 unit/ml thrombin, ionic strength 0.45, and pH 6-20. bound protein. The moderately high reagent concentration required shows that the binding is rather weak. At low concentrations of inhibitor, and at all retarder concentrations, clotting is delayed and opacity is reduced. This opacity result shows that the reagents interact primarily with fibrinogen. This conclusion is also supported by the phenomenon of post-thrombin inhibition, and by the results of physicochemical investigations 10-13 on certain inhibited clotting systems, re- vealing the presence of partially polymerized fibrinogen.The difference between inhibitors and retarders is not just one of degree of effectiveness. The basic difference is well illustrated by fig. 3 and 4, which show the clotting time as a function of concentration for a typical inhibitor, hexamethy- lene glycol, and a typical retarder, glycerol. It will be seen that the curves differ radically in shape. As already mentioned, a certain intermediate in the poly- merization process is found in inhibited systems. Preliminary studies on retarded clotting systems 14 seem to reveal a different intermediate, and this, if confirmed, would further demonstrate the fundamental difference in the two types of inter- ference with clotting.114 CONVERSION OF FIBRINOGEN TO FIBRIN An analysis of the structure of the inhibitor molecules reveals several general principles. It will be profitable to consider the organic and inorganic reagents separately.Among the organic inhibitors, the rather rigid structural requirements suggest that the inhibitor is bound to the protein at or near the reaction site, so that this site is blocked. The reagent structure requires a polar functional group, such as hydroxyl, sulphydryl, amino, or sulphonate, along with a minimum quan- tity of hydrocarbon residue. This is best illustrated among the alcohols. The terminal glycols containing 4-7 carbon atoms are inhibitors. If the quantity of hydrocarbon moiety is reduced, as in trimethylene or ethylene glycols, or if the number of hydroxyl groups is increased, as in mannitol, effectiveness is lost.Moving the hydroxyl groups in from the end of the cliain-compare 1 : 3-butanediol with tetramethylene glycol-removes effectiveness. This observation, along with the fact that the monohydric tetrahydrofurfuryl alcohol is an inhibitor, suggests that the glycols may, in interacting with the protein, assume a cyclic configuration with the two hydroxyl groups functioning essentially as a single unit. Confirmation of this concept would be sought in the behaviour of cyclohexanol; unfortunately, the concentration range is limited by its low solubility, but at the highest concen- tration studied it compared fairly well with hexamethylene glycol in delaying clot- ting.Inhibition was, however, readily obtained with 1 : 2-cyclohexanediol and 0 5 0 100 150 200 Concentration of Qlycerol in g./l. FIG. 4.-Clotting time plotted against concentration of glycerol for 5.0 g/l. fibrinogen, 1.0 unit/ml thrombin, ionic strength 0.45, and pH 6-20. 1 ~Ccyclohexanediol. Further support for this explanation comes from the results on the organic electrolytes, and only a brief discussion will be included here. Among the sulphonates, for example, several inhibitors were found, e.g. ben- zenesulphonate, p-toluenesulphonate, several xylenesulphonates, but neither iso- propylsulphonate, taurine, nor even rn-benzenedisulphonate are effective. In- hibition is clearly not due to the functional group alone, but depends also on the large hydrocarbon residue.This view is further strengthened by the finding of Mihalyi 15 that sodium taurocholate delays clotting by a factor of about 150 at 0-05 M, and that glycocholic acid has a similar action. Glycine and taurine, how- ever, are not inhibitors. The amides must function by a different mechanism. Urea and its derivatives do not require a hydrocarbon residue for effectiveness. It should be noted that of more than a dozen amides studied all were found to be real or potential inhibitors, the latter, e.g. biuret, being limited by solubility. It seems likely that their action is due to hydrogen bonding with the protein, causing very slight and reversible modifications that block or alter the reactive groups. The action of cysteine is probably similar to that of the alcohol reagents, with the sulphydryl group playing a role similar to that of a hydroxyl group.Its possible action as a reducing agent was ruled out ; 3 the same conclusions were reached for sodium ascorbate and sodium sulphite. The inorganic anions, bromide, iodide, thiocyanate, tetrathionate, as well as perhaps the other effective anions would seem to be bound to fibrinogen, increasing the negative net charge, just as does an increase in pH. The augmented repulsionSIDNEY SHULMAN 115 would be expected to diminish the aggregation of fibrils and would thus produce a transparent clot, as observed for low concentrations of these inhibitors at pH 6.2, or for high pH itself. The effect of these reagents on clotting is to delay it markedly, whereas an increase in pH from 6.2 to, say 7.2, markedly hastens clotting.This difference can be readily reconciled with the theory already mentioned, concerning the importance of the state of ionization of the imidazole group in this pH region. As Edsall and Lever 4 have pointed out, the binding of anions at pH 6.2 would still leave the imidazole groups largely in the unreactive acidic form, but the con- comitant increase in negative net charge would retard clotting. As for the action of some of the cations, a different mechanism must be pro- posed. Edsall and Lever,4 by studying the action of guanidine at pH 7.3 and ionic strength 0.135, found that it not only delayed clotting but produced a clot of higher turbidity than the control clot. This is the only reagent so far known to show this behaviour.They pointed out that by these two criteria the addition of guanidinium ions was exactly like the addition of hydrogen ions, that is, a shift to lower pH. This should not present any conflict with the theory of pH effect on clotting; one can postulate that a guanidinium ion can react with an imidazole residue just as does a proton. Independent evidence supporting this hypothesis is yet to be sought. Whether other cation inhibitors can be classed as similar to guanidine, for example, whether the opacity-increasing effect is duplicated, remains to be seen. One reagent that might show this behaviour, and if so, be explained by the same mechanism, is the lithium cation. Its strong interaction with fibrinogen and fibrin is demonstrated not only by the fact that lithium bromide inhibits clotting at a lower concentration than does sodium bromide, but also by the observation 5 that 3 M lithium chloride will dissolve fibrin formed from highly purified protein components, whereas 3 M sodium chloride is ineffective.With respect to histamine, imidazole, and n-butylamine, all of which demon- strate again the importance of a bulky hydrocarbon residue, the inhibitory action may possibly be due to a competition between the free reagent molecules and the corresponding imidazole or lysine side chains in fibrinogen. The result of putting a carboxyl group on the methylamine carbon atom is shown by glycine, which does not prolong but actually hastens clotting. This alteration is similar to the reduction in inhibitory effectiveness resulting from the analogous introduction of a carboxyl group in histamine to give histidine, as well as the loss of inhibition suffered by butylamine on the introduction of a carboxyl group to give lysine. Apparently these molecular alterations cause sufficient interference with the electrostatic attraction between the amino group and the negative protein site that binding is inadequate to cause inhibition. 1 Ferry and Morrison, J . Amer. Chem. SOC., 1947, 69, 388. 2 Ferry and Shulman, J. Amer. Chem. Soc., 1949, 71, 3198. 3 Shulman, Arch. Biochem., 1951, 30, 353. 4 Edsall and Lever, J . Biol. Chem., 1951, 191, 735. 5 Shulman and Katz, Fed. Proc., 1952, 11, 286. 6 Shulman, Herwig and Ferry, Arch. Biochem. Biophys., 1951, 32, 354. 7 Shulman and Ferry, J . Physic. Chem., 1950, 54, 66. 8 Laki and Mommaerts, Nafure, 1945, 156, 664. 9 Laki, Arch. Biochem. Biophys., 1951, 32, 317. 10 Shulman and Ferry, J . Physic. Chem., 1951, 55, 135. 11 Shulman, Ehrlich and Ferry, J. Amer. Chem. Soc., 1951, 73, 1388. 12 Foster, Samsa, Shulman and Ferry, Arch. Biochem. Biuphys., 1951,34,417. 1 3 Ehrlich, Shulman and Ferry, J. Amer. Chem. Soc., 1952, 74, 2258. 14 Ferry and Shulman, unpublished experiments. 15 MihAlyi, Hung. Acta Phusiol., 1948, 1, 179.

