24 CHYMOTRYPSIN AND CHYMOTRYPSINOGEN CERTAIN PHYSICAL PROPERTIES OF CHYMOTRYPSIN AND CHYMOTRYPSINOGEN USING THE DEPOLARIZA- TION OF FLUORESCENCE TECHNIQUE BY V. MASSEY,*$ W. F. HARRINGTON t AND B. S. HARTLEY * Departments of Biochemistry * and Colloid Science,? University of Cambridge Received 31st May, 1955 The work reported here originated in an attempt to determine whether any changes in physical state accompanying the conversion of chymotrypsinogen to chymotrypsin would help in our understanding of the catalytic properties of this enzyme. The fluorescence polarization method of Weber 1 was used in this study because of its sensitivity in dealing with low-molecular-weight proteins. In fact few physical differences between chymotrypsinogen and chymotrypsin were found. The chief difference found in physical properties between the two proteins is a hitherto unreported polymerization reaction of chymotrypsin which does not occur with the precursor under the experimental conditions studied.However, the most important conclusion from the work, which must necessarily remain tentative until values of fluorescent lifetime are determined, is that a group is free in chymotrypshogen which is identical with a part of the active centre of chymotrypsin. * Imperial Chemical Industries Research Fellows, Dept. of Biochemistry, Cambridge University. -f Fellow of the National Foundation for Infantile Paralysis, U.S.A. (1953-4). $ Present address : Edsel B. Forn, Institute, Detroit 2, Michigan, U.S.A.V. MASSEY, W. F. HARRINGTON A N D B .S . HARTLEY 25 EXPERIMENTAL CHYMoTRYPs1NoC;EN.-The chymotrypsinogen used was alcohol-crystallized material obtained from Worthington Biochemicals, New Jersey, U.S.A. CC-CHYMOTRYPSIN was prepared from chymotrypsinogen by activation with trypsin according to the method of Kunitz and Northrop.2 The enzyme was crystallized four times from ammonium sulphate solution at pH 4. DIP-CHYMOTRYPSIN was prepared from a-chymotrypsin by reaction with di-isopropyl fluorophosphonate (DFP), and crystallized four times, according to the method of Jansen et aZ.3 the method of Weber.1 PREPARATION OF FLUORESCENT coNJuGAm.-Protein samples were dialyzed against cold 0.1 M Na2HP04 solution. One-tenth volume of a cold acetone solution of the dye (1-dimethylaminonapthhalene-5-sulphonyl chloride) was added, and the reaction allowed to proceed in the cold, generally for a period of 18 h.The labelled protein was then dialyzed in the cold with changes of the buffer required for the particular experi- ment until the dialysate was free from fluorescence. jugated per molecule of protein was calculated by the following method. The protein concentration was determined by its 280 mp absorption before reaction, corrected for the volume change during dialysis. The conjugated dye concentration was calculated from the extinction coefficient of the dye on these proteins.4 This extinction coefficient has been determined as 3-3 x 106 cmz/mole and is independent of the degree of labelling. l-DIMETHYLAMINONAPHTHALENE-5-SULPHONYL CHLORIDE Was prepared according to CALCULATION OF THE DEGREE OF LABELLING.-The number Of mOleCUkS Of dye Con- CALCULATION OF RESULTS FROM FLUORESCENCE-POLARIZATION MEASUREMENTS be calculated from the equation 1 From the variation of fluorescence polarization with temperature the ratio ph/70 may where 7 0 is the lifetime of excited state of the fluorescence, ph is the mean harmonic rotational relaxation time at the temperature T, B is a term characteristic of the conjugate and determined by the variation of T is the absolute temperature r ) is the viscosity of the solvent.polarization with temperature, If TO is known then ph may be determined. Weber1 has found with conjugates of both serum albumin and ovalbumin that his results are consistent with a value of TO of 1.4 x 10-8 sec. However, our results suggest that with chymotrypsin conjugates the value of 70 can vary with the degree of labelling.Hence all the results are reported in the form ph. 20/70, where ph. 20 is the value of ph corrected to 20" C. A NOTE ON THE INSTABILITY OF CHYMOTRYPSIN-DYE CONJUGATES Most of the work reported here was carried out under pH conditions which favour the autolysis of chymotrypsin. Fortunately the enzyme is inhibited by conjugation. This inhibition is proportional to the degree of labelling and is due to the reaction of the dye with the active centre of chymotrypsin.4 However, at degrees