摘要:

FLORENCE B . SEIBERT 261 SWELLING AND ORIENTATION PHENOMENA WITH NUCLEOPROTEIN FILMS BY E. J. AMBROSE AND J. A. V. BUTLER Chester Beatty Research Institute, Royal Cancer Hospital, London, S.W.3 Received 6th June, 1952 Films of nucleoproteins, made by freeze-drying thin layers of solutions, swell to about six times their area in water, but do not normally disperse completely. This swelling is inhibited by small concentrations of polyvalent cations and at higher concentrations a contraction of the film occurs. The concentration of salts required to prevent swelling is of the same order as that required to precipitate the nucleoprotein from a dilute solution. The swelling properties of the films in alkali halide solutions appear to be controlled by the electrical double layer of the particles, but polyvalent metal cations have specific effects.Dry films have also been made in which the fibres are orientated to some extent. These exhibit a tendency when placed in water and aqueous solutions to return to their original shape. Infra-red and double refraction studies on these films are compatible with an arrangement of /3-polypeptide chains lying parallel to the direction of stretching and with the planes of purine and pyrimidine rings lying perpendicular to this direction. Although natural complexes of nucleic acid and proteins have been isolated from cell nuclei for many years, comparatively few studies have been made of their physical properties and behaviour as colloidal systems. A number of estim- ates of the molecular weight and dimensions of the particles has been made,l and the viscosities of solutions have also been studied? In the course of experiments with dried preparations of certain nucleoproteins which swell considerably on being placed in water, we noted that the swelling was greatly influenced by the additions of salts and other substances.Because of the remarkable and little understood behaviour of chromosomes, consisting, as they do, largely of nucleoprotein materials, this behaviour seemed to merit further study. This has been done by making films of the nucleoproteins by re- moving water in vacuo from thin layers of frozen solutions of the nucleoprotein. Fairly " fibrous " sheets, rather like blotting paper, which swell to about 6 times their initial area when placed in water, are obtained in this way.These films do not usually disperse completely in water, but in the swollen state are rather fragile and can be dispersed by shaking. It has been recogni~ed for some time that, when present in concentrated salt solutions (e.g sodium chloride), dissociation of the protein constituent from the deoxyribonucleic acid occurs.3 Consequently the nucleoprotein recovered by diluting such solutions is not necessarily identical with the original native nucleoprotein. Many of the observations recorded here were obtained with262 SWELLlNG A N D ORIENTATION PHENOMENA nucleoproteins extracted with isotonic salt solution. I n our observations the difference in swelling properties of such native nucleoproteins and those re- constituted after treatment with concentrated salt solutions was not very great.Films have also been made in which the fibre axes are predominantly orientated in one direction and some observations of their swelling as well as their infra-red spectra and dichroism are recorded. EXPERIMENTAL PREPARATION OF NUCLEOPROTEINS.-( 1) Reconstituted nucleoproteins. (A) Fresh bovine thymus glands were frozen and minced and suspended in 2 vol. of 0.9 % NaCl and homogenized in a Waring blender. After 24 h the suspension was centrifuged and the supernatant discarded. The residue was resuspended in 0.9 % NaCl and the oper- ations repeated. (B) The residue was now extracted with 2-3 vol. of 10 % NaCl and centrifuged twice, the residues being rejected. The supernatant was diluted with 10 vol. of water, whereupon the nucleoprotein precipitated.The product was again dissolved in 10 NaCI, centrifuged and precipitated by dilution. It was well washed and thoroughly dialyzed before being freeze-dried. This preparation disperses in water when shaken. (2) Native nucleopvotein.-Preparation as above for stage A. The suspension was centrifuged in the International Refrigerated centrifuge. The supernatant was dis- carded and the top half of the residue resuspended in 200 ml of 0.9 % NaCl by milling in the Waring blender for 20 sec. It was then centrifuged as before and the supernatant discarded. This procedure was repeated more than once. The residue was then sus- pended in distilled water and dialyzed against distilled water overnight. It is then diluted further and freeze dried in small bottles. PREPARATION OF UNORIENTATED NUCLEOPROTEIN FILMS.-The nUCkOprOtein (0.5 %) solution was put in a shallow layer 3 nim deep in a flat dish and frozen.The water vapour was then pumped off in a freeze-drying apparatus, leaving a dry porous film resembling filter paper, and about 1-2 mm thick. When pressed with a flat surface the total thick- ness was diminished and it was used in this form after being cut into approximately 0.5 x 0.5 cm squares. The areas of these were measured before and after swelling by means of a transparent ruler. The films used contain about 1.5 mg/cm* of nucleoprotein. a solution of herring sperm nucleoprotein in distilled water by evaporation. A thin rubber film was cast from a solution in petroleum ether on to a glass slide and the nucleo- protein was cast on the surface of the rubber.The rubber film was divided into strips by cutting with a razor blade, the surface of the nucleoprotein was moistened by breathing and the rubber was then stretched to produce about a 100 % extension. The extended rubber was pressed on to the glass slide in order to maintain it in the extended form. The rubber film was then dissolved away in petroleum ether and the isolated nucleoprotein film was picked up on a clean slide. In order to obtain an orientated film of nucleoprotein which was sufficiently thin for observations in the spectral region 1500-1700 cm-1, it was necessary to keep the material supported on the rubber base. It was found that thin rubber films could be prepared, which were practically transparent in this region of the spectrum.Films of nucleo- protein were therefore prepared on a rubber base for examination in the spectrometer. A similar technique, using somewhat thicker rubber, has already been used for the exam- ination of myosin in the 3 p region of the spectrum4. INFRA-RED DICHROISM AND DOUBLE REFRACTION OF THE NUCLEOPROTEIN.-A Grubb- Parsons model S3 spectrometer was used for the measurements. The selenium polarizer and automatic recording mechanism has been previously described.