ISSN:0366-9033    

DOI:10.1039/DF9531300109

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

ELECTROPHORETIC STUDY OF THE MUSCLE STRUCTURAL PROTEINS BY G. HAMOIR Laboratoire de Biologie gkndrale, Universitd de Likge, Belgique Received 1st May, 1952 Four main protein components of the myofibril, actomyosin, myosin, actin and tropomyosin, have been isolated so far and are fairly well identified. Their electrophoretic behaviour has been investigated mainly in Dubuisson’s department in Likge. The results obtained with each of these components are described and their relation to other electrochemical data discussed. Tables 1 and 2 summarize the present electrochemical determinations made on these proteins. Electrophoresis appears to be more useful for the study of whole extracts or complicated mixtures of muscle proteins than ultra- centrifugation, which is profitably applied only to fairly pure fractions.The comple- mentary character of these two techniques is particularly evident in these studies. The muscle fibre contains two main groups of proteins : the soluble proteins of the sarcoplasm and the structural proteins of the myofibrils. We will limit our present considerations to the latter group. Its main components, actomyosin, myosin, actin and tropomyosin are fairly well known. Some others such as myosin y (Dubuisson 1) and protein Y (Dubuisson 2) have also been described but as they occur in small amounts and their identification is still incomplete, they will not be considered here. The electrophoretic study of these proteins in whole or in fractionated extracts has been carried out since 1941 in Dubuisson’s department in Likge.Papers on this subject have also been published by Bailey,3 Mehl and Sexton,4 Ziff and Moore,s Erdos and Snellman,6 Amberson, Erdos, Chinn and Ludes,7 Spicer and Gergely,8 Mommaerts and Parrish.9 The fairly limited number of publications appears to be due at least partly to the technical difficulties involved : the high ionic strength necessary to keep actomyosin and myosin in solution make the experiments tedious and the study of actomyosin solutions can hardly be carried out in the usual Tiselius cell of 2.5 cm depth and 0-3 cm width because of their high viscosity and their usually marked turbidity. This difficulty was avoided by Dubuisson 10 by the construction of cells of square cross-section (0.6 cm x 0.6 cm) which allow the boundaries to be brought into the optical path without a too noticeable blurring on the descending side.RESULTS MIXTURES OF ACTOMYOSIN AND MYosm.-Preliminary work on electrophoresis of mixtures of rabbit actomyosin and myosin was done in 1944 by Ziff and Moore.4 But the first detailed study of such mixtures was made by Dubuisson 1 in 1946. The electro- phoretic picture observed by him with a preparation made following Greenstein and Edsall 11 is given in fig. 1. Two main gradients were visible on the ascending limb: a faster one separating only after prolonged electrophoresis, remaining very sharp what- ever the length of the experiment and migrating with the turbidity (the a peak), and a slightly slower one showing a more normal boundary spreading (the /3 peak). Their mobilities are given in table 1.The relative area of the cc peak was found to increase in extracts of longer duration which are known to contain 116 No clear separation could be detected in the descending limb. The nature of these two gradients soon became apparent.G . HAMOIR 117 a higher proportion of actomyosin and the two components could be separated by am- monium sulphate fractionation ; the cc peak precipitates first (between 23 and 27 % satura- tion) in good agreement with the lower solubility of actomyosin while the peak is salted out between 27 and 45 % saturation.12 The location of these two peaks in the electrophoretic pattern of muscle whole extracts was described a little later by Jacob.13.14 As shown by fig. 2, they migrate with a speed intermediate between the slow moving myogen (M) and a much less important and more rapid group of proteins whose main component has been tentatively identified by Jacob with myoalbumin (h).The fairly symmetrical aspect of the two patterns of fig. 2 shows that no interaction seems to occur between the median group and the other components. The asymmetry is limited to the 3: and 8 peaks. Their ascending pattern is the same as FIG. 1 .-Electrophoretic pat- tern of a mixture of actomyosin and myosin (rabbit striated muscle) at 1=0.40 and pH 7-1. Upper part : ascending limb ; lower part : descending limb ; duration of the electrophoresis : 114,000 sec ; electrical field : 1.12 V/cni (after Dubuisson 1). I FIG. 2. - Electrophoretic pattern of a rabbit muscle extract at I = 0.35 and pH 7.4.Upper part: as- cending limb; lower part: descending limb ; duration of the electroDhoresis : 50,000 sec ; electrical field : 1-80 V/cm ; M = myogen; /3. = myosin ; cc = actomyo- sin ; h = myoalbumin (after Dubuisson 15). in mixtures of actomyosin and myosin, but on the descending side, the /3 peak seems to split into 81 and 82.15 As in whole extracts components soluble at low ionic strength migrate with approximately the same speed as 8, it is not possible to decide from such diagrams if this behaviour is due to the presence of two 8 myosins or to the interference of other components. In the course of the development of this work, the investigations were extended to muscle of different origin. The results have confirmed the researches made on rabbit skeletal muscle.The electrophoretic patterns of actomyosin and myosin originating from mouse skeletal muscle,l6 from white and red muscle (rabbit) and from heart muscle (horse, rabbit) 7,17,18 do not differ significantly from the pattern of fig. 1 . For cold vertebrates (carp, frog), the formation of actomyosin in the course of the extraction is very rapid and the By lowering the pH of extraction from 7-8 to 5-5-60, extracts could, however, be prepared from carp muscles giving the electrophoretic pattern of fig. 3. The a and peaks of these extracts correspond again to actomyosin and myosin. They have practically the same mobilities as those found previously for rabbit actomyosin and myosin (see table 1). Both peaks are removed peak is not usually observed.l%20,21118 MUSCLE STRUCTURE PROTEINS TABLE 1 .-ELECTROPHORETIC MOBILITIES OF THE MUSCLE STRUCTURAL PROTEINS protein solvent actomyosin sodium phosphates Z = 0.1 + 0.3 M NaCl sodium phosphates Z = 0.15 + 0.25 M NaCl sodium phosphates NaCl myosin sodium phosphates Z = 0.1 + 0.3 M NaCl sodium phosphates Z= 015 + 0.25 M NaCl Z = 0.1 4- 0.25 M K veronal acetate Z = 0.05 + 0.25 M KCl sodium phosphates I = 0.1 + 0.25 M NaCl K phosphate 0.1 M sodium phosphates I = 0.15 sodium phosphates I = 0.15 + 0-25 M NaCl F-ac t i n depolymerized sodium phosphates sodium phosphates Z = 0.15 + 0.25 M NaCl F-actin I = 0.15 tropomyosin sodium phosphates Z = 0.1 + 0.05 M NaCl sodium phosphates NaCl I = 0.1 + 0.25 M total ionic strength pH 0-4 0-4 0.3 5 0.4 0.4 0.3 0.35 0-27 0.15 0.40 0.15 0-40 0.15 0.35 7.2 7.3 7.1 7.2 7.3 7-1 7-1 7.6 7.6 7.4 7-6 7.4 7.4 7-1 mobilities in cmz/V SEX 105 references ascending - 2.7 - 3.1 - 2.9 - 2.5 - 2.9 - 2.6 - 2.8 - 3.66 - 9.3 - 6.3 - 6.4 - 4.6 - 6.90 - 4.30 descending - __ 2.5' - 2.6' - - 2.5* - 2.6 - 2*6* - 3-39 - 9-1 - 6.7 - 6.1 - 4.7 - 6.20 - 3.90 *this value corresponds to the single peak observed in the descending limb with The determinations made by Hamoir are relative to fish proteins ; the others to rabbit mixtures of actomyosin and myosin (see fig.1, 2, 3). proteins. by dialysis against a phosphate + NaCl buffer of I = 0-15 and pH 7.3 (fig. 4). Further- more the a peak can be quantitatively and selectively precipitated by the method recently described by Portzehl, Schramm and Weber22 which allows the separation of acto- myosin and myosin.Although striated muscles of very different properties have been investigated in this comparative work, no variation has been observed in the electro- chemical behaviour of actomyosin and myosin except in the special case of embryonic rabbit muscle.17 This constancy contrasts surprisingly with the behaviour of sarco- plasmic proteins.20 In view of the big differences observed by ultracentrifugation between fish and rabbit actomyosins,20 it seems likely that the electrochemical properties of acto- myosin and myosin are independent not only of the material used but also more or less extensively of the shape of the particles. But the elaboration of methods of isolation allowed one to investigate also solutions of each of these components.ACToMYOSIN.-Fig. 5 shows the very pronounced asymmetry given by a fish actomyosin extracted at pH 5.5 and isolated by the method of Portzehl, Schramm and Weber.22 The ascending boundary remains very sharp indefinitely while the descending one spreadsG . HAMOIR 119 fairly quickly. Viscosity measurements on actomyosin have shown that its solutions are not only highly viscous but also thi~otropic.23~24 Actomyosin must behave as a gel in the U-tube and therefore give rise to this asymmetry. MYosm-The electrophoretic behaviour of myosin preparations devoid of actomyosin was first investigated by Dubuisson.12 But a more detailed study was made later on by Erdos and Snellman 6 2 5 on myosin prepared according to Szent-Gyorgyi.26 The preparation appeared homogeneous in the U-tube for a fairly long period but prolonged electrophoresis revealed a very slow splitting into two components which was not further investigated by the authors.Mobilities were determined in the presence of 0.05 M K veronal acetate buffers of different pH values and with varying amounts of KCl (0.1, 0.25 and 0.5 M). No change was observed on altering the potassium chloride concentra- tion. The influence of the pH is shown in fig. 6 in which the experimental values (circles) are compared with the titration curve (smooth curve) which Dubuisson 27 found for myosin in the presence of some actomyosin and which agrees essentially with the more * FIG. 3.-Electrophoretic pattern of a 10-min extract from carp muscle at I = 0.5 and pH 5.8. Upper part : ascending limb ; lower part : descending limb ; ionic strength 0.35, pH 7.1 ; duration of the electrophoresis: 79,100 sec; electrical field 2-0 V/cm (after Hamoir 20).FIG. 4.-Electrophoretic pattern of a 10-min extract from carp muscle at I = 0.5 and pH 5-5. Upper part : ascending limb ; lower part : descending limb ; ionic strength 0.15, pH 7-1 ; duration of the electrophoresis: 16,500 sec; electrical field: 3.70 V/cm (after Hamoir 20). FIG. 5.-Electrophoretic pattern of a 0.83 % solution of carp actomyosin extracted at I = 1-0 and pH 5-5. Upper part : ascending limb ; lower part : descending limb ; ionic strength 0.35, pH 7.1 ; duration of the electrophoresis : 96,060 sec ; electrical field : 1.74 V/cm. recent titration curve of Mihalyi28 made on pure myosin. An excellent agreement was found between the charge and the mobility of the particle.The isoelectric point of 5.4 given by the pH-mobility curve agrees also with the old determination of Hollwede and Weber.29 The mobility at pH 7.1 is practically identical with the value given by Dubuisson 1 for the /3 peak (see table 1). The influence of Ca2+ and Mgz+ ions on the mobility of myosin was examined by Erdos and Snellman ; 6.24 it is unusually large as shown by the values given in fig. 6 (crosses). Sarkar 30 has recently investigated the precipitation of myosin in the presence of different salts by turbidimetry and followed the migration of the protein in the Abramson micro-electrophoresis apparatus by addition of colloidal charcoal. According to the author, the maximum of precipitation would correspond to the isoelectric state so that, when the potassium chloride concentration is1 20 MUSCLE STRUCTURE PROTEINS increased, the isoelectric point should vary from 5.4 (salt-free myosin) to 6.9 (0.025 M KC1) and should shift back to about 3 (0.8 M KCl).Such behaviour is quite improbable - U x / o ” ’ 4 FIG. 6.-Mobility-pH curvs of rabbit myosin in veronal +acetate buffer and (a) potassium chloride (0.1 to 0.5 M KCl (circles) or (b) calcium or magnesium chloride (0.03 to 0.1 M) (crosses). The smooth curve corresponds to Dubuisson’s titration curve (after Erdos and Snellman 6). Ordinate : left : mobility in cmZ/V sec x 105 ; right : equiv. x lO-s/g myosin ; abscissa : pH. and the experimental evidence given is insufficient : the extent of the coating of the char- coal particles is not considered and the corresponding mobility-pH curves are not given.FIG. 7.-Electrophoretic pattern of a 1.1 % solu- tion of rabbit F-actin incompletely depoly- merized by the use of sodium iodide. Upper part : ascending limb ; lower part : descending limb; ionic strength 0-40; pH 7.44; duration of the electrophoresis : 36.420 sec ; electrical field : 1.6 V/cm F = F-actin ; Fd = depoly- merized F-actin (after Dubuisson 31). 4 I If no account is taken of this last work, the results obtained on actomyosin and myosin are in good agreement. This agreement is, however, sometimes fortuitousG . HAMOIR 121 the mobility values at pH 7.1 of Erdos and Snellman 6 and of Dubuisson 1 , 15 were deter- mined in the presence of different buffers and much higher values were found in Upsala by Amberson et aZ.7 in solutions containing 0-05 or 0-1 M potassium phosphate (see table 1) ; Hollwede and Weber 29 used a myosin preparation contaminated with actomyosin.Accurate determinations of mobility by both electrophoretic methods on pure prepara- tions over a wider range of conditions are desirable. AcTm-The study of the electrophoretic behaviour of actin presents a special interest in view of its well-known ability to occur in two forms : the globular one existing in salt- free solution (G-actin) and the fibrillar one formed by the polymerization of G-actin by salts (F-actin). Dubuisson 31 investigated the electrophoretic behaviour of F-actin and Fd-actin obtained by depolymerization of F-actin with NaI or by dialysis against 0.001 M NaHC03.The results obtained are summarized in fig. 7 and table 1. The method of depolymerization is without influence on the electrophoretic pattern and the two peaks appear to migrate independently in a mixture of the two forms (fig. 7). These results still await a satisfactory explanation for an increase in asymmetry of the particles should decrease the mobility. A suggestion has been made about the nature of the phenomenon by Dubuisson31 who has found that the polymerization is accompanied by a marked acidification of the solution: 1 g of G-actin liberates 45 x 10-5 equiv. H+ on trans- formation into F-actin in the presence of CaC12. The splitting of the ATP combined with actin into ADP during polymerization observed by Straub and Feuer32 would liberate only 33 about 1-5 x 10-5 equiv.H+/g and cannot therefore be the main factor involved. The high mobilities found by Dubuisson disagree also with the amino-acid content of actin given by Feuer, Molnar, Pettko and Straub.34 A comparison of their analytical data with values given by Bailey 35 for myosin shows that the number of the free acidic and basic groups of actin would amount to half the corresponding values for myosin. This seems unlikely in view of the electrophoretic evidence. Although an electrochemical study of F and G-actin seems particularly promising, no further research has been done in this direction. TROPOMYOSIN.-A new component of the myofibril, tropomyosin, was described by Bailey 35 in 1948. He found that this protein was homogeneous by electrophoresis and that its charge was particularly high : its free acidic and basic groups correspond to 45 % of the total residues while the value35 for myosin is 34 %.Its asymmetry is also less than that of myosin, their axial ratios being respectively 33 (at ionic strength 36 0.3 and pH 6.5) and approximately 37 100. From this tropomyosin must exhibit a fairly high mobility. It migrates effectively 1.6 times faster than myosin,38 and its mobility is thus relatively close to that of depolymerized actin (see table 1). Recent research has shown that tropomyosin can exist in two forms differing from that described by Bailey. The first containing 15 to 20 % ribonucleic acid has been called nucleotropomyosin.38r 39 It has been found undistinguishable from tropomyosin by electrophoresis, a behaviour suggesting that the ionic groups of the nucleic acid are not available for chemical reactions.It is possible, however, that the presence of a small gradient corresponding with nucleic acid may have been masked and that the gradient observed was due to pure tropomyosin. A more careful electrophoretic investigation of such preparations is needed. The second form of tropomyosin 40 is devoid of nucleic acid and probably corresponds with a polymer of Bailey’s tropomyosin : it is stable at ionic strength 0.35 and pH 7.1 and sediments, as does nucleotropomyosin, much more rapidly than Bailey’s tropomyosin in the ultracentrifuge ; it depolymerizes on precipita- tion at ionic strength 1 and pH 4.6.Fig. 8 represents the electrophoretic pattern of the polymer form containing a small amount of the faster migrating depolymerized one. Fig. 9 showing the same preparation after incomplete depolymerization indicates clearly the slight change of mobility produced by this phenomenon. The electrophoretic data actually available on actomyosin, myosin, actin and tropo- myosin are summarized in tables 1 and 2. The mobilities given in table 1 are usually mean values of many experiments. Account must be taken for their comparison not only of the nature of the solvent but also of the conductivity value used for the calculation of the electrical field. Higher mobilities are of course obtained when the value corre- sponding to the buffer is taken instead of that of the protein solution.The corresponding variation can easily be evaluated by comparing the conductivity values given by the two solutions before electrophoresis. At I = 0-35 and pH 7.1, the ratio of the conductivity of the buffer to the conductivity of the protein solution (1-2 % protein) amounts to 1.046 (mean of 20 values). The disagreement which could therefore be observed between different authors on account of this factor does not exceed 5 %. The values given in1 22 MUSCLE STRUCTURE PROTEINS table 1 and 2 complete other physicochemical determinations given by Weber 41 in a recent publication on muscle proteins. PROTEIN INTERACTION.-This review would be incomplete if we did not mention some interactions which have been observed between the muscle structural proteins.It appears likely that their very polar character pointed out recently by Edsall,43 and their pronounced FIG. 8 FIG. 9 FIG. 8.-Electrophoretic pattern of a 1.18 % solution of incompletely polymerized carp tropomyosin. Upper part : ascending limb ; lower part : descending limb ; ionic strength 0.35 ; pH 7-1 ; duration of the electrophoresis : 76,320 sec ; electrical field : 1.86 V/cm. FIG. g.-Electrophoretic pattern of a 1.44 % solution of incompletely depolymer ized carp tropomyosin. Upper part : ascending limb ; lower part : descending limb ; ionic strength 0.35 ; pH 7.1 ; duration of the electrophoresis : 77,400 sec ; electrical field : 1-71 V/cm. TABLE 2.-kOELECTRIC POINTS OF THE MUSCLE STRUCTURAL PROTEINS protein solvent 0.01 M acetate + 0.04 M and myosin NaCl 0.1 M acetate + 0.01 M NaCl water mixture actomyosin myosin 0.05 M K veronal acetate water + 0.1 to 0.5 M KCL tropomyosin 0.01 M KC1 0.01 M Na acetate + acetic acid method isoelectric pt references microscopic - 5-0 (42) swellings -5-0 (42) minimum alkali content 5.4 (29) of gel boundary moving 5.4 (6) microscopic 5.4 (30) rate of settling 5.1 (35) of the particles microscopic 5.0 (40) asymmetry would favour adsorption phenomena and interactions.Few anomalies have, however, been observed in the electrophoretic patterns of these compounds or of their mixtures. The absence of a good separation of actomyosin and myosin in the descending limb (fig. 1 and 2) is very probably due to the inevitable initial blurring of the boundary. Some interaction seems to occur, in fish whole extracts, between a component migrating more quickly than actomyosin and actomyosin or myosin : comparison of fig.2 and 4 shows that the area of the fastest ascending gradient increases markedly after the removal of actomyosin and myosin by dialysis at low ionic strength. Fig. 8 shows also by theG . HAMOIR 123 lack of symmetry of the ascending and descending pictures that polymerized and de- polymerized tropomyosin seem to interact strongly ; their close mobilities however, made the study of this phenomenon difficult. A clearer example of interaction is shown by a preparation obtained from the extract represented by fig. 3. The a-peak is first removed following Portzehl, Schramm and Weber.22 The precipitate isolated by dilution of the super-natant is not pure myosin but a mixture of myosin and nucleotropomyosin.Its electrophoretic diagram given in fig. 10 shows that the area and the mobilities of the two peaks observed are very different in both limbs. Similar although less pronounced asymmetries have been obtained by Longsworth and MacInnes 44 in the isoelectric region of ovalbumin with mixtures of this protein and yeast nucleic acid. They have explained this behaviour by assuming the existence in the mixture of a reversibly dissociable complex. FIG. 10.-Electrophoretic pattern of a mixture of carp myosin and carp nucleotropomyosin. Upper part : ascending limb ; lower part : descending limb ; ionic strength 0-35 ; pH 7.1 ; duration of the electrophoresis : 74,640 sec ; electrical field : 1.73 V/cm.The sharp ascending boundary and the slow descending one correspond to the two pure components migrating with their normal mobility ; in the two others, dissociation of the complex occurs and their displacement correspond to the mobility of neither component nor that of the complex. This explanation seems also valid in the present case which differs only by the importance of the asymmetry and by its occurrence between two proteins both carrying large negative charges. CONCLUSION The electrophoretic behaviour of the muscle structural proteins is relatively simple; it does not seem to depend very much on the state of aggregation or on the degree of asymmetry and does not give rise to frequent interactions. These properties, the wide range of mobilities presented by muscle proteins and the absence from muscle extracts of other important components migrating at the level of myosin and actomyosin greatly facilitate their identification in the electro- phoretic patterns of whole muscle extracts.These considerations explain why the electrophoretic method has proved so suitable for the analysis of muscle extracts. However, the general representation obtained by electrophoresis, although giving a fairly true picture of these mixtures from the electrochemical point of view, is a largely over simplified one. The analysis must be completed by the fractionation of the extracts and the ultracentrifugal study of much less complicated mixtures or pure electrophoretic fractions. The pronounced tendency of the muscle structural proteins to exist in different states of aggregation makes ultracentrifugation par- ticularly useful in the investigation of these proteins, but the marked dependence of the ultracentrifugal patterns on the asymmetry and state of aggregation make it unsuitable for the study of complicated mixtures.The complementary character of these two analytical methods seems particularly well illustrated by the example of the muscle structural proteins.124 MUSCLE STRUCTURE PROTElNS I am much indebted to Prof. M. Dubuisson for his advice and the facilities put at our disposal, and to Dr. R. A. Kekwick, who suggested this review, for reading the manuscript. 1 Dubuisson, Experientia, 1946, 2, 258. 2 Dubuisson, Nature, 1950, 166, 11 16. 3 Bailey, Biochem. J., 1942, 36, 121.4 Mehl and Sexton, Fed. Proc., 1942, 1, 125. 5 Ziff and Moore, J. Biol. Chem., 1944, 153, 653. 6 Erdos and Snellman, Biochim. biophys. Acta, 1948, 2, 642. 7 Amberson, Erdos, Chinn and Ludes, J. Biol. Chem., 1949, 181,405. 8 Spicer and Gergely, J. Biol. Chem., 1951, 188, 179. 9 Mommaerts and Parrish, J. Biol. Chem., 1951, 188, 545. 10 Dubuisson, Distkche and Debot, Biochim. biophys. Acta, 1950, 6 , 97. 11 Greenstein and Edsall, J . Biol. Chem., 1940, 133, 397. 12 Dubuisson, Experientia, 1946, 2, 412. 13 Jacob, Biochem. J., 1947, 41, 83. 14 Jacob, Experientia, 1947, 3, 241. 15 Dubuisson, BioL. Rev., 1950, 25, 46. 16 Ravet-HCrion, Rev. Mkdicale Lizge, 1950, 5, 662. 17 Crepax, Biochim. biophys. Acta, 1952 (in press). 18 Gelotte, Biochim. biophys. Acta, 1951, 7, 378. 19 Hamoir, Bull. SOC. chim. biol., 1949, 31, 118. 20 Hamoir, Biochem. SOC. Symposia no. 6 (Cambridge University Press, 1951), p. 8. 21 Godeaux, unpublished results. 22 Portzehl, Schramm and Weber, 2. Naturforsch., 1950, 5b, 61. 23 Bailey and Perry, Biochim. biophys. Acta, 1947, 1, 506. 24 Jaisle, Biochem. Z., 1951, 321, 451. 25 Snellman and Erdos, Nature, 1948, 161, 526. 26 Szent-Gyorgyi, Acta physiol. Scandin., 1945, 9, suppl. 25. 27 Dubuisson, Arch. Inter. Physiol., 1941, 51, 133. 28 Mihalyi, Enzymologia, 1950, 14,224. 29 Hollwede and Weber, Biochern. Z., 1938,295,205. 30 Sarkar, Enzymolugia, 1950, 14,237. 31 Dubuisson, Biochim. biophys. Acta, 1950, 5,426. 32 Straub and Feuer, Biochim. biophys. Acta, 1950, 4,455. 33 Dubuisson and Mathieu, Experientia, 1950, 6, 103. 34 Feuer, Molnar, Pettko and Straub, Hungarica Acta PhysioL., 1948, 1, 1. 35 Bailey, Biochem. J., 1948, 43, 271. 36 Tsao, Bailey and Adair, Biochem. J., 1951, 49, 27. 37 Portzehl, Z. Naturforsch., 1950, 56, 75. 38 Hamoir, Biochem. J., 1951, 48, 146. 39Hamoir, Biochem. J., 1951, 50, 140. 40 Hamoir, unpublished results. 41 Weber, Biochim. biophys. Acta, 1950, 4, 12. 42 Weber, Ergebnisze Physiol. experim. Pharmakol., 1934, 36,205. 43 Edsall, Proc. Roy. SOC. B, 1950, 137, 82. 44 Longsworth and MacInnes, J . gen. Physio1.,1942,25, 507.