of labelling below 1 molecule dye/molecule protein there is still appreciable proteolytic activity. Hence in measurements involving lightly-labelled chymotrypsin the temperature range over which reliable information can be obtained is restricted to 0"-25".At higher degrees of labelling where the inhibition is virtually complete, higher temperatures can be tolerated. However, as a matter of routine all fluorescence polarization readings were restricted to 0-25" as it has been found with these proteins that the dye is hydrolyzed from the protein at an appreciable rate at pH 7-9 when the temperature exceeds 30". This instability is markedly different from that found with other protein conjugates,l andis presumably due to an unstable conjugate of the dye with part of the active centre. Hence, to avoid spurious fluorescence polarization readings the following conditions have to be observed with the labelled proteins used in this study : (i) The conjugate used should be freshly prepared and the fluorescence polarization readings made as soon as possible.26 CHYMOTRYPSIN AND CHYMOTRYPSINOGEN (ii) The conditions used must avoid proteolysis. (iii) The conditions used must avoid hydrolysis of the dye from the protein. As an experimental check that these conditions were fulfilled the lowest temperature reading of polarization was repeated at the end of the experiment after the highest tem- perature reading.Any liberation of low-molecular weight fluorescent compounds due to proteolysis or dye hydrolysis is then easily detected by a lowered polarization reading. RESULTS VARIATION OF ph. 20/70 WITH PROTEIN CONCENTRATION Fig. 1 shows the effect on P~.~O/TO of varying the concentration of 0.50 labelled (i.e. 0.5 mole dyelmole protein) or-chymotrypsin in 0.01 M phosphate buffer pH 7-9.The value of ph.20/70 extrapolated to zero protein concentration is 5. Thus over the range of I 5 10 Protein concentration (rnq/ml) FIG. 1.-The variation of ph.20/70 with protein concentration in 0.01 M phosphate pH 7.9. x 0.50 labelled a-chymotrypsin ; 0 1-56 labelled chymotrypsinogen. protein concentration 0-10 mg/ml there- is a fourfold increase in ph. 20/70. Assuming that TO does not change with protein concentra- tion this would indicate a considerable in- crease in average molecular volume over this concentration range. pH-Dependent dimerization of chymotrypsin has been ob- served by Schwert and Kaufman.5 Although the highest pH used by these authors was pH 6.2, the trend of dimerization with pH which they observed indicates that at pH 7.9 considerable dimerization would not be ac- hieved until higher protein concentrations than those used here.Even if dimerization were occurring under these conditions it would hardly account for such large varia- tions in ph as indicated here. Sedimentation studies in the Spinco ultracentrifuge confirmed that these results were in fact due to considerable polymeri- zation. Fig. 2 shows the Schlieren diagrams of a-chymotrypsin in 0.01 M phosphate buffer, pH 7.9 at several different protein concentrations. It will be noticed that the polymer separates partially from a slower- moving component, although it never sep- arates completely into two symmetrical peaks. The leading edge of the slower component and the trailing edge of the faster component tend to merge together, indicating the interconversion of the two forms during the course of sedimentation. Hence the rate of attainment of equilibrium between the two forms must be rather slow.However, equilibrium must be attained within an hour, since no differences in sedimentation constants or proportions of polymer are found when a concentrated solution of protein is diluted and centrifuged immediately, and when it is allowed to stand for 24 h before centrifuging. It will be noted from fig. 2 that the proportions of the two components vary with protein concentration, so that at low concentration there is only a small proportion of polymer, increasing as the protein concentration increases. The proportion of polymer is not influenced by conjugation with the fluorescent dye, as is shown in fig.3 where the proportion of polymer (obtained by measuring the Schlieren peak areas) is plotted against protein concentration. Fig. 4 shows the effect of protein concentration on the sedimentation constants of the two components. It will be observed that the apparent average size of the polymer0 b C d - - FIG. 2.