% 6 The spectra obtained, for the orientated film described above, with this instrument are shown in fig. l(b). A similar spectrum of a film of thymus deoxyribonucleic acid, prepared by evaporation from aqueous solution is shown for comparison in fig.l(a). The double refraction of the nucleoprotein was measured in the usual way with a polarizing microscope. The maximum value was about - 0.04, the negative sign in- dicating that the polarizability was a maximum perpendicular to the direction of stretching. prepared by freeze drying as described above, swell considerably when placed in water in Petri dishes. There is an increase in thickness as well as an increase of linear dimen- sions, but the thickness was not usually measured. Most of the swelling takes place in PREPARATION OF ORIENTATED NUCLEOPROTEIN FILMS.--These films Were prepared from SWELLING OF THE UNORIENTATED NUCLEOPROTEJN FILMS.-The nUCleOprOtein films,E. J . AMBROSE AND J. A . V. BUTLER 263 the first few hours. The relative area reached after 24 h in a series of alkali chloride solutions is shown in fig.2. It can be seen that the swelling decreases rapidly with increas- ing salt concentrations and a contraction occurs between about 0.1 and 0-5 N. At higher FIG. 1.-(a) Spectrum of deoxyribonucleic acid cast as a film from water, un- orientated. (6) Spectra of herring sperm nucleo- protein, orientated. - - - - - E-vector of plane polarized radiation paral- lel to direction of stretch- ing. E-vector perpen- dicular to direction of stretching. 0 8 7 x @ A 0 FIG. 2. - Effect of concentration of al- kali chlorides on swelling of thymus nucleoprotein films.264 SWELLING AND ORIENTATlON PHENOMENA concentrations of the salts the film begins to swell again, reaching at concentrations above 1.0N a constant area of about twice the initial area.In this more concentrated salt region it can, however, be shown that the nucleoprotein is dissociated at least partly into nucleic acid and protein, which dissolves into the solution, where it can be detected, e.g. by the Sakaguchi test, leaving a rather fragile and easily dispersed film which may be residual nucleoprotein or nucleic acid. The behaviour in solutions of sodium, potas- sium and lithium chlorides are not distinguishable. Fig. 3 shows the behaviour of the films in solutions of calcium and magnesium chlorides. FIG. 3.-Effect of con- centration of calcium and magnesium chlor- ides on swelling of thymus nucleoprotein films. concenhafion (M) 0. ooo/ 0 0002 0 * 0003 0.0004 I I I I FIG. 4.-Effect of small concentrations of metal salts on swelling of " native " thymus nucleoprotein films.-0- alkali chlorides -0- AgN03 -El-- MgC12 -=- Th(N03)4 -@- Lac13 - b----Pb(NO3)2 -@- CaC12 -V- ZnS04 ---.A- FeC13E . J . AMBROSE AND J . A . V . BUTLER 265 The behaviour of similar films in dilute solutions of other salts is shown in fig. 4. The nature of the anion appears to have very little effect, but the effect of the cation increases with its valency. There is not, however, a strict grouping according to the valency, e.g. Ag+ ions are nearly as effective as Ca2+, Zn2+ and Mg2+, and Pb2+ and Mn2+ are nearly as effective as Fe3+ and Th4+. Very small concentrations of the latter-of the order of 4 x 10-5 M-are sufficient to prevent the swelling. The minimum concentrations of salt which just prevent any swelling are given in table 1, TABLE 1 .-EFFECTS OF SALTS ON SWELLING AND PRECIPITATION OF NUCLEOPROTEINS lowest concentration which prevents swelling ( M ) lowest concentration ( M ) whjch precipitates some nucleoprotein - salt herring sperm thymus nucleo- herring sperm thymus nucleoprotein n.p.protein (native) n.p. (0.1 %) (native) (D10) (0-2 %) 0.025 0.01 0.025 0-025 0.00 1 0.001 0.00 1 0.002 0.0005 0.0006 0.0006 0.001 0.001 0*0005 0.000 1 6 0.0005 0*0005 0.0002 - 0.01 0.0 I 0.0 1 0.000 1 0.0002 0.0002 O.OOO8 0.00008 O.ooOo8 00002 0.0003 0.0002 O*ooOo8 0-OOO 1 O.ooOo5 040004 - 0.02 0-1 0.1 0.1 0.0004 0.0004 0.001 0.002 0.0005 0*0002 0.0002 0.0002 04002 0.0004 O.oOO4 o.Oo01 0.0001 0.00004 - 0.04 0-04 0.04 0.001 0.00 1 0-0006 O-Oo08 O.OOO4 0.0004 0-0004 O.OOO4 O.OOO4 0.0002 040008 0~0001 - O-OOOO~ This gives figures for both a " reconstituted " herring sperm nucleoprotein preparation (HS 5 ) and a " native " thymus nucleoprotein preparation (TNP 4).The concentra- tions required by the latter are usually considerably less than those required by the former. There appeared to be no significant difference between the " native " nucleoprotein from calf thymus and that which had been dissolved in 2 M sodium chloride and reconstituted. With very dilute solutions of highly active salts (e.g. such as lead nitrate) there was an initial swelling followed by a contraction to the final state (fig. 5). This is to be ascribed to the amount of metal ions originally penetrating the film from these dilute solutions being insufficient to prevent swelling, so that the initial effect is followed by a slow absorp- tion of ions from the surrounding solution.Table 1 also gives the minimum concentration of the salt which gives rise to an appre- ciable amount of precipitation in a 0.1 % (or 0.2 %) aqueous solution of the nucleo- protein. It is evident that there is a fairly close parallelism between the two sets of figures, i.e. the concentration of salt which prevents swelling is of the same order as that which just begins to precipitate the nucleoprotein from solution. It may perhaps be significant that higher concentrations of salts are required to pre- cipitate the native thymus nucleoprotein than to prevent its swelling, but the reverse is true of the reconstituted herring preparation.The differences noted, however, may not be very significant, as it is difficult to judge when precipitation begins and considerable error is possible. Dilute solutions of urea (0.4 % and more) cause the film to disperse completely in water, but in 0.1 N sodium chloride even concentrated urea solutions (25 %) did not cause any swelling. After being contracted in calcium chloride solutions, the films did not swell when placed in water even after a considerable time. have also been made on the swelling of the orientated nucleoprotein films in various salt concentrations and in the presence of reagents which attack hydrogen bonds. For these experiments small pieces about 0.5 mm square were taken off a single specimen of film, which had a uniform degree of orientation.In this way reasonably reproducible results were obtained. ANISOTROPIC SWELLING OF FILMS OF ORIENTATED NUCLEOPROTEIN FILM.-Observations I266 SWELLING A N D ORIENTATION PHENOMENA The specimen was mounted in a small trough on the microscope and camera iucida drawings were made of the outline of the film at various stages of swelling. The results obtained are summarized in table 2, where the dimensions parallel and perpendicular to In -0- O-0003M -0- 0.00004M -*- Od0008M FIG. / 3 4 I I I I 5.