ISSN:0366-9033    

DOI:10.1039/DF9531300116

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

ENERGETICS AND MOLECULAR MECHANISMS IN MUSCLE ACTION * PART I. OUTLINE OF A THEORY OF MUSCLE ACTION, AND SOME OF ITS EXPERIMENTAL BASIS BY MANUEL MORALES AND JEAN BOTTS Naval Medical Research Institute, National Naval Medical Centre, Bethesda, Md., USA. Received 29th April, 1952 Thermoelastic studies of myosin threads, light-scattering studies on the myosin ATP- system, and a kinetic analysis of myosin ATP-ase are summarized. The results are inter- preted with a theory which assumes that the elastic myosin particle is subject to a con- tractile entropic force and an extensile electrostatic repulsion. In an earlier paper,l we proposed a simple hypothesis about the elementary cycle in muscle action, and later, Hill and one of us discussed the formal thermo- dynamics of such a cycle.2 These papers sought to evaluate all evidence relevant to our argument ; the present communications,t however, are limited to certain recent developments, principally in our own laboratory.At this primitive stage in the formulation of a molecular theory of muscle action, certain concessions are not only becoming modesty, but are absolutely forced upon one by the circumstances. Two such concessions are the following. The theory to be discussed is not unchallenged. In particular, from the protein myosin, here to be regarded as a single functional entity, Straub 3 and the Szent- Gyorgyi school 4 have derived either two sub-units, or else two modifications of the parent material, viz. " myosin " and " actin ".$ " Actin " has been shown 5-7 to undergo a polymerization in the course of which adenosine triphosphate (ATP) is somehow dephosphorylated, and in certain ionic media " actin " and " myosin " have been shown to co-precipitate upon addition of ATP.8 These various ob- servations have been widely verified by competent investigators, and the existence of these phenomena is unquestionable.To the investigators who have discovered and described them it has seemed that these phenomena involving the interplay of chemical reaction with dimensional change, the liberation of free energy by dephosphorylation, etc., must have some very crucial role in the molecular events of muscle action. Yet, despite an intense interest in the subject, no one has pro- posed a physically reasonable mechanism whereby " myosin ", '' actin ", and ATP (acting independently) can form a system capable of generating a time-variable tension.This, we feel, is not a reflection on the imagination of the biochemists concerned, but may be due, rather, to the unlikelihood of building such a system on the basis of polymerizations or co-precipitations. To these philosophical doubts we shall add below evidence which undermines the current interpretation of the " myosin "-" actin "-ATP phenomena. We are, however, scarcely in a * The opinions expressed in this article are those of the authors, and do not necessarily 7 Parts 1 and 2 of this paper. reflect the views of the Navy Department, 01' the Naval Service at large. To distinguish these substances from the myosin prepared by the Weber-Edsall method their names will be enclosed in quotation marks.125126 MUSCLE ACTION position to prove our rival views conclusively, and, despite our misgivings, we find it quite impossible to rule out entirely from the normal action cycle so complicated a set of phenomena as those attributed to " myosin ", " actin ", and ATP. A second concession must be made to physiologists especially concerned with the action of living muscles. Some years ago, in company with most newcomers to this problem, the authors entertained the ideal of explaining actual muscle pheno- mena in terms of a simple molecular model. Since then it has become amply clear that a different and limited objective is more realistic : to specify a reversibly deformable molecular system, constructed out of materials found in muscle.This change of objective is motivated not only by purely practical considerations but by the conviction that in studying condensed heterogeneous assemblies such as actual living muscle, or even the glycerinated psoas preparation, one invites for- midable complications additional to those inherent in the simplest deformable units. However, in studying simple situations one clearly endangers the physio- logical significance of the result. Such a risk is definitely taken in the present work. We consider the particles of myosin to be highly elastic, and to have dimensions governed by the offset between a contractile entropic force and an extensile electrostatic force. * The contractile force is supposed to arise from the fact that the particle gains configurational entropy upon shortening, whether the shortening is by " random " coiling or by coiling into an ordered form.12* 1,13 The extensile force is supposed to arise from mutual repulsions between the excess (i.e.the net) electrical charges of the same sign fixed on the particle. The contractile force may be varied by varying the number and kind of ions adsorbed; thus the adsorption of metal cations, or of protons on free carboxylate and amino groups, algebraically increases the net charge, while the adsorption of anions algebraically decreases the net charge. Among the ions adsorbable on myosin the anion of ATP is very special: (i) it is strongly and specifically adsorbed, by forces in addition to coulombic. (ii) It bears a high net charge (-4, at pH7).14 (iii) It is enzymatically decomposed by myosin into non-adsorbed anions (adenosine diphosphate (ADP) and orthophosphate (P)) of much lower free energy.These simple facts and postulates lead to several predictions. Even in the absence of ATP the particles of myosin should be de- formable by varying the temperature, the ionic milieu, the dielectric constant of the solution, or the pH. The effect of adding ATP should depend on initial cir- cumstances. If the myosin were positively charged and a moderate amount of ATP were added, the absolute value of the net charge would decrease, and the myosin particles should contract. If the myosin were negatively charged and ATP were added, the absolute value of the net charge would increase, and the myosin particles should extend.If the myosin were charged moderately positively and a massive amount of ATP were added the myosin particles then too should extend. In all cases the effect of adding ATP should be spontaneously reversible-rapidly, if the state of the system favours ATP-ase activity, and slowly if it does not. Super- imposed on the foregoing scheme are two effects whose importance in aqueous myosin systems has not been extensively studied : myosin-myosin hydrogen bond- ing (either inter- or intra-particle),ls and myosin-solvent mixing. It has recently been shown,l3 using plausible assumptions, that these effects modify the simple entropic-electrostatic scheme in a favourable way, i.e. increase its similarity to real systems. Of the many experimental suggestions implied above, some have been under consideration in our laboratory, and in the remainder of this paper we wish to summarize those of our results which appear rather conclusive and which seem to support our general hypothesis. * Although our model differs significantly in detail, it adopts the basic scheme proposed by Bull 9 and Riseman and Kirkwood 10 for muscle, and Katchalsky 11 for flexible poly- electrolytes in general.The gist of our model can be stated very simply.MANUEL MORALES AND JEAN BOTTS 127 AN EXPERIMENT DEMONSTRATING THE OPERATION OF AN ENTROPIC FORCE.- Myosin prepared and purified by the methods of Weber and Edsall is soluble in 0-6 M KC1 and very slightly soluble in low (0--05 M) concentrations, at pH 7.0. If a concentrated myosin solution is extruded through a fine jet (moving uniformly in a spiral path) into water, one obtains a myosin " thread " suitable for various studies.16 In such a thread the elongate particles of the myosin are " vulcanized " together into a moderately oriented network.The " vulcanization " is accom- plished by the formation of (presumably) electrostatic linkages and of hydrogen bonds.15 Such threads are rather frail, but if carefully handled and stored at temperatures 0-4" C , they retain their property of shortening with ATP addition, their long-range elasticity, solubility in 0.6 M KCl, birefringence, and ATP-ase activity, for long periods of time. We felt that the macroscopic thermoelastic properties of these threads would give an insight to the thermoelastic properties of their microscopic constituents, i.e.the myosin particles. The system chosen for thermoelastic study was a myosin thread immersed in 0.001 M phosphate buffer at pH 6.8. The state of the system was specified by the thread tension T, temperature T, pressure p , and length E ; the problem was to measure the variations, with length, of the energy U, volume v, and entropy S. It was shown17 that p(31;/3Z)~, was negligible relative to (3 U/31)~, and (3S/31)~, ; consequently the Wiegand-Snyder equation, applied. The results of applying (1) to small extensions (0-10 %) of myosin threads are illustrated in fig. 1. According to this figure the shortening force in a myosin thread is due to the fact that the threadgains entropy upon shortening. A thermo- dynamic experiment of course gives no clue to molecular mechanism, but in view of the structure of the thread it was natural to suppose that the entropy gained on shortening was entropy of configuration.Fig. 1 also shows that the internal energy of the thread gives rise to an outwardly-directed extensile force, the absolute value of which falls with stretch. Stimulated by the contemporary work of Katchalsky 11 and the ideas of Riseman and Kirkwood 10 we were led to speculate that this ex- tensile force was an electrostatic repulsion between charges fixed on the myosin.15 Experimental results similar to the foregoing were reported independently and almost simultaneously by Weber.18 Such results, and the interpretations just given, are basic to our hypothesis. They must, however, be qualified in one im- portant way.Prior to our work, Woods 19 had reported briefly on a thermo- elastic study of myosin films. According to Woods, the entropic tension in his films was negligible, or even negative, whereas the gain in energy with stretch was substantial, and accounted for all the tension exerted by the film. Few details are given by Woods regarding the manner in which he prepared and measured his films, or the tests whereby he excluded denaturation. However, myosin threads can be made to exhibit the thermoelastic behaviour described by Woods provided they are at least once dried under stretch. From infra-red absorption studies and other evidence we feel that this treatment permits extensive " vulcanization " by hydrogen bonds, and thus inverts the thermoelastic behaviour of the myosin, much as real vulcanization inverts the thermoelastic behaviour of rubber.When once- dried threads are placed back into the solution they do not reverse their properties even after an extended time, and they are very sluggish in their response to ATP, possibly because of impaired diffusion of the nucleotide. In seeking a reversibly deformable system, we feel a strong preference for the undried thread. Judging from the 0.15 M K+ concentration supposed to exist in living muscle 20 and the appearance of electron micrographs,21 and, above all, the presence of 90 % water by weight, we feel that the undried thread is also a better model of the myosin in the muscle fibre. Indeed the drastic structural inversion brought about simply128 MUSCLE ACTION by drying under stretch should warn against uncritical structural studies of muscles treated similarly.In summary, we feel that the thermoelastic studies of myosin threads indicate that the positive tension in such threads is entropic, and that the studies are con- sistent with the idea that, in general, the entropic force is offset by an electrostatic repulsion. THE EFFECT OF ATP AND OTHER IONS UPON THE CONFIGURATION OF THE MYOSIN PARTIcLE.-The threads described above when placed unloaded in 0401 M MgC12, contract violently upon ATP addition. At first sight it would seem that the structural changes induced by ATP might also be examined by thermoelastic methods (as indeed has been attempted on similar experimental objects). This approach, however, has many technical and theoretical disadvantages.Firstly, the relatively weak cross-bonding of the threads cannot sustain the high tension I- 6 4 2 0 Exhmion I I I I I I l ~ l ~ l FIG. 1.-The resolution of the tension of undried myosin threads by means of eqn. (1). induced by the adsorption of ATP; as a result, the thread shreds, giving the erroneous (we believe) impression that it has lengthened. Secondly, the system is (at best) in a steady-state with respect to the dephosphorylation of ATP. The thermodynamics of a mechanochemical system in a steady state have not been elucidated, and eqn. (1) cannot apply to such a system except in a very crude way.1 We must, therefore, seek another method of study. The most obvious alternative is to study a population of independent myosin particles, i.e.a solution, employing some method of observation which gives the average mass and shape of the par- ticles during their interaction with ATP. To obtain myosin in solution, however, it is usual to employ 0.6 M KCl as a solvent. In recent years several investigators have interpreted their viscosimetric,Zz ultracentrifugal,2*. 23 and light-scattering 24 measurements as indicating that when ATP is added to a myosin sulutiun the result is a dissociation of the myosin into “ myosin ” and ‘‘ actin ” ; only Jordan and Oster 25 have claimed that ATP coils (rather than dissociates) the dissolved myosinMANUEL MORALES AND JEAN BOTTS 129 I . . L------- . C A = A O sin' (ff/..) + 50c I , a 5 0 11.0 I I/-5 I particles. Prompted by the considerable theoretical difficulties of interpreting viscosity and ultracentrifuge data on asymmetric particles whose shape, charge, and mass may all vary, and noting that light-scattering data have hitherto been analyzed in a very approximate way (by the " dissymmetry " method), Blum under- took a re-examination of the problem.26 Using an incident intensity I,, Blum measured the scattered intensities I at various angles 8 and concentrations c for the systems * : myosin, myosin + ATP (steady state), and myosin + ADP + P (recovery).For each system he computed (cf. fig. 2) the (weight average) particle molecular weight M, the shape functions P (8) and the second virial coefficient B, by taking suitable limits of the equation, KC/I(O, c) = (P-l(S)/M) 4- 2Bc, (2) where K is a factor consisting of independently measured, and of universal, con- stants.This procedure is the extrapolation method of Zimm ; 27,28 it gives (in contrast to the dissymmetry method) a value of M independent of any assumptions regarding particle shape. As shown in fig. 3, Blum found that M was the same (of the order of 20 x 106) for all three systems; in other words, no dissociation into " myosin " and " actin " had occurred. However, the same data, analyzed by the dissymmetry method gave the erroneous impression that ATP addition had caused M to drop to approximately M/2. We feel that Blum's experiments con- tradict, and are more reliable than, earlier light-scattering work of which we are aware. Clearly some reconciliation must be worked out between his result and the seeming implications of the viscosimetric and ultracentrifugal work.This, however, is a debate in which the light-scattering method is, so to speak, on at least equal terms with the rival kinetic methods; the problem is discussed else- where.26 Accepting Blum's result provisionally, we can draw certain other in- teresting conclusions from his work. The shape functions P(8) fit rather well the shape functions recently calculated by Saito and Ikeda 29 for circular cylinders whose length/diameter ratio is finite ; they decidedly do not fit the classical models : * All systems contained 0.6 M KCl and 0.001 M CaC12, and were buffered to pH 7. Protein concentrations were of the order of 0.01 ; ATP concentrations of the order of 10-4 M.130 MUSCLE ACTION sphere, random coil, ellipsoid of revolution, and infinitely thin rod.Using the cylindrical model, however, we find that the result of ATP addition seems to be to extend the cylinder reversibly (from 7000 to 10,OOOA) and possibly to inflate it laterally in some degree. Unlike the assignment of M, the assignment of shape is partly arbitrary, and the result that ATP addition extends the particle must be regarded as preliminary. However, Hayashi and Rosenblueth 30 have recently reported that whereas a myosin film contracts when ATP is added in 0-05 M KCl (as do threads), the film extends (reversibly) when ATP is added in 0.6 M KCl. It would seem that both the light-scattering and the film experiments can be satis- factorily interpreted in terms of the entropic-electrostatic scheme described above.By analogy with Sarkar’s electrophoretic studies 31 (in which Mg2+ rather than Ca2+ was the divalent cation), one would expect that in very dilute M KCl the protein is charged positively, whence anion (ATP) adsorption should lead to contraction ; at FIG. 3.-Graph to show that the molecular weight (reciprocal of the vertical intercept) of myosin in solution is not changed upon ATP addition, but that the particle shape is changed. These are superimposed Zimm extrapolations (illustrated in fig. 2) ; the bottom, nearly horizontal line, is common to all three extrapolations. 0.6 M KCl one would expect the protein to be charged negatively, whence anion (ATP) adsorption should lead to extension. A rough correspondence between the structural state of the myosin particles and the concentration of yet unhydrolyzed ATP was first established by the classical work of the Needham group.32 With the light-scattering method, however, the relationship can be rendered much more precise.For example, one can estimate the (maximum) steady-state rate of de- phosphorylating ATP equally well from optical data 26 or chemical phosphate analysis (see below).33 Still another topic under investigation by light-scattering is the possibility of deforming the myosin particle by changing the number of other ions (Ca2+, Mg2+, Hf) adsorbed upon it. Preliminary results suggest that such reversible deformations (at apparently constant M ) are possible, and that the shape changes induced (for instance, by changing the pH in the range, 7-10) are comparable in magnitude to those induced by adding ATP.Again, these pheno- mena seem to be reasonable consequences of having an adjustable electrostatic field within the myosin particle.MANUEL MORALES A N D JEAN BOTTS 131 SOME THERMODYNAMIC AND KINETIC CONSTANTS OF THE ATP-ATP-ASE SYSTEM.- It was shown by Ouellet, Laidler, and one of us 33 that at pH 7.0 the steady state enzymatic activity V of myosin at total concentration [Eo], and maintained ATP concentration [ATP], was well described by the conventional equation, with k2 as the first-order velocity constant for the decomposition of ATP on the enzyme surface and K (in this case) as probably the actual equilibrium constant of binding ATP to the myosin. Apparent thermodynamic quantities correspond- ing to k2 and K were calculated by applying the usual thermodynamic analysis to rate measurements at different temperatures ; this treatment gave AH^* = 12.4 kcal mole-1 ; A&* = - 8-0 cal mole-1 deg.-1; AH = 8 kcal mole-1 ; and A S = 49 cal mole-1 deg.-l.Thermodynamic quantities derived from kinetic analyses in the simple manner just described are, in general, only “apparent” by reason of (substrate) multi- valency in the enzyme particle, and pH or specific ion effects.34 In the present case, for example, a simple * n-fold multivalency would add - Rln n to A&*, and elsewhere it has been estimated that the foregoing quantities may be subject to “ errors ” as high as 40 % because of pH and salt effects, i.e. they may contain contributions from ionizations amounting to, say, 40 % of the formal quantities written for the net reactions.Despite these uncertainties of detailed interpretation, it appears very likely that the process of binding ATP to myosin under the con- ditions of these experiments (conditions, by the way, which were identical to those employed in the light-scattering work), although strongly exergonic. (4Fo = - 6.6 kcal mole-I), is endothermic, and is accompanied by a large entropy in- crease. These results are in good qualitative agreement with the notion that in this process a highly solvated anion is bound to a negatively charged surface, presumably with a loss of highly oriented water molecules from both anion and surface. With myosin (as opposed to rigid enzymes) still another difficulty is en- countered, viz.that the 4H and 4s of binding may contain substantial contributions from structural modifications of the protein. The issue would be whether the local binding region of the myosin particle has time to extend (see above) between the instant that the dephosphorylation products leave the site and the instant that a new ATP molecule is bound. This issue cannot be settled at present. Our guess is that the relaxation time of the particle is probably too slow for this to happen; moreover, the configurational entropy change accompanying such an extension would be negative, so that the qualitative conclusion that the “ true ” AS of binding is positive would not be affected. We think, therefore, that the local regions do not “ cycle ” mechanically, but that under conditions of this and the light-scattering experiment they are in a steady state of extension.SOME SPECTROSCOPIC STUDIES OF THE NUCLEOTIDES-MYOSIN SYSTEM.-A survey of the ultra-violet 35 and infra-red 3 6 3 7 absorption characteristics of the system in question was made in company with Tarver, Laki and Gergely. It was found possible to follow by infra-red spectroscopy interconversions of ATP, ADP, and AMP, of tripolyphosphate and P, and the dephosphorylation which accompanies actin polymerization. Except for an indication of a fast ultra-violet absorption transient when ATP reacts with myosin in the presence of Ca2+, however, these studies provided no evidence whatever for the idea that myosin is extensively altered (in the sense of covalent bond formation) upon reaction with ATP.This negative result lends a measure of back-handed support to the mechanism of inter- action postulated above. * i.e., no interaction among bound substrate molecules.132 MUSCLE ACTION 1 Morales and Botts, Arch. Biochem. Biophys. (in press). 2 Hill and Morales, Arch. Biochem. Biophys. (in press). 3 Straub, Studies Inst. Med. Chem. Szeged, 1943, 3, 23. 4 Szent-Gyorgyi, The Chemistry of Muscular Contraction (Academic Press, New York, 5 Straub, Biochim. Biophys. Acta, 1950, 4, 455. 6 Laki, Bowen and Clark, J. Gen. Physiol., 1950, 33, 437. 7 Mommaerts, Fed. Proc., 1951, 10, 225. 8 Spicer and Gergely, J, Biol. Chem., 1951, 188, 179. 9 Bull, Quart. Bull. Northwest. Med. Sch., 1946, 20, 175. 10 Riseman and Kirkwood, J. Amer. Chem. Soc., 1948, 70, 2820. 11 Katchalsky, Experientia, 1949, 5, 319. 12 Frenkel, Kinetic Theory of Liquids (Oxford, 1946), p. 485. 13 Hill, J. Chem. Physics (in press). 14 Alberty, Smith and Bock, to be published. 15 Botts and Morales, Fed. Proc., 1950, 9, 15, and J. Cell. Comp. Physiol., 1951, 37, 27. 16 Dubuisson, Arch. Znternat. Physiol., 1943, 53, 29. 17 Botts, Johnson and Morales, J . Cell. Comp. Physiol., 1951, 37, 247. 18 Weber, Abstr., Inter. Physiol. Congr., 1950, 18, 62. 19 Woods, Nature, 1946, 157, 229. 20 Dubuisson, Arch. Inter. Physiol., 1942, 111, 439. 21 Hall, Jakus and Schmitt, Biol. Bull., 1946, 90, 32. 22 Mommaerts, Expt. CelZ. Res., 1951, 2, 133. 23 Johnson and Landolt, Nature, 1950, 165,430. 24 Mommaerts, Fed. Proc., 1950, 9, 207. 25 Jordan and Oster, Science, 1948, 108, 188. 26 Blum, Fed. Proc., 1952, 11, 14 ; Thesis (University of Chicago), to be published. 27 Zimm, J. Chem. Physics, 1948, 16, 1099. 28 Doty and Edsall, Adv. Protein Chem., 1951, 6, 35. 29 Saito and Ikeda, J. Physic. SOC. Japan, 1951, 6, 305. 30 Hayashi and Rosenbleuth, Fed. Proc., 1952, 11,67. 31 Sarkar, Enzymol., 1950, 14, 237. 32 Dainty et al., J. Gen. Physiol., 1944, 27, 355. 33 Ouellet, Laidler and Morales, Arch. Biochem. Biophys. (in press). 34 Botts and Morales, to be published. 35 Tamer and Morales, J. Cell. Comp. Physiol., 1951, 37, 235. 36 Morales and Cecchini, J. Cell. Comp. Physiol., 1951, 37, 107. 37 Morales, Laki, Gergely and Cecchini, J. Cell. Comp. Physiol., 1951, 37, 477. 1947).