-Schlieren patterns of a-chymotrypsin in 0.01 M phosphate pH 7.9. (a) concentration 3.5 (mg/ml) ; (c) concentration 10-4 (mg/ml). (b) Y Y 5.2 YY (4 Y Y 20.5 7 9 [To face 26V . MASSEY, W. F . HARRINGTON A N D B. S . HARTLEY 27 as judged by its sedimentation constant, is dependent on protein concentration up to a concentration of 12 mg/ml. At higher protein concentrations the sedimentation constant decreases with increasing protein concentration, suggesting that in this range the polymer is a single molecular species which exhibits the usual sedimentation-concentration de- pendence.The slower component has an extrapolated sedimentation constant of 2.9 S Pro t in con cen t rat ion ( mq/m I ) FIG. 3.-The variation of the proportion of polymer with protein concentration in 0.01 M phosphate pH 7.9. A 2.6 labelled a-chymotrypsin. X native or-chymotrypsin ; 0 0.60 labelled or-chymotrypsin ; Protein concentqation ( m q / m l ) FIG. 4.-The sedimentation constants of chymotrypsin in 0.01 M phosphate pH 7.9. x native or-chymotrypsin ; A 2-6 labelled a-chymotrypsin ; 0 0.60 labelled a-chymotrypsin ; 0 DIP chymotrypsin. at infinite dilution, while the extrapolated value of the polymer is 8-2 S.These values are possibly maximal and minimal values respectively, owing to interconversion during28 CHYMOTRYPSIN AND CHYMOTRYPSINOGEN the course of sedimentation. This interconversion should cause least error in the sedi- mentation constant of the monomer at low protein concentrations (when most of the protein is in the form of monomer) and least error in the sedimentation constant of the polymer at high protein concentrations (when most of the protein is in the form of polymer). Hence the extrapolated values shown in fig. 4 are not likely to be significantly different from the true values. The value of 2.9 S would correspond to a minimum molecular weight of 23,000 (for a sphere, no hydration) and the value of 8.2 S to a minimum molecular weight of 110,000.Hence the minimum value of polymer unit size at high protein con- centrations is 4.8 times that of the monomer. Fig. 1 also shows the variation of ph ~ O / T O with concentration of 1.56 labelled chymo- trypsinogen in 0.01 M phosphate buffer pH 7.9. Again ph. ~ O / T O is dependent on protein concentration, increasing a little over twofold over the concentration range 0-10 mg/ml. It is evident that the variation is much smaller than with chymotrypsin under the same conditions. The variation is in fact consistent with a dimerization also involving an increase in axial ratio. Sedimentation studies confirmed that chymotrypsinogen dimerized under these con- ditions. Fig. 5 shows the variation of sedimentation constant with protein concentration of native chymotrypsinogen and at two different degrees of labelling. The value of S20, ~~0 extrapolated to zero protein concentration, of 2.70 S, agrees well with values found under different conditions by other workers.6 It should be noted that under these conditions .0 - 5 10 15 2 0 25 Chymofryptinoqrn Concentration (mq/mI) FIG. 5.-Sedimentation constants of chymotrypsinogen in 0.01 M phosphate pH 7.9. 1-56 labelled chymotrypsinogen. 0 native chymotrypsinogen ; A 0.89 labelled chymotrypsinogen ; the chymotrypsinogen sediments to give a single symmetrical Schlieren pattern, indicating that the equilibrium between monomer and dimer is established very rapidly. The extrapolated value of 3.3 S of the high protein concentration sedimentation constants indicates that the dimerization is accompanied by an increase in axial ratio, i.e. the dimerization is probably an end-to-end association.The conditions in these experiments differ in two ways from those employed by pre- vious workers,s where dimerization of chymotrypsin was observed. Not only is the pH higher but the ionic strength is considerably lower. Schwert and Kaufman 5 concluded that the dimerization they observed was independent of ionic strength, but only two ionic strengths were used, 0.2 and 0.5. Here the ionic strength was 0.03. In order to see whether the polymerization of chymotrypsin reported here was due to the high pH or the low ionic strength, the sedimentation studies were repeated with 0.1 M phosphate buffer pH 7-9 (ionic strength = 0-2).A representative Schlieren pattern for chymotrypsin sedimented under these conditions is shown in fig. 6. Now the protein sediments as a single symmetrical component at all concentrations studied. Fig. 7 shows the variation of the sedimentation constant with concentration under these conditions. At high ionic strength dimerization only occurs, compared to the extensive polymerization at low ionic strength.FIG. 6.