-Changes of area of thymus nucleoprotein film with time with small concentrations of lead nitrate. medium 014 M NaCl distilled water 0.14 M NaCl + 0 3 5 M phenol distilled water + 035 M phenol 0.14 M NaCI+ 11 TABLE 2.--NUCLEOPROTEIN SWELLING time interval ~- I -4 % + 140 0 + 350 -40 + 125 + 25 + 190 - 30 + 130 ~ tomia.lday -40% -40% 4-140 +150 + 140 +540 - 30 - 25 + 135 + 70 t-240 - 30 - 20 +130 +140 - - - - double 2 days refraction -40 % persists + 150 I lost - persists - lost - - - persists I distilled water + 1 1 M urea [I + 20 + 50 - - lost I +420 $470 - - I denotes immediately after immersion in medium. Swelling is expressed in terms of the linear dimensions of the film before immersion in the medium. 11 denotes swelling parallel to the direction of stretching. 1 denotes swelling perpendicular to the direction of stretching. the direction of stretching are expressed in terms of the percentage change as compared with those of the original dry film. In table 3 are shown the effects of acid treatment upon nucleoprotein films which have been brought to equilibrium in isotonic salt at pH 6.6.The dimensions are expressed as a percentage of the dimensions of the sample when in equilibrium in 0.14 M salt at pH 6.6 the film was placed in isotonic salt at pH 2 or in acetic acid -I- alcohol hative and then in distilled water. Although the acid treatment appeared to fix the material in its shrunken state the film was still soluble in 11 M urea solution in distilled water.E. J . AMBROSE AND J . A . V. BUTLER TABLE 3 THE EFFECT OF ACETIC ACID -k ALCOHOL FIXAWE 014 M NaCl acetic acid + alcohol distilled water pH 6 6 fixative for 1 day 0 -52% -58% - 58 11 I 0 - 50 THE EFFECT OF HYDROCHLQMC ACID 267 II I 0’14 M NaCl pH 6 6 014 M NaCl P H ~ 1 distilled water 0 -36% no change 0 - 33 no change I[ denotes dimension parallel to direction of stretching expressed as a percentage I denotes dimensions perpendicular to this direction.ELECTROPHORESIS OF PARTICLES OF NUCLEOPROTEIN GEL-The electrophoretic mobility of the nucleoprotein in salt solutions could not be conveniently measured with the usual form of Tiselius apparatus because the material is only soluble in solutions of very low or high salt concentrations. The simple apparatus shown in fig. 5 was used. A piece of frozen dried nucleoprotein was placed in salt solution of the given concentration for a few hours so that conditions approaching those of equilibrium swelling were attained. of length in 0.14 M salt at pH 6.6. G I\ F I \ I I . I \ / I 1’ I U I 4 ‘G ‘5 FIG. 6.--Micro-electrophoresis cell.narrow channel in which particles are observed. plug of filter paper. silver/silver chloride electrode. cell cover. fiduciary marks projected by camera lucida. 0; I 0.5 FIG. 7.-Electrokinetic potential (particles nega- tive) of nucleoprotein gel at various salt con- centrations. The material was then broken up into fine particles and placed in the narrow channel between the two electrode vessels as shown in the figure. The remainder of the cell was filled with salt solutlon of the given concentration and the cell was covered with a slide. The complete cell was immersed in a shallow trough containing some moist filter paper and was covered with a Perspex lid. This arrangement ensured that no evaporation of water, from the edges of the cell cover, took place.The cell was mounted on the micro- scope stage and the gel particles were observed under a magnification of 50 diameters. Two lines were drawn on a sheet of paper and a camera lucidu was used to project an268 SWELLING AND ORIENTATION PHENOMENA image of the lines across the channel of the electrophoresis cell. This arrangement pro- vided a convenient system for measuring the velocity of particles, when the electric field was applied. Observations of the velocity were made for particles 1/5 of the cell width from the wall, a position in which the same electrophoretic velocity can be obtained according to Smoluchowski.7 Because the particle size was large compared with the dimensions of the double layer and the velocity was found to be almost independent of particle size, the formula of Smoluchowski 7 could be applied, viz.: v = E E [ / ~ T ~ where V is velocity of particle, E, applied field, 7, viscosity of medium. E, dielectric constant of medium, 4, electrokinetic potential, The relationship between salt concentration and electrokinetic potential for sodium chloride is shown in fig. 5. The discontinuity of the curve between 0.25 N and 0.5 N sodium chloride appears to be due to the dissociation of the nucleoprotein into nucleic acid and protein. It was found that no arginine could be observed by the Sakaguchi test in the salt solution at 0.25 N, but a distinct colour was observed at 0-5 N. DISCUSSION INTERPRETATION OF INFRA-RED SPECTRA AND OPTICAL PROPERTIES OF THE FILMS.-The infra-red spectrum of orientated fibres of nucleic acid has been in- vestigated by Fraser and Fraser9 using polarized radiation.In the region from 1600-1800 cm-1 they have observed absorption bands at 1695 cm-1 and at 1655 cm-1. These modes have assigned to stretching vibrations of the C=O and C=N bonds of the purine and pyrimidine rings. They consider that the transi- tion moments for these modes would all occur within the planes of the purine and pyrimidine rings. Two kinds of fibres of nucleic acid have been prepared by Wilkins et al.8 The first kind, which are obtained by a moderate extension, are optically negative and give an extremely sharp X-ray diffraction pattern. The second kind, which are obtained by a high degree of extension, are optically positive and give an amorphous X-ray diffraction picture.Fraser and Fraser 9 find that, with the first type of fibre, the C-0 and C=N bands of purine and pyrimidine show a maximum of absorption with the E-vector of the plane polar- ized radiation perpendicular to the fibre axes; in the second kind of fibre the dichroism is reversed. An examination of the spectra in fig. 1 will show that these absorption bands in the nucleoprotein film are showing the first kind of dichroism. These results appear to indicate that the purine and pyrimidine rings of nucleic acid lie at right-angles to the direction of stretching. It will be seen that the relative intensities of the bands at 1695 cm-1 and at 1655 cm-1 are not identical in the nucleic acid spectrum of fig. 1 (a) and in the nucleoprotein spectrum of fig.1 (b). It has been found that the relative intensities of the bands depend upon the hydrogen ion concentration of the solution from which the film of nucleic acid is prepared. There are, however, two fairly strong absorption bands present in the nucleo- protein spectrum which have not been found in nucleic acid. Those are the bands at 1640 cm-1 and at 1540 cm-1. 