ISSN:0366-9033    

DOI:10.1039/DF9531300125

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

132 MUSCLE ACTION ENERGETICS AND MOLECULAR MECHANISMS IN MUSCLE ACTION PART 11-STATISTICAL THERMODYNAMICAL TREATMENT OF CONTRACTILE SYSTEMS BY TERRELL L. HILL Naval Medical Research Institute, Bethesda, Md. Received 29th April, 1952 Several possible models of the fundamental elastic element in muscle are illustrated. It is suggested that phase changes combined with relatively small alterations in the state of charge (or ionic strength) may account for the " razor edge " nature of muscle con- traction. However, it does not appear necessary to invoke phase changes to obtain con- siderable alterations in length of an elastic fibre at constant tension, as a result of a reason-TERRELL L . HILL 133 able change in the state of charge of the fibre. Various alternative methods of calculating the electrostatic free energy are examined.The different methods give results of the same order of magnitude in the example chosen. 1. INmoDucTIoN.---In an earlier paper 1 several models of an elastic fibre were discussed. Our purpose here is to illustrate by a few numerical examples some of the properties of the models, in particular charge effects and phase changes. Also, an analysis of the computation of the electrostatic free energy is given which is somewhat more detailed than in the earlier paper.1 It should be stated explicitly that the models considered are not supposed to be conceivable models (nor represent the behaviour or properties) of a muscle fibre but rather conceivable models on a molecular level of the fundamental elastic element of a muscle fibre.This elastic element must presumably " drag " with it water, other plastic and elastic material, etc. Also, the " elastic " element itself may exhibit non-equilibrium behaviour (e.g. in the a-/3 model 2), which we have not included. 2 . MODELS OF ELASTIC FIBREs.-In this section we consider four models, giving first an analytical treatment followed by a few examples and discussion. Deriva- tions of many equations have already been given 1 and will not be repeated here. (i) Network with free lateral swelling.-Consider a swollen cross-linked network in the form of a cylinder of radius R and length 1. There is no restraint on the lateral swelling of the cylinder. There are charges along the molecular chains so that the net charge is equivalent to n* charges E .A solution of electrolyte with Debye-Huckel constant K surrounds and penetrates the cylinder. We restrict ourselves here to the case where R > 1 / ~ and the linear Debye-Hiickel approximation can be used. Then one finds 1 that the length-tension relation is given by where V(M) is obtained from and (a) ANALYTICAL. t = a - (l/V.2), l/cc = ( 4 2 ) + q [ v / ( l - v ) ] 2 - Z [ ( K v * / 2 ) + v + In ( 1 - v)], (1) (2) t = do/vkT, tc = 1/10, q = 2nn*W/D~2vVokT, T = tension (strictly, force), 10 = rest length of unswollen (" dry ") polymer, v = number of molecular chains (between cross links) in network, D = dielectric constant of solvent, 2 = number of statistical units in a molecular chain, PVO = volume of a statistical unit, vo = volume of a solvent molecule, v = volume fraction of polymer, K = van Laar " heat " of mixing constant.VO = Zvofh = volume of unswollen polymer, In the next two models we shall find that for sufficiently large values of K (" poor " solvent for the polymer) a phase change can occur, which appears as a " loop " in the length-tension curve. This is a consequence of the property of polymer solutions with large3 K to split into two phases. We merely remark here that for large K (putting q = 0 for simplicity as the phase change is not an electrostatic effect), v in eqn. (2) is practically independent of a and is given ap- proximately by (recall that 2 is of the order of 100-1000) - In (1 - v) = ( K f ? / 2 ) $- V . (3) Thus, for large K, z, in eqn. ( 1 ) is essentially constant and no loop occurs in the length-tension curve.134 MUSCLE ACTION (ii) Network with fixed radius R*.--This is the same model as above except that the radius is kept constant, for example by rigid rings attached at intervals along the outside of the cylindrical network, or by some other means.In this case length changes are accompanied by proportional volume changes, and the thermodynamic work term is 1 (4) where Y is the volume of the cylinder and F the Helmholtz free energy. The two terms contributing to 7, combined, make a phase change possible. The first term is the usual deformation tension increasing monotonically with length while the second term is proportional to the chemical potential of the solvent 1,4 (to increase the volume, solvent must be taken in).The second term thus increases (q = 0 for simplicity again) with length, starting from negative values, until it passes through a positive maximum,4 and then falls, approaching zero asymptotically. Instead of eqn. (1) and (2), one fmds 1 rdl = [(3F/bl)T, y + nR*2(3F/3 V)T, Jdl, where 6* = R*/Ro and Ro is the " rest radius " of unswollen polymer. the phase change are related by From 3t/3a = 32t/3ci2 = 0, the values of 2, K and a at the critical point for Critical constants are given in table 1 for q = 0, 6* = 1 and q = 0, 6* = 2. (iii) The " accordion " modeL-This model consists of a number of cylindrical t n FIG, 1 .-The " accordion " model (3). In unit (a) the chains are ordinary " random flight" chains. In unit (b), the entire path must lie within a single unit.units, arranged longitudinally (fig. l), each one of which contains v polymer chains. The cross-linking takes place only in mathematical planes at the top and bottom of each unit (fig. 1). Other polymer molecules or structures, not participating in the elasticity of the system, must run laterally in these boundary planes and participate in the cross-linking in order to keep the cylinder with a constant radius R. R is taken as large enough that R > 1 / ~ and also that edge effects may be neglected (or a mem- brane around the cylinder could prevent " bulging '' of the polymer molecules). Parts of a given chain may lie inside of neighbouring units (fig. la), but by cancellation the average polymer density in a unit will be unaffected by this. An alternative model with essentially the same properties would result if the boundary planes were considered physically impenetrable and only configurations of the type shown in fig.lb counted. We need consider the thermodynamic properties of only one unit. The mixing and electrostatic free energy expressions obtained previously 1 are applic- able with V/Vo = 1/10 = a. For the deformation free energy we use the usual statistics of a single polymer chain based on a probability of occurrence of a given end to end distance I of the form P(1) = const. exp (- 312/2ZL2), (8)TERRELL L . HILL 135 TABLE 1 Critical constants, q = 0, model (2) - LT 1 1-5 2 2.5 3 3-5 4 4.5 5 6 7 6 * = 1 6* = 2 Z 12-50 43.3 110 233 436 750 1208 2710 5310 K 2-7 20 2.057 1.759 1.592 1 -484 1.410 1.356 1 -28 1 1.232 t Z 31.5 181.2 3.59 612 4.63 1559 5-64 3330 6.67 6300 7.67 8-67 9.71 11-72 13.67 K 1 -524 1.291 1.202 1.154 1-125 1.105 t 1.75 2-82 3-86 4.86 5-88 6.76 where L is the length of a statistical unit. for large extensions.) Then where Also, According to eqn.(8), 1 = 0 is the most probable value of I, but the incompress- ibility of the dry polymer (volume Yo) fixes the actual initial length at lo. It is reasonable to assume that a is of the order of magnitude of unity, for example, a = 2 (i.e. this would be the value of a if the chains are formed and attached to top and bottom of units with their most probable three-dimensional end-to-end length). The value of 20 and hence a will be reduced if the chains are formed in the above way in the presence of solvent.As in the preceding model, the volume is not an additional independent external variable. On combining the various free energies,l from r = (3F/31)T we find (Eqn. (8) can, of course, be modified AFdef. = vkTa(cc2 - l)/2, (9) a = 3102/ZL2. Vo = ZV/&I~ = 7cR210. 4 K 1 t = a u - - Eqn. (10) is similar to eqn. (5) with 6* = 1, as might be expected, and has similar properties including a phase change. The critical point is given by Z = 3cc2(a - 1)2 a - - [ (a - 2q 1)4 1 ' K = l + - 1 a3 2q cc - 1 + 2[" + m 3 - J Critical constants are given in table 2 for q = 0. TABLE 2 Critical constants, q = 0, model (3) LT Zla K 2.0 2.5 3.0 3.5 4.0 4.5 5-0 5.5 6.0 12.0 42.2 108 230 432 744 1 200 1840 2700 2.667 2.037 1.750 1-587 1.481 1.408 1.354 1-313 1.280 tla 3.68 4-70 5.7 1 6.72 7.72 8-73 9.73 10-74 11.75I36 MUSCLE ACTION Incidentally, as regards the critical point in the Flory-Huggins theory and in eqn. (7) and (12), K - 2q Z(1 - v)3 plays the role with q > 0 that K plays with q = 0.(iv) The a-/3 model.-For present purposes we need add nothing further to the earlier analysis,l but we describe the model for the benefit of the reader. Sup- pose we have a sheet of C polypeptide chains. In the a structure 5 each amino acid residue forms a hydrogen bond with its xth neighbour above and below in the same chain. According to Pauling and Corey,6 x = 3 for myosin and keratin. In the relatively extended p structure 5 a given residue forms a hydrogen bond with each of its two nearest neighbour residues on adjacent chains. In intermediate states we assume that in a given chain residues occur in groups or units of x, all residues a or all p within a unit, and that a and ,B units are scrambled statistically along the chain.This model can now be treated by a modification of the well-known quasichemical approximation in statistical mechanics, taking into account hydrogen bonds between vertical nearest neighbour a units and horizontal nearest neighbour p units. Steric contributions to the interaction energies between units and the complication of imperfect lateral alignment owing to /3 units being twice the length of a units can also be included in the analysis. With reasonable hydrogen bond energies a phase change (a loop in the length-tension curve) is predicted as is common in two and three dimensional nearest neighbour interaction problems in statistical mechanics. This is in agreement with the experimental measurements of H.J. Woods 5 on the length-tension relation in once-dried myosin strips, in which at a certain tension there is observed a sudden large increase in length at practically constant tension. This represents a change, as shown by X-rays,s from vertical a-a crystallization to horizontal P-p crystallization. (b) EXAMPLES AND DIscussIoN.-The principal objective here is to see whether reasonable changes in the state of charge (by adsorption or desorption of ions 1) of an elastic fibre are sufficient to cause considerable changes in length at constant tension, as required for example in the Morales-Botts 7 theory of muscle action.The earlier work of Riseman and Kirkwoods and Kuhn, Katchalsky and co- workers 9 should of course also be mentioned in this connection. The first three models will be considered first since the electrostatic free energy calculation is the same in these cases. For a single symmetrical electrolyte 1 with ions of charge f E and molar concentration c1, where there is a charge E at every X-th statistical unit along the chain. If there are y monomers (e.g. amino acid residues) per statistical unit, then it is easy to see that we also have where 2’ = Zy is the number of monomers in a chain, P’VO = /3vo/y is the volume of a monomer and there is a charge E at every X’ = yX-th monomer. That is, the value of q does not depend on the size of the statisticaz unit. As an illustration of the magnitude of q suppose we take (with a myosin chain in mind) X’ = 9, @‘ = 4.7,Z’ = 300 (mol.wt. N 35,000), vo = 18 ml/mole and c1 = 0.15 M. Then q = 72.9. By varying Z’, c1 and X’, values of q in the range 0-100 or higher are easily obtained. The limits of the Debye-Huckel linear approximation must of course be kept in mind 1 (see also For values of K we turn to the work of Rowen and Simha.10 From adsorption isotherms of water on proteins, near the saturation pressure, they estimate K = 2-3 for silk, 2.1 for wool, 1.8 for serum albumin, 1.6 for collagen and 0.8 for salmine. These values may be compared with those in tables 1 and 2. It will be noted that phase changes are to be expected. If K represented a pure heat effect, positive values of K would correspond to an endothermic heat of mixing water and protein (with highly polar sites already filled).ls 11 Actually, from temperature coefficients, q = z’/4c1vop‘xJ2, (14) 3).TERRELL L .HILL 137 Rowen and Simha show that the heat part of K is exothermic but that there is also an entropy contribution making the net value of K appear endothermic as a " heat " of mixing. Fig. 2 shows several length-tension curves for the " accordion " model 12 with 2 = 459 and K = 0 and 1.59. The top curve is a critical curve (table 2). Charges (q = 10,20) act against the tendency for a phase change to occur. With K = 0, only a small change in length occurs at constant tension for Aq = 20. The effect is larger with K = 1.59 and is especially large just below the critical tension where the length almost doubles.Note the much greater swelling (e.g. at t = 0) in the K = 0 case. It should perhaps be pointed out that since the elastic element can be surrounded by water, the fraction of protein in the element may be quite different than in the muscle fibre as a whole, and also the element can increase in volume I I -6 - v - 4 FIG. 2.-Length-tension curves according to model (3), with q constant. on stretching (taking in water) without changing the volume of the muscle fibre containing it. Fig. 3 shows a series of curves on both sides of the critical curve, for 2 = 400 and K = 2 (q crit. = 296). Near t = 0, Aq = 40 gives an increase in length of 55 %, but at higher tensions a very small Aq can give an extension of a factor of four or more.In principle, this is possible for a single unit 13 with an infinitesimal Aq, but with many units, not all exactly alike, the flat region will become somewhat smoothed out. In fig. 2 and 3, q has been kept constant along a length-tension curve. A more complicated case of greater interest 1 is to consider explicitly the adsorption from solution of an ion with charge opposite to that on the fibre. We then require, for example, a length-tension curve at constant concentration c of the ion in solu- tion. The value of q will change along such a curve. As an example, suppose in fig. 3 that the q = 40 curve is due to fixed charges + E on the fibre at c = 0. Let the ion being adsorbed have a charge - 4~ (e.g. ATP) and suppose there is Langmuir adsorption of these ions on sites just sufficient in number so that with all sites filled (c = a), q = 0 (the net charge n* is used 1 to determine q-see also E138 MUSCLE ACTION 9 3).Let 8 be the fraction of adsorption sites filled. Then in eqn. (10) we put q = 40 (1 - @12, where @(a) is determined from the adsorption isotherm1 The constant 7.695 is fixed by the choices of parameters already mentioned and one additional: we take X(S = 0) = 9.618 (this gives q =-: 40 with /3 = 10 and r ~. I \ I \ I \ \ \ \ \ 72 I \ I \ Z =400, K ‘2, o = 2 FIG. 3.-Length-tension curves according to model (3), with q constant. c1 = 0.1 5 M). The constant f depends 1 on the partition function for adsorption of an ion on a site and on the standard free energy of the ions in solution. In fig. 4 we show curves for fc = 0 (q == 40), 0.07 and CCI (q = 0).Fig. 3 and 4 are FIG. 4.-Length-tension curves (compare fig. 31, with concentration c of adsorbed ion qualitatively similar. In fig. 5, calculated for this same case from eqn. (10) and (15), the length of the fibre at constant tension is plotted against adsorbate ion concentration. At a certain “ critical ” concentration (estimated from fig. 4) there occurs a sudden threefold contraction of the fibre. By working on either side of such a discontinuity (even though smoothed out a little) it is possible for very small changes in, for example, ATP concentration (or pH, Mg2+, Ca2+, salt concentration-since q depends on K, etc.), in the neighbourhood of the ‘‘ critical ” concentration appropriate to a given tension, to bring about sudden constant.Variation in t3 along a length-tension curve is included.139 and very large contractions and extensions of the elastic element. (The elastic element, we recall, must presumably carry along with it considerable extraneous material.) These considerations may give a clue to the rather obvious “razor edge ” nature of muscle contraction.14 Fig. 6 shows the adsorption isotherm accompanying fig. 5. TERRELL L. HILL FIG. 5.--Ciange of length with concentration c of adsorbed ion, at constant tension, corresponding to fig. 4. Fig. 7 contains several other illustrations, all for 2 = 100 and q = 0 or 72.9 (see above). Curves la-d show Aq effects according to the first model (eqn. (1) and (2)). The first pair refer to K = 2 and the second pair to K = 0.With FIG. 6.-Amount of ion adsorbed as a function of ion concentration, at constant tension corresponding to fig. 4. K = 2 there is, for example, a respectable 90 % increase in length at f = 0, while this increase is only 20 % for K = 0. Curves 2a and 2b pertain to the second model (eqn. (5)) with 6* = 1 and K = 2. Curves 3a and 36 have been computed from the third model (eqn. 10)) with K z= 2. Very large charge effects are predicted in these latter two models. The a-13 model, for different reasons, shows in many ways the same general type of behaviour as the second and third models so we give two illustrations only.1 40 MUSCLE ACTION In fig. 8, la and 2a are typical length-tension curves 1 according to the a-/3 model, in the absence of charges.The location Curve l a happens to be a critical curve. FIG. 7. - Length-ten- sion curves with q con- stant. Curves 1 abcd- model (1); curves 2 ab-model (2) ; curves 3 ab-model (3). =0, K.2 ~72.9, K = 2 '0, K + O = 7 P q K = O of t' = 0 depends on the value of J a ~ , which in turn is determined by the relative intrinsic stability of a and /3 units (t'='~(Zp- la)/ CkT, where Is and 1, are lengths of /3 and a units, respectively). Curves 16 and 2b show, as an example, the result of introducing a charge E at every ninth residue, with c1 = 0.15 M and T = 310" K, using the dimensions in Pauling and Corey 6 and eqn. (102) of ref, (1) for the electrostatic term. As before, a re- latively large effect occurs near or in a phase change region (la -+ lb). We may note two differences between the cc-p model (4) and models (2) and (3).First, because of Jap, the phase change region in model(4) can occur at either low or high ten- sions instead of at rather high tensions only (unless a is small ; see tables 1 and 2). This could be an important advantage of this model in a muscle, if phase changes are actu- ally made use 0f.15 In fact, by changing the order of side chains, and hence Jao (through steric effects, etc.), it is conceivable that a group of elastic elements could be available with the proper J a ~ to give the desired sensi- tivity in each range of tension. Second, the maximum change in length in the a-p model is about a factor of two, so that a phase change region would almost be a necessity in 2.0 1"" LL J.0 order to give a change in length approaching FIG.8.-Length-tension ac- this order of magnitude (as a result of a 'Ording to (4), with amount of charge constant. l a and 2a without charge; l b and 26 with change in the state of charge). Although we have emphasized in this = fraction of units in c( paper the interesting possibilities of phase form. changes, it should be pointed out that model (1) appears adequate (recall, for example, the 90 % change in length in l a --f lb,TERRELL L. HILL 141 This agrees with the conclusion of fig. 7) without invoking a phase change. other investigators.7-9 compare, in a single example, several different calculations of the electrostatic term in eqn. (2), (5) and (10) and in similar equations in ref. (1). We restrict the discussion here to macroscopic (dimensions very large compared to 1 / ~ ) and iso- tropic (density of polymer segments uniform) systems-the case of interest in the present problem.A single polyelectrolyte molecule in solution is an example of a non-macroscopic, non-isotropic system for which similar but modified considera- tio ns apply.1 Also, for present comparative purposes, we omit correcting the electrolyte concentration and dielectric constant for the volume fraction of poly- mer,l and simply consider that the charges “ on the polymer ” are distributed uniformly in a macroscopic region (the “ fibre ” or “ gel ”) of the electrolyte solu- tion. These charges and the ions of the electrolyte are treated as point charges. (a) ANALYTICAL.-(i) Donnan equilibrium-linear.-This amounts 1, 16y l 7 simply to expressing the neutrality in the gel, 3.CALCULATION OF ELECTROSTATIC FREE ENERGY.-In this S&iOn We wish to Outside of this region is electrolyte solution only. where i refers to ions of the electrolyte, and the net charge is equivalent to n* charges E in the volume V of gel. On linearizing eqn. (16), solving for $, calculating the work W of charging in the presence of electrolyte and then p’ G - 3 W/3 V, one obtains L15 p’ = = n*zkT/4cl Vz (single 5 E electrolyte), or p = (4q V$/n*2kT)p’ = u2. (17) (eqn. (1 7) defines p ) . without linearizing leads to 1 (ii) Donnan equilibrium-non-linear.-For a single 41 E electrolyte, eqn. (1 6 ) Eqn. (17) and (18) are the results used in the earlier paper 1 and in 5 2. (iii) Fixed charges in a lattice-linear.-If there are n charges E at fixed points in the gel and we linearize the Poisson-Boltzmann equation for this problem, the potential $ at any point inside or outside the gel is (because of linearity and point charges) the sum over c[exp (- KY)]/DI“ for all the n charges, where r is the distance of a given charge from the point. Consider a lattice with Zi i-th neigh- bours a distance ri from a given lattice point.Then one finds where the yi are geometrical factors determined by yi3 = nri3/V. An example with two kinds of charges is included below. There is, incidentally, here a term - E ~ K M / ~ D in W, owing to the potential of the ion atmosphere at each charge, which does not appear in the Donnan method. However, this term is independent of V and does not contribute to p .(iv) Fixed charges smeared uniformly-linear.-Instead of a lattice, suppose the n fixed charges are smeared with uniform density. After integrating c[exp (- ~r)]/Dr over the macroscopic gel to obtain $, one is led again to eqn. (17), as might be expected. The same new term - e2~n/2D occurs again of course in W.142 MUSCLE ACTION (v) Charges part of " electrolyte "--linear.-In the present application charges are attached to polymer chains. One limiting model is to assume the charges are immobile, as in (3) and (4) above. The opposite limiting model, which we consider here, is to assume that the charges behave as species of electro- lyte ions (restricted to the inside of the gel). The correct situation is intermediate and inseparable from the polymer configuration part of the problem.Consider the Poisson- Boltzmann equation for the potential $ in the neighbourhood of a particular j-th charge when all (polymer) charges (the electrolyte ions are kept fully charged) are in the state of charging h (0 < h < 1 ) . Far from the particular charge let $ + $0 and average neutrality obtains (the potential outside the gel is $ = 0) : Suppose there are nj charges mje in the volume V. Putting $' = $ - $0 and linearizing, where v2$' = K A ~ $ ' , It will be noted that KO is a function of V. j-th charge is * n - d d = *o(h) K A ~ f K2 + Ko2h2, The non-self potential at the particular - (mj€hKn/ 0). (25) The work W of charging all the (polymer) charges simultaneously is then and the term in braces = 1 + 1 &(?) 4 + .. . The first term in eqn. (26a) is the same 1 as in (1) and (4) above. The second term may also be compared with (4) (the leading term in the series agrees with (4)). Finally - !(z)z{((:)2 3 + 1)" - 111;- (27a) Note that retaining only the leading term in the series does not give just p = v2.TERRELL L. HILL 143 An analogous discussion can be given using eqn. (20) in nonlinear form (to obtain z,ho) but linearizing eqn. (22). The results are algebraically complicated and will be omitted. (b) EXAMPLES AND mscussIoN.-In fig. 9 we compare p(l/er) calculated in the different ways above, in a special case : a charge E on every ninth residue of myosin (see 9 2), T = 310" K and c1 = 0.15 M. Curve 1 is for eqn. (17), curve 2 for eqn. (18), curve 3 for eqn.(19) and curve 5 for eqn. (27a). In eqn. (19), zi and yi were chosen for a simple cubic lattice.18 The summation was carried through the first four neighbour shells (32 charges) and integration (with a smeared, uniform density) was used for the remaining charges. In eqn. (27a), n = no = n* and ( K O / K ) ~ = n/2clV. It will be seem from the figure that the simple eqn. (17) gives the largest electrostatic termp but that the order of magnitude is the same in all cases. The qualitative results of 3 2 would not be affected by using an alternative electrostatic computation instead of eqn. (1 7). Eqn. (17) is least satisfactory when several types of charges are present. As an example, consider the previous case with the same total number of charges but FIG.9. - p = - (3W/3 V ) x const. plotted against l/v = V x const., according to computation methods (l), (2), (3) and (5) in a special case. Curves 111 and V (- p ) calculated from methods (3) and (5) with net charge = 0 but total charge + 0. la1 numbers of + E and - E. Then 25 rz* = 0 and p' = 0 for the Donnan methods (1) and (2) (also (4)). But p' < 0 for methods (3) and (5) because of attraction between oppositely charged ions.19 In the simple cubic lattice we assume alternate positive and negative ions, sum over the first four shells and integrate over the rest (separate integrations for positive and negative charges are required as different numbers of the two types of charge are included in the first four shells). p must be redefined in both (3) and (5) (since n* = 0) by replacing n* in the defining eqn.(17) by no. Then eqn. (27a) becomes (n* = 0) Curves I11 and V in fig. 9 give - p ( l / v ) . may be negative if n&* is large enough. more negative on neutralizing the charge. Even with a considerable net charge (n* =I= 0), according to eqn. (27a), p' But in this case it will in general become144 MUSCLE ACTION In the a-8 model the charges are smeared (n* thus comes in) on a surface and the non-linear Poisson-Boltzmann equation solved. A two dimensional lattice (see (3) above) could be used here but only with the linear differential equation (i.e. thesum over E [exp (- ~ r ) ] / D r is used again). In conclusion we may indicate the type of calculation of Wused in other work. Hermans and Overbeeck 16 used a linearized Poisson-Boltzmann equation based on eqn.(16) for spheres, not necessarily large. The linear Donnan method (1) was given as a special case (large spheres). Kimball et al.17 suggested that in the Hermans and Overbeeck problem a better approximation than linearizing is to put 021) = 0 in the Poisson-Boltzmann equation, thus obtaining eqn. (16) (with n*/ V a function of r in general) for spheres, not necessarily large. Our use 1 of eqn. (16) (non-linear Donnan) thus amounts to a special case of the method of Kimball et al. Katchalsky et a120 do not indicate in their preliminary short note on gels how the electrostatic calculation was carried out, but one may surmise from an earlier paper by Katchalsky 21 that the electrostatic problem was coupled explicitly into the polymer configuration statistics, by using E [exp (-.KY)]/DP for pairs of charges in a single polymer chain of the network. This type of coupling is of course a desirable feature, but in a gel, consideration of interactions within a singular molecular chain only is not. The writer is indebted to Dr. M. F. Morales, Dr. K. J. Laidler and Dr. S. L. Friess for very helpful suggestions. 1 Hill, J , Clzem. Physics, 1952 (in press). The problem of the size and shape of poly- electrolyte molecules in solution is treated by the same methods in Hill, J . Chem. Physics, 1952 (in press). 2 Alfrey, Mechanical Behaviour of High Polymers (Interscience Publishers, New York, 1948) ; Burte and Halsey, Textile Res. J., 1947, 17, 465. See also, for application to muscle, Butchthal and Kaiser, Dan.Biol. Medd., 1951, 21, no. 7 ; Polissar, Amer. J. Physiol., 1952, 168, 766. 3 In the Flory-Huggins theory of polymer solutions, Huggins’ p = K/2. 4 Flory and Rehner, J. Chem. Physics, 1943, 11, 521. 5 Astbury, Pruc. Roy. SOC. B, 1947, 134,303 ; Astbury and Dickinson, Proc. Roy. SOC. 6 Pauling and Corey, Proc. Nat. Acad. Sci., 1951,37,261,729 ; Nature, 1951,168, 550. 7 Morales and Botts, Arch. Biochem. Biophys., 1952 (in press). 8 Riseman and Kirkwood, J . Amer. Chem. SOC., 1948, 70,2820. 9 Katchalsky, Kunzle and Kuhn, J. Polymer Sci., 1950,5,283 ; Katchalsky, Experientia, 1949, 5, 319 ; Kuhn, Experientia, 1949, 5, 318 ; Kuhn, Hargitay, Katchalsky and Eisenberg, Nature, 1950, 165, 5 14. B, 1940, 129, 307. 10 Rowen and Simha, J . Physic. Chem., 1949, 53, 921. 11 Hill and Rowen, J, Polymer Sci., 1952 (in press). 12 Models (2) and (3) are very similar in properties. Since model (3) seems more realistic, most examples concern it. 13 A single unit must be at one end or the other of the phase change (in an approxi- mate theory of the present type). With many units in a chain, intermediate (average) lengths are possible. 14 One gets the superficial impression that a muscle fibre is a very delicately balanced system ready for a sudden, precipitous change (contraction) as a result of some small alteration in environment. Szent-Gyorgyi uses the term “ razor-edge ” in a similar connection (actin-myosin combination), Science, 1949, 110, 41 1. 15 A small Aq can give a large A1 at low or high tensions on model (4), but only at fairly high tensions on models (2) and (3) (unless a is small). But a large Aq can give a large A1 at low or high tensions on models (2) and (3) (see, for example, fig. 7). Astbury 5 believes, of course, that the #3 --f cc transition (model (4)) itself, though a property of myosin, is not involved when a muscle fibre contracts. Pauling and Corey,6 on the other hand, assume that this transition is involved. 16 Hermans and Overbeeck, Rec. trav. chim., 1948, 67, 761. 17 Kimball, Cutler and Samelson, J . Physic. Chem., 1952, 56, 57,TERRELL L. HILL 145 18 Slater, htroduction to Chemical Physics (McGraw-Hill, New York, 1939), p. 386. 19 The linear Donnan method overestimates p both with charges on the gel and with charges neutralized by opposite charges. The error in A1 will thus tend to cancel. 20 Katchalsky, Lifson and Eisenberg, J . Polymer Sci., 1951, 7, 571. 21 Katchalsky, J. Polymer Sci., 1951, 7, 393.