-Schlieren diagram of a-chymotrypsin in 0.1 M phosphate pH 7.9. (concentration 18 mg/ml). [To face 28V. MASSEY, W. F . HARRINGTON AND B . S. HARTLEY 29 VARIATION OF Ph.20/70 WITH THE DEGREE OF LABELLING It has been shown in the previous section that the variation in ph. 20170 with protein concentration is due to aggregation of the particular proteins studied. Weber 1 has found with fluorescent conjugates of ovalbumin and serum albumin that p h / ~ o is independent of the degree of labelling.With chymotrypsin, however, at any constant protein con- \ O X \ ~ 3.5 - 3.0 - (Sved bergs) I 1 5 10 15 2 0 2 5 Protein concentration (mq/ml) FIG. 7.-Sedimentation constants of chymotrypsin in 0.1 M phosphate pH 7.9. 0 a-chymotrypsin, x DIP chymotrypsin. Deqrec of labelling (molt dye/mol protein) FIG. 8.-Variation of ph. 2 0 / ~ ~ with degree of Iabelling (0.01 M phosphate pH 7.9, protein concentration 1-0 mglml). X cx-chymotrypsin ; 0 DIP chymotrypsin ; A chymotrypsinogen. centration ph. 20/70 varies quite markedly when the degree of labelling is greater than 1 molecule of dye per molecule of protein.Results for a protein concentration of 1 rng/ml are shown in fig. 8. Also shown in this figure are results for labelled chymotrypsinogen and label Izd DIP chy mot rypsin (i .e. crystal line di-isopropylfluorophosphonate-reacted30 CHYMOTRYPSIN AND CHYMOTRYPSINOGEN chymotrypsin which has then been conjugated with the fluorescent dye). It is evident that there is no significant difference between these three proteins in the variation of ph. 20/-ro with degree of labelling. The possible significance of this observation concerning the active centre of chymotrypsin will be considered in the discussion. A possible explanation for this variation was that the coupling of the dye to the protein resulted in physical changes, which became apparent only at high degrees of labelling.However, such an explanation is unlikely in view of the fact that the sedimentation characteristics are unchanged on coupling with the dye, even at high degrees of labelling. This can be seen from an inspection of fig. 3,4 and 5, which show that the proportion of polymer and the sedimentation constants are unchanged on conjugation. It thus seems unlikely that any major physical changes have accompanied conjugation. The most feasible explanation for this variation is that ph. 20 remains unchanged, and that the in- crease in ph. 20/70 found as the degree of labelling increases is due to a decrease in 70. Reaction of 1-dimethylaminonaphthalene-5-sulphonyl chloride with amino acids and amino acid derivatives has shown,4 that as well as reacting with free amino groups, this dye will also react with imidazole, sulphydryl and phenolic hydroxyl groups.If the lifetime of excited state of the dye depends on the group with which it reacts, and if different groups have reacted at different degrees of labelling, then such variations in 70 with degree of labelling would be expected. DISCUSSION THE VALUE OF 70 OF FLUORESCENT CONJUGATES OF CHYMOTRYPSINOGEN, CHYMO- TRYPSIN AND DIP CHYMOTRYPSIN The value of ph.201~0 extrapolated to zero protein concentration of 0.50 labelled chymotrypsin is approximately 5 (fig. 1). From previous sedimentation and diffusion data 5 s 7 it is known that the molecular weight of chymotrypsin is 21,500-24,000 and that the axial ratio is approximately 4. The value of p0.20 for a sphere of molecular weight 23,000 can be calculated from the equation1 po = 37 V/RT, where Vis the molecular volume.For a sphere of molecular weight For an ellipsoid of axial ratio 4, molecular weight 23,000 and 30 % hydration, the mean harmonic rotational relaxation time at 20" C, ph. 20, would be 5 x 10-8. As the zero concentration value of ph. &O is 5, then if the ph. 20 were 5 x 10-8, TO would be 1.0 x 10-8. Assuming a constant value of ph. 20 of 5 x 10-8 the variation of TO with degree of labelling is shown in fig. 9. Such a variation, occurring to appreciable extent above degrees of labelling of 1 molecule of dye per molecule of protein would be consistent with the preferential reaction of the dye with one specific group giving a higher TO than a non-specific reaction with other groups which give a lower 70.Weber 1b has found with conjugates