1640 cm-1 is the frequency that has come to be associated with the peptide C=O group of an extension of p configuration of polypeptide chain.10 The C=O band associated with the folded form occurs at about 1660 cm-1. This result suggests that the protein associated with nucleic acid in the nucleo- protein is in the p configuration. *It is difficult to decide the dichroism of this band owing to the proximity of the band at 1655 cm-1.The band at 1540 cm-1 in the nucleoprotein also appears in polypeptides. This band shows a maximum of absorption with the E-vector parallel to the fibre axes in /%polypeptides and a maximum with the vector perpendicular to the fibre axis in folded polypeptides.10 The dichroism of this band is seen to be of the first kind in fig. 1 (b). WhereE. J . AMBROSE AND J . A. V. BUTLER 269 allowance is made for the base line in the two records, the dichroic ratio is found to be about 1.5. The effects, though small, are reproducible. The frequency of this absorption band is somewhat high for a P-polypeptide but it has been found that the N-H mode is more sensitive to the strength of the N-H . . . 0-C hydrogen bond than is the C=O band at 1640 cm-1.The frequency of the C-0 band has so far been found to give a reliable indication of the type of chain configuration present. The infra-red data therefore provide reasonably conclusive evidence for the presence of p-polypeptide chains in these films. It might possibly be considered that this result was an artefact produced by the stretching process. Evidence against this possibility is given by experiments which have been made with protein nucleates, materials which were insoluble in water and prepared by mixing nucleic acid and protamine. When films of this material are stretched they show infra- red dichroism of the purine and pyrimidine rings which is of the kind observed by Fraser and Fraser in highly stretched nucleic acid. In this case the somewhat looser combination between nucleic acid and protamine apparently allows the nucleic acid chains to be stretched into the second configuration.The protein still appears to be in the /3 form in this case. It does not appear, Lherefore, that the stretching process has led to a serious distension of the molecular chains with the specimens whose spectra are shown in fig. 1 (b). It may be of interest to note that the C=O stretching frequency for films of clupein has a calue of 1665 cm 1, a frequency corresponding to the rx configuration. With the dry materials it appears to be a combination with nucleic acid which stabilizes the ,8 configuration in the protamine. SWELLING OF THE FILMs.--Since they exhibit no anisotropy the unorientated films are a mass of fibres lying in all directions.When placed in water they expand up to a certain point and then remain in equilibrium. The film in this state is rather fragile and can be broken up and dispersed by shaking. The fact that the expansion comes to a stop shows that there are attachments between the fibres which prevent further expansion or complete dispersion of the film. These links are broken by the action of urea or lithium chloride which bring about complete dispersion in time. The attachments must therefore be ascribed to attachments between the fibres due to hydrogen bonds which permit a considerable amount of expansion. They might be due for example to small quantities of protein which have become detached from the nucleoprotein particles, which are themselves soluble in water, or to a small quantity of nucleoprotein which has become denatured in the process of making the films.A film made from a nucleoprotein solution which had not been clarified by centrifugation and which contained a considerable quantity of insoluble protein did not swell at all in water, although it was able to swell in 5 % urea. This supports the idea that the limited swelling in water is due to protein links between the fibres. It has been shown that comparatively small concentrations of salts prevent the dispersion. The fairly close correspondence between the salt concentrations preventing expansion and those which precipitate the nucleoprotein solution, indicated that both phenomena are due to the same cause. With the chlorides of sodium and potassium it would appear that this is due to change of the double layer potential round the particles with increasing ionic strength.as has been demon- strated by the electrokinetic experiments. The effect of polyvalent cations is, however, much greater than would be expected from the increase of ionic strength and it is apparent that these ions have a special effect apart from that due to their contribution to the ionic strength. The relative magnitudes of the concentrations of a series of cations required to precipitate the nucleoprotein is not unlike the series of flocculating concentrations of negatively charged lyophobic colloids, but there is much overlapping. The nucleoprotein, however, can hardly be re- garded as a lyophobic colloid since aqueous solutions once formed appear to be perfectly stable.The addition of minute quantities of polyvalent cations appears I*270 SWELLING AND ORIENTATION PHENOMENA to bring about a change in the equilibrium state of the nucleoprotein and they could do this by cross linking between two negatively charged groups of adjacent fibres, e.g. R . COO- . . . MZr . . . -0OC . R ; or by forming co-ordination links between two adjacent -NH2 or similar groups, e.g. R-NH2 . . . M2f . . . H2N. R. The fact that silver ions have a considerably lower coagulating concentration than alkali metal ions indicates that such a process may occur. More complex types of interaction, such as have been suggested between peptides and various metal ions to account for the activation of the peptidases by such ions 11 may also contribute.The observations made on dry films of stretched nucleoprotein using polarized infra-red radiation and the polarizing microscope indicate that at least a partial orientation of the long fibres has been produced. The observauons are compatible with an arrangement of extended or 13-polypeptide chains, lying parallel to the direction of stretching, with purine and pyrimidine rings having planes perpen- dicular to this direction. These results are not in conflict with the suggesLion of Astbury and Bell on the basis of X-ray diffraction data and density data. The periodicity of 3-5A observed by these authors along the fibre axis is similar to that observed in /3 protein films. The orientated nucleoprotein films (table 2) swell much more in the direction perpendicular to the fibre axis than parallel to it.At first sight this suggests an expansion at right-angles to the fibre