ISSN:0366-9033    

DOI:10.1039/DF9531300132

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

TERRELL L. HILL 145 ASPECTS OF POLYMERIZATION IN PROTEINS OF THE MUSCLE FIBRIL BY T.-C. TSAO AND K. BAILEY Biochemical Laboratory, Cambridge Received 12th May, 1952 Molecular studies are reported for the three main proteins of the myofibril, tropo- myosin, myosin and actin. The structure of the tropomyosin particle is discussed and aspects of its polymerization compared with that of actin. The much larger myosin particle has been studied largely from the standpoint of the subunits it contains, and the conditions under which they are liberated by fragmentation of the whole molecule. The proteins of the myofibril are being studied in many laboratories from differ- ent aspects, sometimes from the enzymic standpoint, sometimes from the physico- chemical, and all studies are largely directed to an understanding of the nature of the interaction of the contractile proteins with each other and with adenosine triphosphate.It has become quite clear, from the very different approach of Weber’s group in Germany,l and from Marsh’s study,2 carried out with the authors’ group in Cambridge, that ATP serves two distinct functions: its presence is a necessary condition of relaxation phenomena, and its splitting, that of contraction. In Cambridge it has been shown that muscle contains an inhibitor of the latter process, and that the stimulus is somehow connected with the release of this in- hi bition. So far, these are factual, descriptive statements for which no meaning exists on a molecular level, and it seems all-important at present to understand the size, shape and make-up of the components of the myofibril before we can begin to propose theories of the mechanism of contraction.We shall deal with the three proteins which as far as we know make up the whole, or at least the bulk, of the myofibril : tropomyosin, myosin and actin ; and inasmuch as molecular studies in all three cases cannot be divorced from the interesting features of polymerization phenomena, we shall discuss mainly this aspect. TROPOMYOSIN.~-T~~S protein, which exists in skeletal, cardiac and smooth muscle, has several unusual properties. Though soluble in aqueous solvents after isolation, it is not readily shed into the muscle press juice, nor into salt ex- tractants which are capable of withdrawing the myosin component. It resembles actin in that it remains largely with the stroma protein.It is readily obtained if muscle fibre is first treated with ethanol and ether, and then extracted, not with water, but strong salt solution. The work of Hamoir 4 with fish muscle suggests that in sifu it may exist as a ribonucleic acid complex, which would explain the necessity for salt as inducing a process of metathesis. But the treatment with organic solvents suggests also complex-formation with lipid. In many properties, tropomyosin is like myosin ; many of the amino-acids are present in comparable amount, many of the physicochemical properties are similar, and like myosin the intramolecular pattern is of the a-keratin type. Analytically, the absence of tryptophan, and the presence of larger amounts of lysine and146 POLY MERIZATlON I N PROTEINS glutamic acid, certainly distinguish it from myosin, as does the lower particle weight (53,000 against 850,000).The total charged groups represent 45 % of the total residues, of which 27 % of the total residues are anionic. No protein is known with a higher mixed valence than this. Although tropomyosin is more soluble in water than myosin, it nevertheless has globulin properties, which suggest that the charge distribution is not symmetrical. This fact, coupled with the asymmetry of the molecule, probably explains why tropomyosin solutions freed of salt become so very viscous, due simply to the end-to-end aggregation of particles. That the ag- gregation is purely electrostatic is shown by the fact that the addition of salt to salt-free sols causes an immediate drop in viscosity, and this can be very large, from yrel of 50 to one of about 3 at I = 0.1 ( c = 0.8 %).It would be interesting to know the dipole moment of such a molecule. The particle weight of tropomyosin under various conditions 5 is given in fig. 1, i.e. (i> in neutral salt solutions of varying ionic strength, (ii) in neutral urea and (iii) in acid at pH 2. The conditions in (ii) and (iii) are such as to induce FIG. 1 .-Particle weight of tropomyosin under various conditions. depolymerization and were expected to reveal whether the particle which undergoes polymerization in neutral salt solutions is capable of fragmenting into subunits. It can be seen that the average particle weight by osmotic pressure measurements in salt solutions tends towards that in the depolymerizing solvents.That the primary particle, which we shall call the monomer, does not contain subunits, received confirmation in other ways. From the intrinsic viscosity, obtained under varying conditions, it was deduced that the monomer of 53,000 possessed an asymmetry (unhydrated) of about 30. From the X-ray data and chemical analysis, and accepting the Astbury model for the cc-fold, it is possible to calculate the asymmetry of a single, double or triple chain model, which works out at about 78, 26 and 13 respectively. Only the double chain model thus seemed acceptable, and this was all the more favoured when end group assay of the N-terminal amino acids showed none to be present.6 A double chain model involving spiralization as in the Pauling-Corey hypothesis7 gives less good agreement with viscosity data (about 19), but we would not suggest that our data can distinguish between them. The simplest interpretation from all the evidence was a cyclic chain of a configuration, disposed like a deflated bicycle tube hanging from a point on its circumference. The length is 385A approximately and the width 14.5A.T .- C . TSAO AND K . BAILEY 147 The reasons for the polymerizability of tropomyosin are not far to seek. The trend of the viscosity measurements as salt is removed shows (table 1) that up to the trimer stage the process is end-to-end. But if salt-free sols are dried down on a glass plate to facilitate aggregation, and the aggregates washed off on to a supporting film, electromicrographs show very large fibrils, 3000-6000 8, long and 200-300 8, wide.This shows that side-to-side aggregation is also possible, and will, of course. be encouraged by the preliminary end-to-end process. The first stages must involve the aggregation of particles which are essentially bipolar at neutral pH, and such linear aggregates are then capable of aligning side by side. TABLE 1 . -SHAPE OF RABBIT-TROPOMYOSTN PARTICLES IN SOLUTION axial ratio (Simha equation) medium intrinsic viscosity viscosity increment hydrated solvent and ionic strength unhydrated (25 % water) 6.5 6.5 6.5 6.5 6-5 2.1 6.5 12.0 NaCl (0.1) NaCl (0.2) NaCl (0-3) NaCI (0-6) NaCl (1.1) HC1 (0.3) urea (0-3) NaOH (0.3) I -40 1.00 0.70 0-59 0.57 0.523 0.523 0.39 197 53 45 141 44 37 99 35 30 83 32 27 80 31 26 74 30 25 74 30 25 55 25 21 Still unexplained is the fact that after dissolving in concentrated urea and dia- lyzing, solutions of tropomyosin are much more viscous than before, and after aggregation by drying on a glass plate, form tremendously long aggregates (2-6,~) which have a fairly uniform width.As we have seen, urea does not fragment the monomer, but it does induce some change, because although tropomyosin treated with acid at pH 2 or alkali at pH 12 will crystallize isoelectrically at ionic strength 0.4, it will not do so after contact with urea. What significance the aggregation of particles by electrostatic forces holds for questions which relate to the biogenesis of natural fibre molecules is not known.It could be the initial orienting mechanism which is later implemented by stronger secondary valence forces along the length of the aggregates, or even by covalent bonds. MYOSIN.-Turning now to myosin, the best data on the particle weight are those of Weber’s group: 8 s;;O = 7.1 x 10-13, D = 0.87 x 10-7, whence M r= 860,000 i 30,000, a value checked by osmotic pressure measurements.9 Conventional interpretation of the frictional ratio in terms of shape suggests a molecule about 2300 A long and only 23 8, thick. It was observed that solutions of myosin tend to aggregate in a series of steps, that of s = 15 x 10-13 being particularly stable. This aggregation, which Weber calls denaturation must, from the large increase in sedimentation constant, involve an increase in the dia- meter of the myosin particles ; but whether it is a physical process, or due perhaps to the formation of intermolecular disulphide bonds between the many cysteine residues which myosin contains has not been explored. What significance, if any, this type of aggregation has in muscle is not known.For the present, we are more concerned with the make-up of the particle of 850,000 molecular weight. These undoubtedly are built up of smaller units, as was first indicated by Weber and Stover 10 and later by Snellman and Erdos,ll and it is only by following the time course and conditions of fragmentation that we can hope to obtain in- formation on the mode of biogenesis. If myosin is dissolved in concentrated urea, fragmentation (at room temperature) is quite a slow process.When kept for a month and ethanol added in small aliquots, the main fraction (I) precipitates between 20 and 35 % (v/v) ethanol, but thereafter a small fraction (In continues148 POLYMERlZATION I N PROTEINS to precipitate up to 70 % ethanol. After removal of urea, fraction I is a syneres- ing gel, insoluble in water and salt, fraction I1 a water-soluble protein, which when separated from some contamination with fraction I, constitutes 4-5 % of the original myosin. Fraction I1 is not split off from myosin immediately after dis- persing in urea, though the exact time course of its liberation has not been followed in the very early stages. After storage for 6 months, the behaviour towards ethanol has changed : no fraction appears until the ethanol concentration is 65 %, so that the now-modified fraction J and fraction I1 come out together, and the whole of the protein is water- soluble.No further change can be detected between 6 months and 2 years, the threshold concentration of ethanol and the intrinsic viscosity of the system remaining constant. At this steady state, fractions I and 11 can be separated by ammonium sulphate fractionation, and it is then found that fraction I1 con- stitutes 8 % of the total protein. Though by the action of urea fraction I1 is broken off the myosin particle, there is no evidence that fraction I, whether in its water-insoluble or -soluble form, is depolymerized. The particle weight of the soluble form in water at pH 7 determined by the polarization of fluorescence technique of Weber,R 16 is very large indeed, and its sedimentation constant in urea approximately 8.7 x 10-13, though the material is rather polydisperse.However, depolymerization of fraction I readily occurs when the pH is changed to a value between 10 and 1 1 , a range in which the lysine €-amino groups are presumed to have lost their charge. The average particle weight both by osmotic pressure and polarization techniques is then about 170,000 (table 2). On this evidence, the change of solubility of fraction I cannot be due to a depolymerization process. Whatever the initial action of urea, there would seem to follow a slow intramolecular rearrangement of groups, such that the polar side chains are freer to interact with water, or have lost their asymmetry of distribution, or both. The assay of N-terminal groups in native myosin by Sanger’s method 6 does not allow more than a total of about 1 group in 500,000, and this in reality com- prises three different amino acids.It was thought that these were assignable to an impurity, and that myosin, like tropomyosin, might be built up of cyclopeptides. After long treatment in urea, or in alkali at pH 10.7 (table 2) the proportion of N-terminal groups does not increase, showing that fragmentation is not due to the rupture of covalent bonds. The bulk of the N-terminal amino acids are in fact found in fraction 11, and amount to a total of 1 group in 16,000; this value agrees with that obtained by physical methods (table 2). From the physical evidence given above it seems likely that fraction I1 is an intrinsic component of the myosin particle.First of all, electrophoresis and sedimentation show the myosh used to be monodisperse, and the slow liberation of fraction I1 by urea does not sug- gest that it is a loosely held impurity; nor is it due to the presence of actin, which interacts specifically with myosin, because the average particle weight of actin treated with urea under comparable conditions is quite large (about 74,000). The overall picture that we have of the myosin particle is one consisting of 4-5 units, probably cyclic, of average particle weight 170,OOO, associated with 4-5 open polypeptides of fairly low average particle weight (about 15,000). These latter are split off only by the action of concentrated urea leaving the larger units, which we may regard as the framework, still associated.In alkali, however, the framework itself breaks up into its component parts, and if the open chain components have not already been split off by urea, they remain combined with the larger units. It is premature to speculate upon the types of linkage which exist between the various subunits in the myosin molecule, but the depolymer- ization which accompanies the loss of charge of the +amino groups suggests that these latter play a part in holding the framework together. A similar mechanism has been put forward for serum albumin.12fraction and depoly- merizing conditions myosin: in 6.7 M urea, 0.06 M phosphate, pH 6.5, kept for 6-24 months at room temp. myosin FRACTION I1 ethanol-urea frac- tionation after 1-2 months in conc.urea ammonium sulphate fractionation after 6-24 months in conc. urea FRACTION I not separated ethanol-urea frac- tionation after 1-2 months in conc. urea ammonium sul- phate fractionation after 6-24 months in conc. urea T . - C . TSAO AND K . BAILEY TABLE 2.-DIMENSIONS OF MYOSIN FRAGMENTS average particle wt. + axial solvent ratio by no O.P. 1 lP viscosity 6.7 M urea, 0.6 M phosphate, pH 6-5 0.1 M borate, pH 10.7 0.06 M phosphate, 0.2 M KCI, pH 6.5 water, pH 7 6.7 M urea, 0.06 M phosphate, pH 6.5 6-7 M urea, 0.1 M phosphate, 0.1 M KCI, 0.05 M thio- glycollic acid, pH 7.0 water, pH 7 0.1 M borate, 0.1 M KCI, pH 10.7 170,000 - 14,000 - - 17,000 122,000 - - > 300,000 165,000 - - 175,000 149 -30 1 terminal amino- group/900,000 g protein -40 1 terminal amino- group/500,000 g protein -10 1 terminal amino- group/ 16,000 g protein - * ~ ~ ~ = 8 .7 x 10-13 (C = 0.4 %) -40 still including 4 % of fraction I1 -30 - f O.P. by osmotic pressure measurements. * kindly measured by Dr. A. G. Ogston. 1 / p by fluorescence polarization measurements. AcTm-The mechanism of the polymerization of G-actin to F-actin has been the subject of numerous speculations 13 both from the chemical and physico- chemical standpoints. The characteristic properties of F-actin are the high, anomalous viscosity, flow birefringence and high sedimentation rate, which have all been interpreted as due to the linear aggregation of globular units into long filaments. An apparent confirmation of this mechanism has been afforded by the fibrils seen under the electron microscope.14~ 15 A more detailed study of the polymerization by Weber’s fluorescence-polarization method 16 seems to suggest that such large units do not exist in solution, and alternative explanations may need to be sought for the properties given above.Whatever the final interpretation, we are mainly concerned at present with the dimensions of the primary unit, and of its participation in a monomer-dimer transformation. The actin used was prepared by a new method, and unlike the Straub pre- paration, was electrophoretically homogeneous. Various preparations were subjected to osmotic pressure measurements in 0.6 M KI, which is sometimes used as a depolymerizing agent in protein chemistry.17 The results were found150 POLYMERIZATION I N PROTEINS to be somewhat erratic, indicating in some experiments the presence of a 74,000 unit, at other times one of 140,000, and in very dilute solution the transition of the latter to the former with progressive dilution.An analysis of the condi- tions employed in these experiments has not entirely explained the results, but did suggest that the presence of ATP encouraged the formation of the monomeric state. This was confirmed by the application of Weber's polarization-fluorescence technique. If typical F-actin preparations are depolymerized in simple buffers at pH 2-2 or 10 or in neutral 0.6 M KI, or if G-actin 18 with or without the addition of traces of Mg ion are examined, the polarization of fluorescence indicates a rota- tional relaxation time p corresponding to the dimer.G-actin in neutral urea or in buffer (without urea) at pH 11, or at pH 8 in presence of ATP, possesses relaxation times intermediate between that of dimer and monomer. The lowest relaxation time is obtained in presence of a chelating agent (versenate) at temper- atures above 20" C ; preliminary measurements of the intrinsic viscosity suggest an axial ratio greater than 10, and this order of asymmetry combined with the value of p = 13.8 x 10-8 (at 25" C) gives a particle weight of 70,000, corresponding to the monomeric form indicated by the osmotic pressure measurements. Below 20" C in presence of versenate, the monomeric form passes over to the dimeric, and a similar change of state with temperature occurs in presence of ADP or hexametaphosphate.The above results suggest rather strongly that the units of the dimeric form of actin are held together by a divalent metal, linked possibly through the nucleo- tide prosthetic group. ATP, ADP, pyrophosphate and polyphosphate all com- pete for the metal and cause dissociation, versenate, an excellent chelating agent, being most efficient. The dissociation which is effected at high pH is probably due to electrostatic repulsion. DISCUSSION The polymerization-depolymerization phenomena brought about under such diverse conditions in these three proteins of the myofibril are interesting to compare. The features which are most important in such comparison are the reversibility of the process, the types of primary unit involved, and their structural features.In the case of tropomyosin, the polymerization is freely reversible and electrostatic in nature, and the units participating are possibly cyclic in structure and possess no subunits. Myosin, on the other hand, appears to con- tain two types of subunit, one possibly cyclic and the other an open chain, each requiring different conditions for their liberation ; in contrast with tropomyosin, this process is slow and irreversible. It is not easy to prove that the open chain components of the myosin particle are an intrinsic part of the molecule, and in reality it is difficult to decide the meaning of purity with large molecules which possess a specific physiological function. It must be remembered that myosin possesses an enzyme character, and as an enzyme also possesses a uniquely high molecular weight.Whether the enzyme property resides in the " framework " or in the small complement of open chains is a very relevant question, and might influence our views on what constitutes the myosin particle as a physiological unit. Quite independently of such considerations, the forces which hold the framework together, and the open chains to the framework, seem to be of different character. Whereas salt effects the disaggregation of tropomyosin, with actin it elicits all the properties which ar rather typical of polymeric molecules-high, anomalous viscosity and flow birefringence. Though we have cause to question the accepted structure of F-actin, it seems fairly certain that it is formed in two separate stages ; 19 first, the primary unit or monomer, itself asymmetric, passes to a dimeric state in which divalent metal ions are important.These ions may act as a bridge throughT . - C . TSAO AND K . BAILEY 151 the ADP or ATP prosthetic group which have been shown always to occur in actin preparations. It does not seem probable that they act by modifyingthe charge on the protein through adsorption : mercury in organic combination with one valency free at once diminishes the viscosity of F-actin and prevents the formation of the latter from G-actin. (We do not consider that mercury acts by combination with SH groups, or that SH groups are important in the polymer- ization of actin.20) The formation of F-actin, whatever its nature, involves only the interaction of the dimeric form.The formation of the dimer, o r of the monomer from the dimer, is a relatively slow process under the conditions so far studied, and in time scale is comparable to the formation and depolymerization of the mercury-linked dimer of serum albumin ; 21 it differs very notably from the rapid transformations observed in tropomyosin. We are very much indebted to Mr. G. S. Adair, F.R.S., and Dr. G. Weber for help with methods and for valuable discussions. The research has been financed by grants from Prof. W. T. Astbury, F.R.S., and from Imperial Chemical Industries, to whom we are greatly indebted. 1 Weber and Portzehl, Adv. Protein. Chem., 1952, 7, 161. 2 Marsh, Biochim. Biophys. Acta (in press). 3 Bailey, Biochem. J., 1948, 43, 271. 4Hamoir, Biochem. J., 1951, 50, 140. 5 Tsao, Bailey and Adair, Biochem. J., 1951, 49, 27. 6 Bailey, Biochem. J., 1951, 49, 23. 7 Pauling and Corey, Proc. Nut. Acad. Sci., 1951, 37, 235. 8 Portzehl, Schramm and Weber, 2. Nuturforsch., 1950, 5b, 61. 9 Portzehl, 2. Nuturforsch., 1950, 5b, 75. 10 Weber and Stover, Biochem. Z., 1933, 259,269. 11 Snellman and Erdos, Biochim. Biophys. Acta, 1948, 2, 650. 12 Weber, Biochem. J., 1952, 51, 155. 13 summarized in ref. (1). 14 Jakus and Hall, J. Biol. Chem., 1947, 167, 705. 15 Rozsa, Szent-Gyorgyi and Wyckoff, Biochim. Biophys. Acta, 1949, 3, 561. 16 Weber, Biochem. J., 1952, 51, 145. 17 Szent-Gyorgyi, J. Biol. Chem., 1951, 192, 361. 18 This does not imply an identity with the actin which is obtained by direct extraction of acetone-treated muscle fibre; it is obtained by dialysis of polymerized actin against 0.002 M NaHC03. 19 Feuer, MolnAr, Pettko and Straub, Hung. Physiol. Actu, 1948, 1, 150. 20 Turba and Kuschinsky, Biochim. Biophys. Actu, 1952, 8, 76. 21 Hughes, J. Amer. Chem. Soc., 1947, 69, 1836.