of serum albumin and ovalbumin that a value O f To of 1.4 x 10-8 is consistent with his polarization data and independent determinations of rotational relaxation time.8 The considerably lower values of TO suggested above may be due to an error of extrapolation of ph. 20/70 to infinite dilution in fig. 1. The value of 70 calculated from this extrapolation is likely to be minimal owing to the curvature of the ph. 201~0 plot. Furthermore the cal- culation of theoretical ph. 20 from sedimentation and diffusion data could yield a lower ph. 20 value than the true value. In this calculation a perfect ellipsoidal shape was assumed. Deviations in shape from a perfect ellipsoid would be ex- pected to increase ph.Hence the values of 70 shown in fig. 9 probably represent minimal values. However, the above assumptions do not affect the relative values of 70 shown in fig. 9. 23,000, p0.20 = 2.14 x 10-8. THE POSSIBLE SIGNIFICANCE OF To CHANGES CONCERNING THE ACTIVE CENTRE OF There is good evidence for believing that the primary reaction of the dye with chymotrypsin is a specific labelling of a group in the active centre. Fig. 10 shows the inhibition of enzymic activity of chymotrypsin produced by varying CHY MOTRYPSINV. MASSEY, W. F . HARRINGTON AND B . S . HARTLEY 31 degrees of labelling with the fluorescent dye, showing stoichiometric inhibition by the first molecule of dye. That this inhibition is caused by a specific reaction with a group in the active centre has been shown 4 by reacting the dye with chymo- trypsin in the presence of a competitive inhibitor.Under these conditions the inhibition caused by any particular degree of labelling is considerably reduced. It is clear from fig. 3 and 4 that the active centre cannot be concerned in the polymerization of chymotrypsin at low ionic strength, since no differences in sedi- mentation constants or proportions of polymer are found between labelled and unlabelled chymotrypsin. If the mechanism postulated in the previous section to explain the variation in ph. ~ O / T O with degree of labelling is correct, then the first group to which the dye conjugates is probably the same in chymotrypsinogen, DIP chymotrypsin Deqrce of labelling (molr dye/mol protein) FIG 9.-The postulated variation of TO with degree of labelling.The values of 70 shown on this curve are calculated from fig. 1 and 8, to keep the value of ph. 20 6xed at 5 X 10-8 sec. I00 c 0 .- & E 5 0 .- x c .- 0-0 Deqree of labelling (mots d y e / mot protein) FIG, 10.-The inhibition of esterase activity of a-chymotrypsin produced by conjugation with dye. and a-chymotrypsin. This is clear from fig. 8 where no significant differences in ph. 20/70 between the three proteins can be detected. Hence it appears likely that the enzymically-inactive precursor of this enzyme, chymotrypsinogen, as well as the chemically-inhibited enzyme, DIP chymotrypsin, contain a specific group (capable of chemical reaction) which is essential for the catalytic activity of the active enzyme. Hence the tryptic activation of chymotrypsinogen to a-chymo- trypsin must involve the liberation of another group also essential to activity. This hypothesis finds support in the experiments of Doherty and Vaslow 9 who showed that chymotrypsinogen, as well as chymotrypsin, will bind one molecule32 ACTIVATION OF TRYPSINOGEN of a substrate per molecule of protein. Thus one might conciude that the primary site of reaction of the dye with these proteins is a group concerned with binding the substrate to the active centre, and that another group (the DFP-reacting site) 10 is necessary for the activation of this enzyme substrate complex. Our thanks are due to Mr. Boon for carrying out the sedimentation experiments. 1 Weber, (a) Biochem. J., 1952, 51, 145 ; (b) Biochem. J., 1952, 51, 155. 2 Kunitz and Northrop, J. Gen. Physiol., 1935, 18, 433. 3 Jansen, Nutting, Jang and Balls, J. Biol. Chem., 1949, 179, 189. 4 Hartley and Massey, unpublished. 5 Schwert and Kaufman, J. Biol. Chem., 1951,190,807. 6 Schwert, J. Biol. Chem., 1951, 190, 799. 7 Smith, Brown and Laskowski, J. Biol. Chem., 1951, 191, 639. Hartley and Kilby, 8 Oncley, Chem. Rev., 1942, 30, 443. 9 Doherty and Vaslow, J. Amer. Chem. SOC., 1953, 75, 928. Vaslow and Doherty, 10 Schaffer, May and Summerson, J. Biol. Chem., 1953, 202, 67. Oosterbaan, Kunst Biochem. J., 1954, 56, 288. J. Amer. Chem. SOC., 1953, 74, 931. and Cohen, Biochim. Biophys. Acta, 1955,16,299.