axis, indicating a repulsion between parallel fibres. In the isotonic salt solutions there is a contraction in the direction of stretching and an expansion perpendicular to this direction. The result of this is that after coniact with the solution the films return to approximately their original shape before stretching. This apparent relaxation is not, however, com- plete as a considerable degree of double refraction is preserved. In water, expansion initially takes place almost entirely perpendicular to the direction of stretching. The result of this is to restore approximately the original shape. This is followed by a considerable overall expansion in which the initial shape is very approxim- ately retained. It must be concluded from these experiments that the material is capable of undergoing a considerable degree of elastic deformation and is fixed or frozen in the deformed condition by rapid drying.Immersion in distilled _water leads to an initial relaxalion accompanied by some anisotropic swelling. Later the swelling becomes isotropic and the birefringence is lost. The effect of dilute solutions of sodium and potassium chloride is reversible, but after treatment with calcium chloride very little swelling occurs when the film is placed in distilled water. This suggests that adsorbed Ca2+ ions are lost very slowly, if at all. After treatment of the films with acid mixtures such as are used in fixing tissue sections no swelling occurs in water.However, strong urea causes the dispersion of the films treated in this way which suggests that the inability to swell is due to hydrogen bonding between the fibres induced by the acid media. On the other hand phenol or urea do not cause dispersion in isotonic salt solutions which supports the idea that in this case insolubility is due to the effects of the salts on the double layer interactions. The denaturation which is produced by an acid medium appears to be ana- logous to that observed in treating globular proteins with acid. With insulin the denaturation appears to involve a change from intra-chain hydrogen bonds. With the nucleoproteins we already have a p-protein with inter-chain hydrogen bonds associated with the nucleic acid in the middle.It is possible that the micelle is arranged so that most or all of the hydrogen bonds between peptide -NH -CO groups, and purine and pyrimidine -CO and NH2 groups are satisfied within the rnicelle. Denaturation of the nucleoprotein would then lead to a disturbance of this delicate state of equilibrium.E. J . AMBROSE AND J . A . V. BUTLER 27 1 We are most grateful to Dr. A. Elliott of the Courtauld Research Laboratory, Maidenhead, where the measuremenls with polarized infra-red radiation were carried out by one of us (E. J. A.); also to Dr. C. H. Bamford of the same laboratory. We are indebted to Mr. P. Gilbert-Avis for assistance with the preparative work and to Miss E. M. Press for help in making the films. This investigation has been supported by grants to the Royal Cancer Hospital and Chester Beatty Research Institute from the British Empire Cancer Campaign, the Jane Coffin Childs Memorial Fund for Medical Research, the Anna Fuller Fund, and the National Cancer Institute of the National Institutes of Health, U.S. Public Health Service. 1 Carter, J. Amer. Chem. Soc., 1941, 63, 1960. Stern, Biol. Brill., 1945, 89, 190. Van Winkle and France, J. Physic. Chem. Carter and Hall, J. Amer. Chem. Soc., Hammersten, Biochem. J . , Expt. Cell Res., suppl., 1947, 1, 97. 1948, 52, 207. 1940, 62, 1194. 1924, 144, 383. 2 Frick, Biochim. Biophys. Acfa, 1952, 8, 202. 3 Mirsky and Pollister, J. Gen. physiol., 1946, 30, 117. 4 Ambrose, Elliott and Temple, Nature, 1949, 163, 859. 5 Elliott and Ambrose, Nature, 1947, 159, 641. 6 Elliott, Ambrose and Temple, J. Chem. Physics, 1948, 16, 877. 7 Smoluchowski, 2. physik. Chem., 1918, 93, 129. 8 Wilkins, Gosling and Seeds, Nature, 1951, 167, 759. 9 Fraser and Fraser, Nature, 195 1 , 167, 761. Petermann and Lamb, J. Biol. Chem., 1948, 176, 685. JOAmbrose and Elliott, Proc. Roy. SOC. A, 1951, 76, 208. 11 Smith and Bergmann, J. Biol. Chem., 1941, 138, 789. Smith, J. Biol. Chem., 1946, 163, 15; 1948, 173, 571 ; 1949, 180, 1209 ; Proc. Nut. Acad. Sci., 1949, 35, 80.

ISSN:0366-9033    

DOI:10.1039/DF9531300261

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

E. J . AMBROSE AND J . A . V. BUTLER 27 1 HYALURONIC ACID, CHONDROITIN SULPHATES AND THEIR PROTEIN COMPLEXES * BY KARL MEYER Department of Medicine, Columbia University College of Physicians and Presbyterian Hospital, New York Received 26th May, 1952 Surgeons, and the Edward Daniels Faulkner Arthritis Clinic of the In this paper the evidence for the existence of protein complexes of hyaluronic acid and also the chondroitin sulphates of hyaline cartilage and connective tissue is reviewed Most of the experimental data for hyaluronic acid are best explained by assumption of reversibly formed polar complexes. (The existence of secondary non-polar forces is not excluded.) The irreversible loss of viscosity observed during isolation, for example, may be a result of depolymerization of the carbohydrate chain by mechanisms not now understood, rather than the irreversible cleavage of a native protein complex.The data on chondroitin sulphate A in cartilage are also best explained by the assumption of polar linkages with protein. The chondroitin sulphates of connective tissue, however, are stably linked to protein in the native state. It has been assumed for many years that the mucopolysaccharides occur in nature as protein complexes which, although poorly defined chemically or physically, have been classified according to their origin in the older literature 1 *Supported in part by a grant from the Helen Hay Whitney Foundation, and the U.S. Public Health Service.272 HYALURONIC ACID COMPLEXES as vitreous muciii, syiiovial much, chondromucoid, osseomucoid, etc.The title of this section obviously presupposes the existence of mucoproteins. The author, some years ago, defined mucoproteins or mucoids as proteins containing mucopolysaccharides, i.e. hexosamine containing polysaccharides, in firm chemical union, that is, linked by a covalent bond to a peptide.2 Conjugated proteins of this type are ovomucoid, the various mucoproteins of serum, the acid muco- protein of submaxillary gland and the neutral mucoids of gastric mucosa. The separation of mucopolysaccharide and peptide has been achieved in some of these by action of hot alkali or by prolonged enzymatic hydrolysis. Whether the bonds (of unknown nature) between mucopolysaccharide and peptide have been broken after such treatment has not been established. From consideration of the various methods of isolation of hyaluronic acid and of the chondroitin