ISSN:0366-9033    

DOI:10.1039/DF9531300145

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

T . - C . TSAO AND K . BAILEY 151 ELECTROCHEMICAL PROPERTIES OF LUPIN SEED PROTEIN BY MISS E. M. PETRI AND A. J. STAVERMAN Plastics Research Institute, T.N.O., Delft Received 28th April, 1952 After a short survey of the literature on protein from the lupin seed (lupinus luteus) the isolation and chemical analysis of this protein are described briefly. The preparation and chemical modification of membranes from the protein are described, a method of examining the electrochemical properties of such membranes is given, together with the results obtained with this method on various membranes of this protein. It is found152 LUPIN SEED PROTEIN that the examination of membranes is a very suitable method, not only to determine the electrochemical behaviour of a given protein but also to understand the exact nature of chemical modifications such as are used in practical applications of proteins.1. INTRODUCTION.-AS lupin is used in the Netherlands on a fairly wide scale in the fallowing of uncultivated soil and in the improvement of soils which lack nitrogen, the seed of lupin may be considered as an easily accessible raw material from Dutch soil. In view of the paucity of data in the literature on the properties of the protein obtainable from this seed, we thought it worth while to embark on a rather extensive research programme concerning this protein. This paper con- tains the first results of our work in this field. The literature does not give many results of a systematic character. After Von Ritthausen 1 had proposed the name of conglutine for the protein from lupin seed, Osborne2 drew attention to the specificity of this protein.He separated the protein into two components, a- and P-conglutine with differcnt solubilities in solutions of ammonium sulphate, and published the results of an elementary analysis of both. Amino acid compositions of the total protein from the yellow lupin (lupinus luteus) were given by Abderhalden and Herrick,3 Onslow,4 and Heiiirich.5 Their results are incomplete and are in disagreement. Thomson 6 discovered the conditions for gelation of alkaline lupin seed globulin solutions, and Husfeld and Zang 7 patented a spinning procedure for the protein. Recently Russian sources 8 have stressed the possible value of the protein, while some data are also published on proteins from the blue and the white lupin (lupinus augusto- folius 9 and lupines albus 10).2. IsoLATroN.--From the nitrogen content of 6.3 % we concluded that the seed of the sweet yellow lupin (lupinus luteus) contains about 40 % protein. We isolated this protein according to the method of Osborne; 24 the seed was milled, freed from oil, extracted with NaCl solution and freed from salt by dialysis. We obtained yields of 30-35 % (calculated from the nitrogen content) of an apparently pure protein (16.5 % N), soluble in salt solutions. The constant chemical com- position and the linear plots of the solubility confirm the high purity. When high purity was not required we isolated in 85-92 % yield a fairly pure product (N = 15 %), soluble in alkaline solutions, by means of precipitation at pH 4.3, from solutions prepared by extraction of the seed at pH 7. By dissolving the protein in 0.1 saturated solution of ammonium sulphate and increasing the concentration of this salt consecutively to 0.55, 0.65 and 1.0 satura- tion, we obtained, in complete accord with Osborne,2b two crystalline proteins, presumably the ammonium salts of a- and 6-conglutine with nitrogen contents of 17.42 % and 18-36 %.Owing to denaturation the yields were small (respectively 8 % a-conglutine and 9 % P-conglutine expressed as ;< starting material). 3. CHEMICAL coMPosmoN.-Our investigations on the chemical composition of the lupin seed protein will be published in detail elsewhere. In this paper we confine ourselves to a survey of the results of some investigations which are of interest in an understanding of the physicochemical properties of the protein.Elementary chemicaE composition.*-The analysis yielded the following results : C = 48-2; H = 7.2; S = 1 . 1 ; N -= 16.5; 0 = 25.1 ; ash = 1.7. Amino-acid composition, qualitative.-By paper chromatography H. Peters of (i) rather large quantities of glutaminic acid, argenine and leucines, (ii) fair quantities of aspartic acid, lysine, proline, glycine, alanine, valine, (iii) small quantities of histidine and tyrosine, perhaps a trace of threonine. * We are indebted to Dr. H. W. van Deinum of the Netherlands State Mines Laboratory our laboratory found the following amino-acids : phenyl alanine, serine and cystine, or performing this analysis.MISS E .M. PETRI AND A . J . STAVERMAN 153 Table 1 summarizes our own and earlier results of quantitative amino acid determinations. Microbiological determinations * are indicated by m.b.11 TABLE 1 content in E - 100 g nations 4 alanine argenine aspartic acid cysteine cystine glutamic acid glycine histidine isoleucine leucine lysine methionine phenylalanine proline serine threonine tryptophan tyrosine valine 3I 12.6 10.8 0.0 4.1 26.3 2.1 6.1 9.1 4.2 0.0 3.9 4.1 5.2 I 2.4 0.0 2.9 3.7 - - = 5 % m.b. * 10 % m.b. - no SH-groups - S content - m.b. - 2.5 % m.b. 5-10 % m.b. 5-10 % m.b. 5 % m.b. i 5 % m.b. 5 % m.b. uncorrected oxidation 12 - - - m. b., chromatography for destruction 5 % m.b. - m.b., chromatography 5-10 % m.b. 5 % m.b. - 2.5 2.5 1.9 8 10.9 10.9 12.6 5 3-0 3.0 5.4 - - - - 3 - - 4-7 6 1-1 1.1 1.3 4. ELECTROCHEMICAL PROPERTIES OF MEMBRANES.-It occurred to US that in many of their applications proteins are in the insoluble state and very often chemi- cally modified by formaldehyde, chromium salts and the like, whereas conventional methods of physicochemical characterization usually deal with the unmodified protein in the soluble state.We found it desirable, therefore, to extend our programme of physicochemical techniques using one method particularly applicable to the insoluble protein. For that purpose we found a very useful method in the investigation of the electrochemical behaviour of membranes. It is well known 33 that the permeability of a membrane for positive or negative ions largely depends upon the degree of dissociation of the ionic groups in the membrane material.As proteins possess both acid and basic groups their permeability may possibly be changed within wide limits by changing the pH of the medium. Jn view of the large content of both acid and basic amino acids reported in 0 3 for the lupin seed protein, this protein could be expected to be suitable for testing the method. Both expectations have been confirmed by experiment. PREPARATION OF MEMmANEs.-In the preparation of membranes from conglutine we were guided by the gel-concentration plots of Thomson 6 which we completely confirmed. The problem in preparing fibres or membranes from a soluble natural protein is to treat the protein chemically in such a way that the molecule unfolds without strong degradation of the long chain molecule.If one treats the protein in different concentrations with different amounts of caustic soda during a specified time at a specified temperature a region is found in the protein-NaOH-concen- tration plot where gels are formed. In this region the conditions for unfolding *All microbiological determinations were performed by Dr. A. C. v. Linden of Netherlands Nutrition Institute at Amsterdam. We are much indebted to Dr. v. Linden for his work.154 LUPIN SEED PROTEIN without too strong degradation are fulfilled. For the preparation of both fibres and membranes the borderline of this region represents the most suitable conditions in solutions. Working along these lines we found that a solution of 22 % unpurified con- glutine in 1.5 % caustic soda and 0.5 % sodium sulphile after a reaction time of 20 h at 20" C was a good medium for casting membranes in a coagulation bath, containing 5 % sulphuric acid and 10 % formaldehyde.ELECTROCHEMICAL EXAMINATION.*-There are various ways of characterizing the permeability properties of a membrane.14 One may pass a known volume of a solution through the membrane and measure the contribution of each of the components of the solution to the flow (mechanical transport numbers). One may also pass a known number of coulombs through the membrane and measure the contribution of the components to this electric current (electric transport numbers). Several other phenomena generally designated as electrokinetic phenomena may also be used to characterize the permeability relations of a membrane.As we were mainly interested in the function of the polar groups, we decided to characterize our membranes first by measuring the dialysis potential, which is m : membrane 12 mV mcfcr e : ca/omc/ elecl'rodei FIG. l.--Cell for the measurement of dialysis potentials of membranes. the potential measured between two KCl solutions of 0.1 and 0.01 N. As is well known, by this method essentially the Hittorf transport numbers of the ions were measured. A cell was constructed as shown in fig. 1. The membrane rn separates the two solutions of 0.1 and 0.01 N KCI. Nitrogen is bubbled through the cell in order to ensure homogeneity of the two phases and to avoid as much as possible the polarization of the liquid in the immediate neighbourhood of the membrane and to avoid the ingress of C02.By means of two identical calomel electrodes with satured KC1 salt-bridges e the potential difference between the two half cells was measured. By filling the cell with buffered of various pH values we obtained graphs of the dialysis potential against pH.t In strongly acid solution the membrane will assume a positive potential with respect to the penetrating liquid and it will transmit negative ions far more easily * The authors are indebted to Dr. Bergsma of this Institute for advice and help in ?Prof. Overbeek (Utrecht) drew our attention to the fact that a more rigorous In the measurements. interpretation of experiments with electrodes without liquid junctions is possible. later measurements we adopted this procedure.MISS E.M. PETRI AND A . J . STAVERMAN 155 than positive ions. Therefore, chlorine ions pass through the membrane from the concentrated to the dilute KCl solution until a potential difference between the two solutions has been set up which retards the passage of chlorine ions and ac- celerates that of the potassium ions until a stationary state is set up. This potential difference makes the concentrated solution positive with respect to the dilute solu- tion and we assign a positive sign to this potential difference. It is clear that in a medium of very high pH in which all basic groups are uncharged and all acid groups dissociated the membrane will assume a negative charge and the potential difference between the cells will have a negative sign. Two phenomena appeared to us to be of interest in these measurements : (i) the absolute magnitude of the potential difference obtainable at the extreme (ii) the pH value at which the potential difference vanishes.(i) From thermodynamics the potential difference between 0.1 and 0.01 N KCl at room temperature cannot exceed 52mV, this value being the theoretical value for complete impermeability to all ions of one and the same sign. Therefore, the proportion of the absolute magnitude of the experimental potential difference to 52 mV gives a good indication of the extent to which the membrane is imper- meable to ions of one sign. (ii) At the pH value where the dialysis potential is zero, potassium and chlorine ions pass through the membrane at equal rates. We propose the designation of “ iso-electric point ” for this pH value and we consider it as the merit of this method that it enables us to define and measure the iso-electric point of completely in- soluble, and eventually strongly modified, proteins.We must note, however, that the theoretical definition of the iso-electric point is the pH at which the protein molecule has no net charge. Every experimental method aiming at measuring this pH is subject to certain errors, characteristic of the method, and a complete understanding of the method requires a careful quantitative estimate of these errors. So our method of determining the pH at which K+ and C1- ions pass the membrane at equal rates may be expected to give a good first approximation,ls as it is based upon the well-known equality of the velocity of these ions in water.However, in a second approximation, we should take account of the selective action of the membrane due to its charge and that due to its structure, from which a small differ- ence between measured and theoretical might originate. The influence of the buffer on the experimental potentials should also be considered, although we have found that the buffer affects the diffusion potential of the KCI solution only slightly (1 mV). We have avoided the weakness of the method of Baxter 16 who did not perform the electrical measurement on the buffered solu- tion in which his protein was conditioned. We do not think that Baxter’s pessimistic views on his method apply to ours. We used a 0.02 M veronal- acetate buffer. As we shall see, the change of this dialysis iso-electric point upon chemical modification of the membrane is so marked that this quantity can be considered as a useful characteristic quantity to describe membranes.CHEMICAL MoDmCATIoNS.-We first investigated how the mechanical and the electrochemical behaviour of the membrane was affected by treatments with (a) formaldehyde, (b) trimethylene bromide, (c) chromium salts, ( d ) thorium salts. The purpose of (a) and (b) was to achieve cross-linking and at the same time to increase the basicity by changing primary into secondary amino groups. The metal ions, chromium and thorium, were used with a view to blocking the carboxylic groups and to prevent their dissociation. pH values ;156 LUPIN SEED PROTEIN RESULTS * The results are given by fig.2, 3, 4 and 5 . The symbols used in the figures have the following meaning : S, coagulated in sulphuric acid ; SF, coagulated in sulphuric acid + 10 % formaldehyde ; - , after-treatment with : xCvy x% solution of chromium alum for y h (room temperature) ; xC,,70, x % solution of chromium alum for y h at 70" C ; xFu, x% solution of formaldehyde for y h (room temperature) ; xTh,, x% solution of thorium nitrate for y h (room temperature) ; xTm,, x% solution of trimethylene bromide for y h (room temperature) ; 9 a comma is used to separate consecutive treatments. FIG, 2.-Dialysis potentials against pH of different membranes. FIG. 3.-Dialysis potentials against pH of different membranes. From fig. 2 it is seen that treatment with formaldehyde or 1 % chromium alum scarcely affects the electrochemical behaviour of the membrane.The iso-electric points of all these membranes were around the pH 5 and the selectivity corresponded to 40 mV * In collaboration with Mr. R. W. van Hoesen Korndorffer.MISS E. M . PETRI AND A. J . STAVERMAN 157 for positive membranes and to 32 mV for negative ones. From fig. 3 we see, however, that intensification of the treatment with chromium alum, either by increasing the concentration to 5 % (curve 4), or the temperature to 70 "C (curve 5), or by addition of 1 % thorium nitrate (curve 6), resulted in a marked displacement of the iso-electric point to values up to 11. The reproducibility of the curves is fairly good with a few exceptions as is shown by the two curves 6. From curve 7 we note that an after-treatment with formaldehyde in watzr not only reduced appreciably the pH of the iso-electric point but also decreased the absolute magnitude of the selectivity.This can be understood if we assume that the chromium ions discharge the carboxylic groups whereas formaldehyde destroys the basis activity of the amino groups, so that curve 7 is concerned with a nearly neutral membrane. In fig. 4, which deals with membranes treated with formaldehyde on precipitation in the acid bath, we observe that formaldehyde does not destroy the basicity of the amino groups provided it reacts with the protein in acid solution. From a comparison of curve 3 of fig. 4 with curve 5 of fig. 3 one might even conclude that increased basicisty was brought about by the formaldehyde treatment although we must take note of the rather poor reproducibility at high pH.FIG. 4.-Dialysis potentials against pH of different membranes. However, from the results shown in fig. 3 and 4 one may conclude that the reaction Presumably between formaldehyde and a protein takes different courses at different pH's. at low pH a reactor of type (1) proceeds RNH2 + CH2O -+ RNHCH2OH (14 followed by RNHCH20H + RNH2 -+ RNHCH2NHR (1b) leading to a slight increase of basicity of the amino group and at the same time to a marked increase of mechanical strength due to the cross linking reaction ( l b ) . On the other hand in pH - 7, a reaction of type (2) occurs : RNH2 + CH20 -+ R-N=CH;! by which the basicity of the amino groups is destroyed and the mechanical strength is decreased.Fig. 5 shows that the effect of trimethylene bromide is not markedly different from that of formaldehyde. However, it may be that differences will become apparent after treatment with chromium in order to screen off the carboxylic ions. Indeed, if there is any difference between the action of formal and trimethylene bromide it would be expected to appear in the basicity of the basic groups, but such difference can appear only at high pH where all carboxylic groups are dissociated provided they are not blocked by metal ions. (2)158 LUPIN SEED PROTEIN In conclusion we note that- (i) the examination of membranes provides a useful tool for the understanding of (ii) chromium reacts with the carboxylic groups ; (iii) formaldehyde reacts with the amino groups, according to different mechanisms, such as (1) and (2), depending on the pH at which the reaction proceeds ; (iv) lupin seed protein is a useful protein for investigations of this kind owing to its high content of both acid and basic groups ; (v) from the fact that the pure protein shows an iso-electric point of about pH 5 and that rather high concentrations of chromium are needed to shift the point markedly, it follows that the number of carboxylic groups surpasses that of the basic groups in the pure protein.This is also borne out by the amino acid analysis. the electric state of an insoluble, pure or modified, protein ; 40 2. \ \ 4 e l 5 02 SF 0 3 5 -2Tm, ~4 SF-ZTm, PH 8 -20 -3 0 -4 0 -2 - - - _ _ _ FIG. 5.-Dialysis potentials against pH of different membranes.EXPERIMENTAL UNPURIFIED LUPIN SEED PROTEIN.-TO extract the oil, 500 g ground lupin seed was extracted for 5 days with petrol ether. After grinding the residue in a ball mill with 1 1. water for 20 h, the paste was extracted by stirring with 11.5 1. water for 3 h. To maintain the pH at 7, 4 80 ml 2 % NaOH was added successively. After centrifuging, the solution was brought to pH 4.3 by passing in S02. The precipitated protein was centrifuged, washed quickly, successively with acetone and ether, filtered by suction and partly dried by passing air through the suction funnel. Afterwards the protein was spread on filter paper and completely dried in the air. The extraction after grinding in the ball mill and the following procedures must be carried out in one day.The grinding in the ball mill has to be started 20 h before the extraction. Yield 180-200 g protein, N = 4 15 %. LUPIN PROTEIN MEMBRANES.-A solution of 22 % lupin seed protein (N = 15 %), 1.5 % NaOH and 0.5 % Na2S03 was prepared by stirring the protein with some water to get a suspension and then adding to this suspension a NaOH solution of such con- centration that the concentration mentioned above was finally reached. After a reaction time of 20 h at 20" C the viscous solution was spread in a thin layer on a glass plate. The plate was placed for a few minutes in 6 % sulphuric acid containing 10 % formaldehyde, at room temperature. After drying for some hours in the air the membrane was soaked in water and then removed from the plate.The membrane was washed with distilled water until no SO4 ions could be detected in the washing water. The membranes (SF) must be kept in a moist atmosphere. MoD1FICATIONS.-Membrane(s) prepared without formaldehyde in the coagulating bath.-If no formal was added to the coagulating bath, a Nylon tissue was used forMISS E. M . PETRI AND A . J . STAVERMAN 159 srrengthening the membrane. This tissue was spread with the viscous solution on the glass plate before coagulation. Treatment of the membranes with chromium alum.-The following solutions were used : code 1 c6 1 % chromium alum and 0.1 % NaHC03 for 6 h at room temp. 1 % 9 , ,, 0.1 % Y Y 20 Y, 39 1 G o 5 % ,, Y Y 0.4 % Y Y 6 3, Y 9 5 c6 1 % 9 , Y , 0.1 % 9 , 6 9 , 70" C 1 C670 5 % ,, ,Y 0.4 % Y, 1 9 , 70" C 5 C170 Treatment with Th(N03)4.--1 % Th(N03)4 weakened the unmodified membrane in such a way that they could not be used. The membranes (Th 6) modified with chromium alum could withstand the treatment with this solution for 6 h. with 2 % trimethylene bromide and 1 % pyridine in acetone solution for 6 h. After treatment with 10 % formal for 6 h membrane (10 F 6) was obtained. TREATMENT WITH TRIMETHYLENE-BROMIDE.-The membranes (2 Tm 6) were treated 1 Ritthausen, J . prakt. Chem., 1881, 25,422. 2 Osborne and Campbell, (a) J. Amer. Chem. SOC., 1896, 18, 609 ; (b) J. Amer. Chem. Osborne and Harris, (c) Amer. J . Physiol., 1905, 13, 436; SOC., 1897, 19, 454. (d) J. Amer. Chem. SOC., 1903, 25, 323. 3 Abderhalden and Herrick, 2. physiol. Chem., 1905, 45,479. 4 Onslow, Tlle Principles of Plant Biochemistry, vol. 1 (Cambridge, 1931). 5 Heinrich, Bodenkunde Pfanzenernahrung, 1941, 23, 9 1. 6 Thomson, J. Sac. Dyers Col. (Symp.) 1946, 173. 7 Husfeld and Zang, D.R.P., 1942, 722, 266. 8 Feditor, Sernenswodstro, 1946, 7-8, 40. 9 Dunn, Camien, Shankman and BIock, Arch. Biochem., 1948, 18, 195. 10 Nehring and Schwerdtfeger, Arch. Tierernuhrung, 1950, 1, 295. 11 Henderson, J. Biol. Chem., 1948, 172, 15. 12 Shinn and Nicolett, J. Biol. Chem., 1941, 138, 91 ; 139, 687. 13 Meyer, Sievers and Teorell, Helv. chim. Acta, 1936, 19, 649, 655. 14 Staverman, Trans. Faraday SOC., 1952, 48, 176. 15 SoIIner, J. Physic. Chem., 1945, 49, 47, 171, 265. 16 Baxter, J. Colloid Sci., 1947, 2, 495.