sulphates, it is apparent that the stability of their protein complexes is of a different order of magnitude than that of the mucoproteins proper.It is the object of this paper to discuss the question of the nature of the inter- action of protein and hyaluronic acid and the chondroitin sulphates. It will be clear from the following that at the present time few definite answers can be given to the questions raised. HYALURON~C Acn-Hyaluronic acid occurs either in dissolved form as in vitreous humor, synovial fluid and some tumour fluids, or in form of gels as in umbilical cord, in certain mesodermal tumours and in dermis.3 Methods for the preparation of the acid, free of protein and of other accompanying substances (within reasonable limits) have been described.3 Hyaluronate prepared from various mammalian sources shows no significant differences in elementary analysis, in optical rotation, in the proportion of its component monosaccharides (glucos- amine and glucuronic acid), in the degree of hydrolysis by various enzymes or thus far in the nature and quantities of products which can be obtained on frac- tionation of enzymatic hydrolysis products.4 Physical properties, however, especially viscosity, differ among preparations, depending on the source and the method of purification of the polysaccharide.The viscosities are highest with samples prepared from umbilical cord and from group A haemolytic streptococcus, followed in order by tumour fluid, synovial fluid and vitreous humor.The latter appears to be also most polydisperse, although no detailed information of the molecular weight distribution of the acid from different sources has been published. Isolated hyaluronate has a lower viscosity in equivalent concentrations than that contained in the native fluid. Such fluids on dilution and acidification pre- cipitate as long fibres, the so-called mucin clots. Hyaluronate isolated by most procedures, especially from synovial and vitreous fluids, on addition of serum and acidification, precipitates in flocculent form. However, highly viscous hyaluronaie under the same conditions yields mucin clots.5 After incubation of native hyaluronate with 0.01 to 0.005 units of purified testicular hyaluronidase,3 the fluid no longer gives a mucin clot and precipitates in flocculent form.Isolated hyaluronate under these conditions shows no loss in turbidity formation and a negligible loss in viscosity. The " mucin " formed on acidification of synovial fluid has been shown to be a polar compound formed by the free amino groups of protein with the COOH groups of polysaccharide acid. In salt solutions or on alkalinization, these salts dissociate.6 From electrophoretic studies no evidence other than of dissociating complexes between hyaluronate and protein has been obtained. The mobility of isolated hyaluronate is identical with that of the fastest component in native fluids.7~ 8 Ogston and Stanier 9 9 10 on the basis of viscometry and sedimentation have concluded that synovial fluid contains a definite protein complex of hyaluronate which on isolation apparently breaks down irreversibly.The relatively highKARL MEYER 273 viscosity of the native fluids may perhaps equally well be explained by specific interaction of the native hyaluronate molecules, similar to that postulated for certain desoxypentose ribonucleic acids, or depolymerization by hydrolytic or oxidative processes of the carbohydrate chain during isolation, or, as on addition of enzyme, by cleavage of a few centrally located hexosaminidic bonds. CHONDROITIN suLPHAms.-We distinguish three different chondroitin sulphates, designated as A, B and C.11 The chemical structure of these compounds is at present unknown, although various structures have been proposed in recent years for chondroitin sulphate of hyaline cartilage.In contrast to hyaluronate which occurs both in fluids and in structural elements, the chondroitin sulphates do not occur in fluids." The physical properties of native chondroitin sulphates, especially their molecular size and the nature of the complexes in which they may occur in the matrix are very difficult to ascertain. In the older literature, chondroitin sulphate had been thought to occur as a protein complex, presumably as an ester which had to be hydrolyzed by alkali during isolation.1 Morner had, however, shown many years ago that a small fraction of chondroitin sulphate could be extracted by water.12 Following the study in our laboratory of artificial protein salts of hyalurinic acid, similar studies were carried out with chondroitin sulphate.13 On the basis of this work, a method for the preparation in excellent yield of chondroitin sulphate from finely ground defatted cartilage powder, based on extraction with 10 % CaC12, was presented.14 This procedure has been adopted by other investigators.15 The viscosity of isolated chondroitin sulphate varies greatly with the procedure adopted.A high pH or high temperature leads to loss of viscosity. The molecular weight of chondroitin sulphate (A?) has been reported 1s as 260,000 (birefringence of flow of carefully prepared chondroitin sulphate). Molecular weights of 15,000 to 43,000 obtained by an osmometric method have recently been reported.16 The lower figure corresponds to alkali-extracted, the highest to CaC12-extracted material.It appears quite clear that the method of treatment greatly influences the particle size of the isolated mucopolysaccharide. Isolated chondroitin sulphate was shown to react at low pH stoichiometrically as a dibasic acid with proteins.13 The quantity of chondroitin sulphate bound by the protein corresponded to the proportion of basic amino acids contained in the protein, and was also equivalent to the quantity of acid dyes bound by the protein. The ratio of hexosamine to total N of the artificial mucoids prepared from chondroitin sulphate and gelatin closely approximated the corresponding ratio for cartilage powder. It was suggested, for this and other reasons, that chondroitin sulphate in cartilage exists largely as a salt of the mucopolysaccharide with collagen.Electrophoretic studies of extracts of cartilage have been reported by Blix8 and by Partridge.17 Blix found no evidence for existence of protein complexes in aqueous extracts In Partridge's experiments with extract prepared at elevated temperatures, he observed the expected two components in the descending limb of the electrophoresis cell, corresponding to chondroitin sulphate and protein derived from degraded collagen. In the ascending limb he found, in addition, a new distinct zone, which he attributed to a dissociating complex between protein and carbohydrate. Costal cartilage has recently been shown18 to bind Na+. Ca2+ or Ba2+ in quantities equivalent to the chondroitin sulphate present its measured by the sulphate content.It was concluded that the chondroitin sulphate above pH 3.5 in this reaction behaved as a divalent acid. One may interpret this experiment to indicate that the acidic groups of chondroitin sulphate as present in cartilage are capable of dissociation. * Recently from the highly viscous contents of a cyst obtained on operation of a chondrosarconia there was isolated a sulphated polysaccharide ; from analysis and from its behaviour toward enzymes this appeared to be chondroitin sulphate A unaccompanied by other fractions.274 HYALURONIC ACID COMPLEXES Einbinder and Schubert19 have studied the binding by rat tail and bovine Achilles tendon of chondroitin sulphate (and hyaluronate) as compared to the binding of acid dyes. Chondroitin sulphate in these experiments had an equivalent weight of one (rather than one-half) repeating unit, and hyaluronate was bound only at a pH below 3.The authors called attention to the fact that collagen reacted with the anions in a manner very different from that of ~001.20 The explanation for the differences seems to lie in the fact that collagen at acid pH swells and is transformed irreversibly into degraded collagen. Further, the solubilities (and not impossibly the chemical natures) of the collagens of tendon differ markedly from those of cartilage. A comparison, therefore, of the behaviour of the two may not be justified. The difference in solubility may be caused by the large concentration of mucopolysaccharide in cartilage (ca. 20 to 40 % of the dry weight) while the amount of mucopolysaccharide in tendon is small (ca.1 %). It can be concluded that the nature of the complex of chondroitin sulphate and protein in hyaline cartilage remains to be elucidated. However, it may be said that none of the observations so far reported are in contradiction to the picture of a salt-like combination of polysaccharide and protein. The presence of primary covalent links between the two appears, at any rate, to be excluded. In cartilage, chondroitin sulphate B is absent. This substance has been isolated from skin,21 and later from tendon, heart valves and aorta.11 In skin, chondroitin sulphate is found together with hyaluronic acid, and in the other tissues the second mucopolysaccharide is chondroitin sulphate C. Chondroitin sulphates B and C are firmly bound to proteins which are distinct from collagen.It has been con- cluded from histological work that these protein + chondroitin sulphate complexes are contained in the interfibrillar ground substances. Extracts of bovine or porcine Achilles tendon with 0.02 N Ca(OH)2 (half-saturated lime water) on repeated precipitation yield mucoids from which the mucopolysaccharide cannot be separ- ated by either electrophoresis or treatment with 10 % CaCl2, in contrast with the behaviour of hyaline cartilage. A mucoid fraction prepared from heart valves at pH 8.5 in barbiturate buffer ( I = 0.1) had a mobility of 8-1 x 10-5, while the mobility of the chondroitin sulphates at the same condition is 12.5 x 10-5. The mucopolysaccharides are split off on treatment at 4" C with 0.33 N NaOH or on digestion with pepsin at pH 2 followed by tryptic digestion.The nature of the protein complexes, as well as the chemistry of the mucopolysaccharides has been studied very incompletely . On the biological level, the interaction of mucopolysaccharides with proteins may be of importance among other things in fibrillogenesis and in the organization of other structural elements, especially in growth and differentiation. The role of mucoids in the reconstitution of collagen has recently been described.22 It was shown in this work that soluble collagen gave, after dialysis, either long spac- ing (2000 A) or regular (640 A) axial striations, depending on the concentrations of serum mucoprotein fractions added (these mucoproteins contain neither sulphate nor uronic acid and are true neutral mucoids).In general, hyaluronate or the chondroitin sul phates on addition to dissolved collagen gave immediate precipit- ates devoid of cross striations. However, one of the samples of chondroitin sulphate A used gave the 2000A axial orientation.22 In contrast to the fibrils produced by mucoprotein, this cross striation appeared immediately, without prior dialysis. The difference between this sample and others appears to be only the higher molecular weight of the former. Anothcr exampIe of mucopolysaccharide 1- protein interaction is that de- scribed by Cohen.23 According to this author, macromolecules of very large size such as haemocyanine, liver particles and some viruses are precipitated by heparin, chondroitin sulphate or hyaluroiiate at neutral reaction.Some of the viruses in the presence of the mucopolysaccharides were transferred into para- crystals differing from their ordinary form. Hyaluronate of a high degree of polymerization was active in much lower concentration than either heparin orKARL MEYER 275 chondroitin sulphate (cp. ref. (3), p. 354). Enzymatically depolymerized hyal- uronate was inactive in this reaction. At present no satisfactory explanation can be given for these phenomena or for the orientation effects observed with soluble collagen. sUMMARY.--The evidence for the existence of reversible polar complexes of hyaluronic acid in native fluids is reviewed. Data on cartilage are reviewed and explained by the assumption of polar linkages with protein. In contrast, the chondroitin sulphates of connective tissue are stably Iinked to protein. 1 Levene, Hexosarnines and Mucoproteins (Longmans, Green & Co., London and 2 Meyer, Advances in Protein Chem., 1945, 2, 249. 3 Meyer, Physiol. Rev., 1947, 27, 335. 4 Rapport, Meyer and Linker, J. Amer. Chem. Soc., 1951, 73, 2416. 5 Meyer, J. Biol. Chem., 1948, 176, 993. 6 Meyer, Cold Spring Harbor Symposia, 1938, 6, 91. 7 Meyer and Chaffee, J. Biol. Chem., 1940, 133, 83. 8 Blix, Acta Physiol. Scand., 1940, 1, 29. 9 Ogston and Stanier, Biochem. J., 1950, 46, 364. 10 Ogston and Stanier, Biochern. J., 1951, 49, 585. 11 Meyer and Rapport, Science, 1951, 113, 596. 12 Marner, Skand. Arch. Physiol., 1889, 1, 210. 13 Meyer, Palmer and Smyth, J. Biol. Chem., 1937, 119, 501. 14 Meyer and Smyth, J. Biol. Chern., 1937, 119, 507. 15 Blix and Snellman, Ark. Kemi, Min. Geol. A, 1945,19. 16 Mathews, Fed. Proc., 1952, 11, 255. 17 Partridge, Biochern. J., 1948, 43, 387. 18 Boyd and Neuman, J. Biol. Chem., 1951,193,243. 19 Einbinder and Schubert, J. Biol. Chem., 1951, 188, 335. 20 Steinhardt, Fugitt and Harris, J . Res. Nat. Bur. Stand., 1942, 28, 201. 21 Meyer and Chaffee, J. Biol. Chem., 1941, 138,491. 22 Highberger, Gross and Schmitt, Proc. Nat. Acad. Sci., 1951, 37,286. 23 Cohen, J. Biol. Chem., 1942, 144, 353. York, 1925).