ISSN:0366-9033    

DOI:10.1039/DF9531300151

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

MISS E. M . PETRI AND A . J . STAVERMAN 159 GENERAL DISCUSSION Dr. G. A. Gilbert (Birmingham University) said: Any attempt to measure the relative concentrations of loose complexes or association products by sedimenta- tion experiments raises the question of the effect of sedimentation on the equilibria under study. This problem must have been discussed many times, but may well be mentioned again. The problem does not arise, of course, if association and dissociation occur extremely slowly, since the different molecular species then sediment almost independently. In the present instance, however, Dr. Johnson and Naismith have shown by light-scattering experiments that equilibrium is reached in their system in less than a minute, and that accordingly it corresponds more closely to the case where equilibrium is instantaneous.When this is so, simple considerations show that the concentration changes across the sedimenta- tion boundaries need no longer correspond directly to the concentration of any of the species present, nor the velocity of the boundaries to the velocity of the species. Further, the number of boundaries present is equal to the number of elementary species (except in the trivial case of these having the same sedimentation constant) and is not increased by the presence of complexes in mobile equilibrium with these.160 GENERAL DISCUSSION Thus in the simplest case of a substance A of molar concentration a in reversible equilibrium with its dimeric form D of concentration d, one boundary and not two will be found during sedimentation, because when dimer sediments ahead of monomer the system is thrown out of equilibrium and monomer associates to form more dimer.It might be thought that this would cause a broadening of the boundary, but in fact if equilibrium is instantaneous this is not so and the boundary remains sharp, apart from the effect of diffusion. Merely the velocity of the boundary is affected. This is most easily seen by considering an initially sharp boundary between solution and solvent and giving the cell a velocity equal and opposite to the velocity of sedimentation of the dimer molecules. Monomer then streams past stationary dimer into pure solvent, and as it passes the boundary it associates and builds up the dimer concentration until the latter is in equilibrium with the monomer coming from the undisturbed solution behind the boundary.Looked at in this way the system is exactly analogous to an ideal chromatogram in which solute is moving past stationary adsorbed molecules, and, similarly, a sharp boundary or front is to be expected, again neglecting diffusion. To fmd the velocity of the joint boundary it is perhaps best to bring the boundary to rest with an imposed velocity on the cell of - X , and to take account of the fact that the transport of A to the boundary in the form of monomer is equal to the transport of A away from the boundary in the form of dimer. Then, if the sedimentation velocities of monomer and dimer are respectively sA and sD, a(X - sA) = 2d(sD - x) giving the velocity of the joint boundary as Dilution reduces the ratio d/a and so slows down the boundary.Similarly, any change in temperature or solvent which affects the equilibrium constant of associ- ation changes the velocity of the boundary. If two substances A and B are present in equilibrium with complex C in accordance with the equation A + B + C, two boundaries and not three should be found if equilibrium is instantaneous. The slower boundary is due to one of the elementary components, the faster boundary to the complex and the other component together with a concentration change of the first component. Let this faster, joint, boundary be brought to rest by a velocity - Ximposed on the cell. Then since the velocity sc of the molecules of complex may be supposed greater than the velocities sA and sB of the components, a pseudo-steady state is achieved at this boundary with complex streaming forward from the boundary at a rate proportional to c(sc - X ) and A and B streaming back to it at rates proportional to a ( X - sA) and b ( X - sB) respectively. According to whether a(X - sA) is greater than or less than b(X - sB), A or B passes through the joint boundary in part and forms the slower boundary. The slower boundary is thus not necessarily formed by the slower of the two components. For argument let A reach the joint boundary in excess and so form the slow boundary.Then for no accumulation of B, which is arriving at the joint boundary in the free form and leaving as complex, Xz-= (asA + 2dsD)/(a + 2d). b(X - sB) = S C ( ~ - X ) giving for the velocity of the joint boundary X = (CSc + bSB)/(C + b).The concentration a’ of A in the region between the slower boundary and the faster boundary follows from the equation a(x - SA) = a’(X - sA) + c(sc - x) obtained by considering the transport of A to and from the joint boundary.GENERAL DISCUSSION 161 Changes in conditions which alter the equilibrium constant of association, or dilution which affects the relative values of a, b and c will change X and also the relative areas of the two peaks in the sedimentation diagram. This simple example suggests that a smooth shift in sedimentation velocity with change of conditions is as important a sign of complex formation as a change in relative areas. Dr. P. Johnson (Cambridge University) said: As Dr. Gilbert suggests, dis- sociation processes may cause considerable complication of sedimentation patterns and in the work reported a detailed analysis would necessarily involve equilibrium considerations.However, no such analysis has yet been attempted, the absence of diffusion data alone making this impossible. Light scattering measurements show clearly the occurrence of reversible association and dissociation processes and the effect of changes in ionic strength and pH on the weight-average molecular weight. Where the equilibrium is dis- placed far to the dissociated (e.g. I = 0.5, pH - 7.6) or associated form (e.g. I =-- 0.1, pH w 6) sedimentation must occur almost normally and sedimentation constants will refer accurately to the particular molecular species occurring. Thus the end products of the reaction are accurately characterized by sedimenta- tion.However, under conditions where two or more components occur together in equilibrium, the disappearance of a component over a boundary, common to normal sedimentation, may be prevented and observed sedimentation constants may differ from ideal values for the stable component. These effects may, in some circumstances, be expected to be small at the most rapid boundary where the component of largest sedimentation constant is sedi- menting in the presence of all the slower components. In certain conditions ( I = 0.1, pH = 9-4) it has been observed that excessive boundary spreading and very poor resolution occurs and rapid equilibria have been suggested. In others, however, the sedimentation constants were not far from those expected for association products of the completely dissociated form, and the agreement of weight average molecular weight from sedimentation patterns (using peak areas) with light scattering values suggests that the effects of the equilibria have not obscured the main features of the reaction chain in the sedi- mentation diagrams.This, is somewhat surprising in view of the rapidity of the establishment of equilibrium as indicated by light scattering, but in this connection it should be remembered that protein concentration is much less important than ionic strength in affecting the equilibrium, and this may simplify the situation. The present system is by no means exceptional in this latter property, for which no satisfactory explanation is available.Dr. L. Nanninga (Leiden) said: In connection with Schulman and Harrap’s paper, by adding the reagent after mixing fibrinogen and thrombin it was con- cluded that the inhibitor affects fibrinogen, In the time before clotting, however, the inhibitor can react with thrombin, even in the presence of fibrinogen. The inhibitor should be mixed with thrombin and the mixture checked on fibrinogen after different times. It is stated that inorganic anions seem to be bound to fibrinogen. Several authors have, however, demonstrated that anions (for instance, ferricyanide) affect thrombin. This is a “ progressive ” inhibition (dependent on time of in- cu ba t ion). Dr. K. H. Gustavson (Stockholm, Sweden) said: The determination of the dialysis potential of membranes of proteins at different pH for localizationof the isoelectric point of insoluble proteins, and particularly of modified proteins, certainly is a welcome addition to the few methods available for such determin- ation.Since I have for years been studying the reactions of chromium salts with insoluble proteins, principally collagen of skin, including the effect of various chromium salts on the electrochemical state of collagen, the data on the lupin protein in combination with chromium salts are especially interesting.162 GENERAL DISCUSSION The displacement of the isoelectric point of collagen towards higher pH values (from pH 5 to pH 7-8) by means of irreversibly fixed cationic basic chromium salts (chlorides and sulphates) has been proved by three difierent methods: (i) the fixation of cationic and anionic dyestuffs by the chromed protein, (ii) the electro- phoresis of suspensions of chromed hide powder in a microcell and (iii) by the electro-kinetic method of Neale.1 These results as well as the ones presented by Petri and Staverman, prove a reaction of cationic chromium complexes with the carboxyl ions, probably leading to a crosslinking of adjacent protein chains by means of the polynuclear complexes, the carboxyl groups being attached directly to the chromium atoms of the complex.The choice of buffers is very important in this determination. In buflcrs containing anions of high complexing potency, as for instance, acetate, the complexing agent will react with the fixed sulphato- chromium complexes. The penetration of the acetate groups into the chromium complexes may result in the formation of non-ionic and anionic complexes which in turn will affect the binding between chromium and collagen, the cationic protein groups being inactivated.Thus, in determination of the isoelectric point of collagen tanned by means of cationic sulphato-chromium complexes, this point corresponds to the pH range 7-8, in systems not buffered or in solutions with buffers containing anions of low complexing power, such as borate. However, with acetate buffers, in concentrations of 0.03-0.05 N the isoelectric point determina- tion will give pH values about 4. It is of interest to note that the use of acetate and phosphate buffers in examining chromed collageii for determination of its isoelectric point is the reason for some peculiar results reported by some American workers recently.They find that small amounts of cationic chromium incorporated with collagen displace the isoelectric point towards the acid side, whereas larger amounts of chromium combined with collagen tend towards a slight shift of the isoelectric point towards higher pH values. After tanning skin proteins by means of anionic oxalato-diol-chromiates, the isoelectric point is found to be in the range 3.5-4.0. In this case the pH corresponding to the isoelectric state of the chromed proteins is not affected by the type of buffer used. The reason is that the oxalato group is an exceedingly powerful complexing agent. Hence the acetate is not able to penetrate into the chromium complex. It is possible that the results reported in the paper under discussion, i.e.that small amounts of fixed chromium do not affect the isoelectric point of collagen, is due to the penetration of acetate into the complex, reversing its charge which should be expected to move the isoelectric point towards the acid side. If the two opposing reactions, the shifting towards higher pH values caused by inactivation of the anionic groups (carboxyl) by the original cationic chromium complexes against the shift towards lower pH values due to co-ordination of acido-groups in the chrome complexes and the participa- tion of cationic protein groups in the secondary binding, are of the same order of magnitude, no change in the isoelectric point would be expected.With larger amounts of chromium combined with the protein, the former influence will dominate as evident by the shift of the isoelectric point of collagen towards higher pH values. It should be very interesting to hear from Dr. Staverman, if he has observed similar effects of the buffers on the electrochemical function of the mem- branes of his chromed protein. As to the action of formaldehyde (F) on the lupin protein, the reaction mechan- isms given in the paper, in a i l attempt to explain the different properties imparted to the protein by its combination with F in acid solution on the one hand and at neutral reaction on the other hand, appear too schematic. The mechanism of F fixation by proteins apparently differs with the nature of the protein. Thus, for collagen, crosslinking is indicated to be most strongly developed in the pH range 7-8.The F fixation at low pH values requires exceedingly high F concentrations 1 Gustavson, Adv. Protein Chem., 1949,5,391 ; J. Amer. Leather Chem. ASSOC., 1952, 47, 425.GENERAL DISCUSSION 163 and extended time of interaction and still the stabilizing effect is slight. The role of the amide group in the fixation of F by vegetable proteins would be expected to be prominent in acidic solution, as the work of Fraenkel-Conrat and others have shown, No information of the content of amide groups (ammonia) is given for the lupin protein. With collagen, it is evident that the F fixation in acid solution involves a slow fixation of F by the €-amino groups of lysine, whereas the F fixed in the other extreme of tanning, at pH values greater than 11 (probably mainly by means of the guanidyl group) does not contribute to the stabilization of the protein lattice by crosslinking. Finally, it is interesting to find that the isoelectric point of the lupin protein after inactivation of part of the carboxyl ions by chromium and of part of the amino groups by F is at pH 7.The data reported on the lupin protein as to the effect of various agents on its electrochemical behaviour show the same trend as the corresponding findings from investigations of collagen. Dr. A. J. Staverman (Delft) said : We think that Dr. Gustavson's remark about possible complex formation between chromium and the acetate buffer is a very valuable one. So far our later findings completely confirm Dr.Gustavson's remarks. We realize that there are many possibilities to explain our results with formaldehyde and that the reaction schemes given in our paper are just two of them, which we considered most probable, The reason why we did not include a reaction with amide groups is that our membranes have been treated with 1.5 % caustic soda for 20 h at room temperature, so we supposed that amide groups should be absent. Recent experiments, however, showed this assumption to be erroneous. Dr. M. G. ter Horst (Leeuwarden, Netherlands) said: In connection with the paper of Petri and Staverman it might be useful to mention the results of some experiments we did a number of years ago. We investigated the reaction of for- maldehyde with proteins over a wide pH range, from slightly alkaline treatments of pH 9 to treatments in very strong acid media, up to 20 % H2SO4.We selected several proteins for our investigation, but most of our work was done on casein. We have been using several methods, of which we want to mention here especially the estimation of the quantity of formaldehyde bound to the casein under standard conditions of temperature and concentration. We found that this quantity of formaldehyde was steadily decreasing in the range of pH 9-1. Actually there was a minimum for treatments in strong acid media, somewhere between 5 and 10 % of sulphuric acid. As far as concerns the reaction on the alkahe side of this minimum, we have carried out several potentiometric titrations, and we were able to show that a considerable part of the bound formaldehyde had reacted with the " free amino groups ".Now in all cases where the measurements are based upon the charge of the molecule, the expression " free amino groups ''I does not give a proper idea of the situation. It has been shown by Harris that the apparent pK's of the amino groups are shifted 2-3 units by the reaction with formaldehyde. Thus the apparent pK of the +amino group of lysine is moved from 10.5 to 8. Arginine has an apparent pK greater than 13 and the corresponding formaldehyde will have a pK greater than 11. That means that the difference between arginine and its formaldehyde complex has a maximum at pH greater than 12. The difference does not show at pH values below 10. We never found any differences due to histi- dine ('pK 6) or to end-groups of the polypeptide chains.So the only amino group that can be traced by the methods mentioned above is the €-amino group of lysine. But even then the difference between lysine and its formaldehyde complex is largest at pH 9-9.5, and if we want to see if formaldehyde has reacted with lysine, we must look for it at pH 9.5. This reaction has nu eflect upon the iscelectric point of acid proteins such as casein or lupin seed protein. We treated casein as mildly as possible with nitrous acid, thus changing the €-amino group into an OH. The difference between the quantity of formaldehyde164 GENERAL DISCUSSION bound by this deaminated casein and that bound by the untreated sample equals the loss of lysine at a ratio of 1 lysine to 1 formaldehyde.If, however, the reacting medium contains 10 % of sulphuric acid or more, both samples bind the same quantity of formaldehyde. Hence we came to the conclusion that in strong acid solution the €-amino group of lysine does not react with formaldehyde. I should like to point out that the same is probably true for lupin seed protein, for this conclusion is in excellent agreement with all the experiments of Petri and Staverman. Dr. P. Johnson (Cambridge University) said : From the electrophoretic diagrams presented in Dr. Hamoir’s paper, 1 see no evidence which excludes the possibility of the reversible combination of actin (A) and myosin (M) to give acto-myosin (AM) : A$- M +AM. Does Dr. Hamoir have other evidence on this point? In view of irreversible effects occurring at very strong salt concentrations, experiments based upon the separation of components under such conditions are of doubtful validity.The evidence, already published,l upon which a reversible reaction of the above type was suggested seems difficult to explain otherwise. In preparative ultra-centrifugation, resuspended actomyosin showed unexpectedly high relative proportions of slow sedimenting components, whilst the supernatant fluids con- tained a considerable proportion of rapidly sedimenting actomyosin. The latter observation refutes the suggestion of actomyosin degradation by the conditions of the experiment. Further, since at higher salt concentrations than 0.6 M KC1, partial dissociation of actomyosin is clearly indicated by sedimentation patterns (and has been suggested by other workers on other grounds), it seems entirely reasonable and in line with other observations to suggest that a partial though lower degree of dissociation obtains at 0.6 M KCl, and lower salt con- cent rat ions.Dr. G. Hamoir (Universite‘ de Li&) said: Johnson and Landolt have suggested that actomyosin is a reversibly dissociating complex (AM f A + M) which should be completely dissociated in 2 M KCl and the equilibrium would be well towards AM at 0.6 M KCl.1 Evidence for the occurrence of such an equilibrium has also been given recently by Laki, Spicer and Carroll,2 with actomyosin prepared artificially from actin and myosin and dissolved in 0.6 M KCl. Such a dissoci- ation, however, has never been observed at the ionic strength of 0.35 or 0.4 of the electrophoretic experiments ; fish and rabbit actomyosins, isolated from the extract, behave homogeneously in the U-tube. Preparations of fish actomysoin investig- ated by ultracentrifugation also give single peak diagrams at ionic strength up to 0.5 and pH 7.Therefore, the equilibrium suggested by the previous authors does not seem to exist in preparations isolated directly from the extract at ionic strengths up to 0.5 and at neutral pH. Dr. A. Wassermann (University College, London) said : In collaboration with Mrs. M. L. R. Harkness, potassium actomyosinate gels were prepared and con- verted, by various ion exchange techniques, into soluble actomyosin of pH 4.0-42. which was characterized by analysis, precipitation tests, electrometric and con- ductometric titrations and by electrophoretic measurements, after reconversion into an actomyosinate.In the absence of salts this reconversion is accompanied by an increase of (i) the influence of rate of shear on the viscosity; (ii) intrinsic viscosity at zero rate of shear ; (iii) flow birefringence. The turbidity under various pH conditions and the influence of salts and excess potassium hydroxide were also studied. All the effects accompanying the conversion of the water- 1 Johnson and Landolt, Faraday Soc. Discussions, 1951. 11, 179. 2 Laki, Spicer and Carroll, Nature, 1952, 169, 328.GENERAL DISCUSSION 165 soluble actoniyosin of pH 4.0-4-2 into the water-soluble myosinate are strongly influenced by small quantities of salt.The results of the various experiments can be interpreted from the point of view of molecular shape changes, the struc- tural muscle protein being regarded as flexible chain-like polyelectrolytes. A different model according to which the actomyosin is taken to be a rigid structure, undergoing a reversible polymerization, is not suitable for an explanation of the essential results. Dr. A. G. Ogston (Oxford Universify) said: Morales and Botts have suggested that the effects of ATP on the viscosity and sedimentation of actomyosin can be interpreted as due to changes of volume and shape rather than to dissociation. There may be some difficulty, however, in reconciling the reduction of viscosity, which would be caused by decreased volume or decreased asymmetry, with the reduction of sedimentation rate, which would be brought about by increases of volume or asymmetry. Some of Dr.Hamoir's electrophoresis diagrams, especially fig. 1, 5 and 10 bear a striking resemblance to those obtained with P-lactoglobulin at pH 4.6. In this case, the electrophoresis results (unpublished) obtained by Mr. J. M. A. Tilley and the sedimentation measurements at 3" to 5" (kindly performed for us by Dr. Creeth) are consistent with formation of a dimer which sediments and migrates more rapidly than monomer. This accounts for the incompletely re- solved descending boundaries and the hyper-sharp front in the ascending limb of the U-tube. There is the interesting difference between the two systems, that actin, in either form, seems to migrate more rapidly than myosin or actomyosin. One would, therefore, expect that actin should migrate ahead of the hyper-sharp ascending boundary if dissociation occurs in this region.In the migration of an equilibrium mixture, three cases may be distinguished, where the equilibrium is established at a rate (i) rapid, (ii) slow, (iii) comparable with the rate of resolution of the boundaries. In the case (i) a single boundary is obtained migrating at a velocity intermediate between those characteristic of the components, but showing an abnormal degree of spreading: this case has been elegantly illustrated by Philpot and Philpot 1 for casein. In case (ii) normal resolution is obtained. Case (iii) is more complex ; an incomplete resolution is obtained. The boundary which is advancing through unchanged solution behaves normally but that which is in a region of changed concentration will be distorted, showing skewing, with a tail in the direction of unchanged solution.Dr. G. Hawoir (Universite' de Li2ge) said : Dr. Ogston has noted that asym- metries described in my paper can also be observed with lactoglobulin prepara- tions and that their ultracentrifugal study at different temperatures shows that this asymmetry is due to an association product stable only at the low temperature of the electrophoretic experiments. The origin of the asymmetry of fig. 8 appears more complicated. Since this paper was written, new experiments have shown that we are not, as we thought at first, dealing here with a true polymerization phenomenon and that some difference in chemical composition must occur be- tween the two peaks of fig.8. The transformation of the preparation of fig. 8 into the one of fig. 9 obtained by precipitation at pH 4.6 and ionic strength 1 results in a lowering of viscosity which is definitely smaller than the one which could be expected in a depolymerization phenomenon. The ultracentrifugation of several preparations corresponding to fig. 8 reveals the presence of a small peak sedimenting at the rate of Bailey's tropomyosin (fast ascending peak of fig. 8) and usually of one much larger sedimenting much more quickly (slow ascending peak of fig. 8). Precipitation of these preparations at pH 4.6 and ionic strength 1 produces a notable increase of the slow sedimenting peak at the expense of the fast one.But here as in the electrophoretic experiments the transformation is A detailed account of this work will be published. 1 Philpot and Philpot, Proc. Roy. SOC. B, 1939, 846, 21.166 GENERAL DISCUSSION incomplete. Furthermore the comparison of the ultra-violet absorption curve of our preparations corresponding to fig. 8 with the same curve determined by Dr. T.-C. Tsao on pure tropomyosin (unpublished) reveals the presence of cofitaminants increasing the absorption at 250-260mp. The slower component of fig. 8 which sediments more quickly than tropomyosin in the ultracentrifuge corresponds to an association of tropomyosin with a non-nucleic impurity which seems to be partially released at I = 1 and pH 4.6. These results show that we are not dealing here, as we thought at first, with a polymerization phenomenon different from the purely electrostatic one described by Tsao and Bailey, but more likely with the occurrence of a conjugated protein of higher sedimentation constant.Work is in progress with a view to elucidating these differences in chemical composition. The two components interacting in fig. 8 are therefore not of identical chemical composition as appears to be the case in the lactoglobulin preparations described by Dr. Ogston. Fig. 8 corresponds to a case intermediate between the one described by Dr. Ogston and the one of fig. 10 in which much bigger differences in mobility are observed corresponding to very different solubilities. Prof. W. T. Astbury (Leeds University) said: The feature that strikes one most in Morales and Botts’s theory of muscle action is the way it takes practically no account of actin, and I feel that the authors are bound in honour to tell us why.Surely, at this stage of muscle knowledge, such a prominent and much-investigated constituent cannot be dismissed so lightly. X-ray and electron microscope studies have now shown that, in dried muscle at least, the macro-period (about 410& which runs continuously along the contractile fibrils is a consequence of myosin chains in parallel with a regular array of actin units ; and in view of only this one finding among many others pointing in a similar direction, it scarcely seems adequate to argue almost as if the actin were there “ just to make it harder ”, as the saying goes.And another point I might make concerns the tensile properties of undried “ myosin ” threads described by the authors. Both these threads and the oriented strips studied at Leeds can afford only indirect evidence on what happens when living muscle contracts under stiniulution, for though it is true that living unstimu- lated muscle, like these undried “myosin ” threads, seems to gain entropy on shortening, present indications are that stimulated muscle is dominated rather by internal energy than by entropy considerations. Dr. J. T. Edsall (Harvurd University) said: I should like to emphasize the crucial importance of the net electric charge on the myosin in the theory of muscular contraction outlined by Morales and Botts. Since ATP is negatively charged, the electrostatic interaction of bound ATP with myosin should lead to contraction if the protein fibre is positively charged, and to extension if the fibre is negatively charged. The isolectric points of myosin and of actomyosin 1 are near pH 5-4; hence, in the absence of binding of other ions, myosin must be negatively charged at pH near 7.However, binding of other ions, and especially of divalent cations, certainly does occur and can shift the isoelectric point to aboke 9,2 although so large a shift occurs only at Ca2+ or Mg2f concentrations which are considerably above the physiological range. Weber and Portzehl 1 observed strong contractions of actomyosin threads in the presence of ATP ; small amounts of magnesium were essential, but the concentrations employed were very low.It seems doubtful whether the net charge on the actomyosin thread would be positive or negative under the conditions of the experiments; if the electrostatic mechanism proposed by Morales and Botts is correct, moderate alterations of net charge on the fibre, produced by varying the pH or the calcium or magnesium ion content of the medium, should have a profound effect on the contractile mechanism. 1 see, for instance, Weber and Portzehl, Adv. in Protein Chem., 1952, 7, 161, especially 2 Erdos and Snellman, Biochirn. Biopliys. Acta, 1948, 2, 642. table VI on p. 197.GENERAL DISCUSSION 167 Since the work of Weber and Portzehl may not be familiar to some who are attending this Discussion, I think it should be pointed out that they favour the view that ATP brings about contraction by utilizing the large amount of free energy released on its hydrolysis. In other words, they consider that the main- tenance of contraction involves a steady state in which fresh supplies of free energy must be constantly fed into the contractile system through the splitting of ATP.They do not attempt to offer any interpretation concerning the mechanism involved. On the other hand, the view advocated by Morales and Botts postulates that the contraction is due to the binding, not to the splitting, of ATP. It is my impression that the experimental evidence is still insufficient to decide between these two points of view. Weber and Portzehl have produced some strong circumstantial evidence in favour of their belief, but I should agree with what I take to be the view of Dr.Morales that neither point of view has yet been rigorously proven by experiment. It is also perhaps worth emphasizing that any theory of contraction must be able to explain the important experimental facts discovered by A. V. Hill and his collaborators during the last few years. Notably it must explain his evidence for the apparently passive nature of the relaxation process.1 It is hardly fair to ask Dr. Morales at this stage to come up with an explanation of all this. Perhaps he can do so, but I think it is only fair, in judging the proposed theory, to wait until there has been more time for the development of theory and the discovery of new experimental facts. Finally I should like to pay a brief tribute to the work of the late Prof.K. H. Meyer in formulating a picture of the electrostatic mechanism of muscular con- traction. In 1929 he stated very clearly2 the conception that muscular con- traction and relaxation might depend on the balance between spontaneous coiling of polypeptide chains, due to an entropy effect on the one hand, and electrostatic forces due to ionization of the protein side-chains on the other. The muscle was pictured as being maximally contracted at the isoelectric point of the protein, and extended by electrostatic repulsion when the protein acquired a net charge. At that time, the only known process in muscle, which could lead to an alteration of net charge, was the production of lactic acid from glycogen. Meyer ac- cordingly attempted to explain muscular relaxation by acidification due to lactic acid. Weber 3 criticized this proposal on the ground that no physiologically possible pH change could supply enough energy to account for the observed effects. Meyer4 offered a few comments in reply and the whole conception was apparently forgotten by almost everyone for many years thereafter.Naturally there was no need for Dr. Morales and Botts to refer to this early work of Meyer in their communication here-they have already made due reference to it in some of their other papers-but particularly because of the recent un- timely death of Prof. Meyer, I think that it is worth while to recall his highly original contribution to this subject. Whether electrostatic mechanisms furnish the correct explanation for muscular contraction or not, Dr.Meyer displayed in this as in other investigations a really pioneering spirit. Dr. M. Morales (U.S. Nav. l e d . Rcs. Inst., Bethesda) said : We suggest else- where 6 that one source of the apparent conflict between deductions from light scattering and deductions from kinetic methods may be the wide difference in concentration at which the two approaches have hitherto been applied. At the comparatively high concentrations used in the kinetic methods, both viscosity and sedimentation constant may show important contributions from inter-particle interactions. We were aware that in calling “myosin” what many current authors call 1 Will, Proc. Roy. Soc. B, 1949, 136, 211, 420. 2 Meyer, Biocliem. Z., 1929, 214,253 3 Weber, Biuckem. -24, 1930, 217, 430.5 Blum and Morales, Arch. Biochem. Biuphys. (in press). 4 Meyer, Bioclzem. Z., 193G, 217, 433.I68 GENERAL DISCUSSION " natural actomyosin ", we were confusing further an already confused terminology. Our original reason for so doing was our feeling that the essential known facts about the interaction of the material with ATP could be interpreted assuming that the material was a single entity, with no appeal to its copolymeric nature.1 This by no means implies a denial of the existence of " actin " and its many interesting properties, once isolated. The recent analyses of Kominz and Eaki 2 have encouraged us to think that we adopted a fortunate vocabulary, since their work suggests that the current " pure myosin " of most authors is a 1 : 1 co-polymer of actin and tropomyosin, thus questioning the physiological significance of the material (" reconstituted actomyosin ") obtained by adding more actin to this co-polymer .Prof. Astbury is quite right in remarking that both his pioneer work and our subsequent work on thermoelastic properties of myosin films, threads, etc., pretends to give information about the " resting " state only ; however, in formulating a complete theory, this is very essential information none the less.3 In the absence of an " irreversible thermodynamics " treatment, we are quite circumspect about such attempts as have been made or could be made to study the thermoelastic properties of myosin-ATP systems. In our formulation the internal energy (for instance, the electrostatic con- tribution) of the system plays a very crucial role; it is the fault of our writing if we have succeeded in concealing this fact.Due to the excellent work of such laboratories as Likge and Upsala, much is already known about the net electrical state of myosin in rather concentrated (0.6 M) salt solutions. Before precisely formulating or testing any " electro- static " hypothesis, however, it will be necessary to extend such work to dilute solutions of KCI and of divalent cations, and it is well to bear in mind that the net charge need not be uniformly smeared. The charge on ATP as a function of pH has been very expertly measured by Alberty et a/.,4 but here again it is necessary to extend these measurements to solutions containing ions which interact with ATP. Several laboratories, including our own, are concurrently working to fill these various gaps. Our own suggestions (our paper and ref.(3)) that electro- static processes are involved rest on the evidence mentioned in this paper and on such circumstances as the necessity of explaining how ATP can, at one salt concentration, cause contraction, and at another, cause extension. Another new indication that electrostatic processes are involved comes from the interesting experiments of Ethier and Laidler,s who measured ATPase activities as a function of dielectric constant E, and found a strong linear dependence of the free energy of binding and of the free energy of activation upon I/€. Elsewhere 3 we have discussed the relation of OUI formulation to those of other authors. So far as we know, Wohlisch and Meyer were the first to propose, respectively, the " entropic " and " electrostatic " mechanisms for muscle action. Much more recently, these general concepts have been modernized and combined by Bull and Riseman-Kirkwood, as well as by the Katchalskys and Kuhn. The latter authors have also provided ingenious demonstrations with models. Our own efforts have been directed at inquiring whether the actual system, myosin t salts + ATP, behaves in accordance with these general ideas. Dr. G. Hamoir (Universite' de Li2ge) said: In connection with the remarks made by Tsao and Bailey about the extractibility of tropomyosin, we should like to mention some unpublished results. Tropomyosin can easily be prepared from fish muscle, without previous dehydration with organic solvents, by extraction of the minced muscle with phosphate solutions of ionic strength 1 and pH 4.8 (pH 1 Blum and Morales, Arch. Biochenz. Bioplzys. (in press). 2 Kominz, Laki and Hough, to be published. 3 Morales and Botts, Arch. Biochem. Biophys., 1952, 37, 283. 4 Alberty, Smith and Bock, J. Bid. Chern., 1951, 193,425. 5 Ethier and Laidler, to be published.GENERAL DISCUSSION 169 of ex these only .tract, 5.2). conditions go slightly The yield, so far as we can judge it, seems to be good. With of ionic strength and pH, however, fish myosin and fish actomyosin into solution.1 Rabbit muscles treated in the same way behave similarly but the yield in tropomyosin is much lower. It seems therefore that the linkages occurring between tropomyosin and other components of the myofibril can vary widely. If tropomyosin seems to behave usually as a stroma protein, i.e. a protein of very low extractibility, it can also behave as the most extractible component of the myofibril. 1 Dyer, French and Snow, J . Fish. Res. Bd. Can., 1950, 7, 585. 'F

ISSN:0366-9033    

DOI:10.1039/DF9531300159

出版商:RSC    

年代:1953

数据来源: RSC