ISSN:0366-9033    

DOI:10.1039/DF9531300271

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

KARL MEYER 275 COMPOSITION AND PROPERTIES OF HYALURONIC ACID COMPLEX OF OX SYNOVIAL F'LUID BY A. G. OGSTON AND J. E. STANIER Department of Biochemistry, University of Oxford Received 9th April, 1952 Isolation of hyaluronic acid by ultrafiltration gives a material with unchanged physical properties ; methods involving precipitation affect the physical properties. The material obtained by filtration contains about 27 % of protein ; most of the rest of its weight is accounted for by glucuronic acid and acetyl glucosamine (molar ratio 1.25 to 1) and ash. Study of sedimentation, viscosity and streaming birefringence indicates that native hyalu- ronic acid complex is a random-coil of nearly spherical shape and very large hydration. The interpretation of diffusion data is discussed. The bearing of the physical properties on the problem of its biological function is considered briefly.The study of the hyaluronic acid present in synovial fluid was undertaken with a view of throwing light upon its biological function in this and other sites.1-3 Its presence free in solution, and its overwhelming effect (compared with that of other components) on certain of the properties of the fluid, enabled a line to276 HYALURONIC A C I D COMPLEX be taken which has not often been followed, namely, of determining those pro- perties which are specific attributes of a particular material before attempting to separate it, and of using these as a control on procedures for separation. The investigation of the hyaluronic acid has also presented special problems in the interpretation of physicochemical data.This has led to the view that hyaluronic acid behaves as a random-coil in solution : 2 it is believed that this is the first native biological material to which such behaviour has been ascribed. EXPERIMENTAL PREPARATIoN.-After collection, ox synovial fluid was cleared by centrifuging and dialyzed against a buffer containing NaCl 0.2 M, KH2PO4 0.023 M, Na2HPOs 0.077 M. Dilutions were made with the same buffer. Separation of the hyaluronic acid was effected by use of the following methods : (i) Precipitation of " mucin ", by acidifying with acetic acid, followed by washing the precipitate with dilute acetic acid and water. The mucin was redissolved in water or buffer. (ii) Precipitation of hyaluronic acid by ethanol at - 20°, as described by Ropes et aZ.4 This was applied to redissolved mucin, or to native fluid, with or without addition of alkali.(iii) Electrophoresis : a sample was made for comparison with those made by other methods. (iv) Filtration through sintered glass of average pore diameter 1 to 1-5 p, followed by repeated (8-25 times) re-solution in water or 0-2 M NaCl and re-filtration. In this way the free protein could be completely removed (fig. 1). ANALYsIS.-The foIlowing methods were used : total nitrogen by micro Kjeldahl, dry weight, glucosamine,~ glucuronic acid,6 ash, sodium and calcium by flame photometry. As subsidiary methods, refractive increment 7 was used to measure total colloid and ultra- violet absorption to measure protein. Qualitative estimates of amino acids, after hydrolysis, were made by paper chromatography PHYSICAL METHoDs.-Sedimentatin in the ultracentrifuge.-The hyaluronic acid in synovial fluid gives a hyper-sharp boundary (fig.l), which is visible at concentration down to 7-5 mg/100 ml. The rate of sedimentation varies rapidly with concentration, the plot of l/s against c being linear at concentration less than 30 mg/100 ml. J. P. Johnston (personal communication) has shown that the rate of sedimentation of hyaluronic acid is affected by the presence of protein, through change of concentration of the hyaluronic acid by the boundary anomaly effect of Johnston and 0gston.s This has not seriously impaired the use of sedimentation rate as a control, since the ratio of the concentration of protein to that of hyaluronic acid in the ox fluid was always fairly constant.Comparison of the total refractive increment was made with that contributing to the boundaries of free protein.9 Diflusion.-Measurement by the Gouy method 10 proved diffcult, because. of the high viscosity; a specially designed cell was used.' Values for D around 1 x 10-7 were obtained for hyaluronic acid inixed with free protein, in the concentration range 200 to 20 mg/100 ml. Viscosity.-Routine control of the viscosity of samples was performed with an OstwaId viscometer of 2 ml capacity and water-time 11 sec. More extensive measurements were made with a Couette viscometer: this allowed measurements to be made over a wide range of concentration and up to velocity gradient 50 sec-1.The results obtained with the Couette viscometer are represented by lines of log ((qrel-- 1)/+} plotted against 4 at various values of the velocity gradient (4 = unsolvated volume fraction) : the divergence of these lines gives a measure of the degree of non-Newtonian viscosity.2$3 Streaming Birefringence.-Both the angle of isocline and the degree of birefringence of synovial fluid were measured over a range of concentration and velocity gradient.:! RESULTS AND DISCUSSION PREPARATIVE m-moDs.-The ability of a method to yield unaltered material was judged by comparing the results of sedimentation and viscosity measurements on solutions of separated material and on synovial fluid at the same concentrationsA B FIG. 1 .-Ultracentrifuge diagrams ; A, synovial fluid ; B, hyaluronic acid prepared by ult rafil t rat ion.[To ,fuce page 276277 of hyaluronic acid. Of these properties, the rate of sedimentation proved to be the least sensitive to treatment of the material, the most sensitive being the non- Newtonian viscosity.3 This showed that precipitation of mucin by acid and pre- cipitation of hyaluronic acid with ethanol both produce considerable change in the material (fig. 2, table 1). Filtration leaves all these properties unaffected, provided that it is not continued too long and that the protein is not completely removed. A . G . OGSTON AND J. E . STANIER '" I / ,2 ,3 ,4 ,5 ,6 ,7 ,8 ,9 y mdieui TABLE 1 .-VALUES OF LOG {(vre1-1)/4} FOR SYNOVIAL FLUID AND HYALURONC ACID SOLUTIONS: 4 = 7 x 10-4 fluid 29 K 36 A 37 A 53 54 55 redissolved much 35 K hyaluronic acid precipitated with ethanol : from mucin from alkaline fluid from neutral fluid hyaluronic acid by filtration log {(?lFe~-1)/4j at velocity gradient 0 4*6? 4-97 4.97 4-65 4.86 4-80 4.2 1 4.49 4-67 4-34 4.75 03 3.87 4.2 1 4-06 3.92 4*00 3.99 3.88 4.10 4.08 3-83 3.95 A log 0.80 0.76 0.9 1 0.73 0.86 0.81 0.33 0.39 0.59 0.51 0.80 CoMPOSITION.-F~tratiOn and electrophoresis remove free protein but leave a proportion of protein which is difficult to separate from the hyaluronic acid.Removal of this protein by any means causes the properties of the material to change. Analysis of the boundary diagrams in sedimentation and in electrophoresis, and273 HYALURONIC ACID COMPLEX of the Gouy fringes in diffusion, obtained with various materials, showed that a similar proportion of protein migrates as a complex with hyaluronic acid in each case (table 2).It is concluded that this protein forms an essential part of the hyaluronic acid complex in v i v a The carbohydrate contains glucuronic acid and glucosamine in a molar ratio approx. 1-25,3 contrary to the findings of Meyer, Smyth and Dawson.11 These components, together with ash make up 93 % of the dry weight (table 3). Thz distribution of amino acids in the protein closely rzsembles that in serum albumin. TABLE 2.-ESTIM4TION OF PROPORTION OF BOUND PROTEIN FROM ANALYSIS OF SEDIMENTA- TION, DIFFUSION AND ELECTROPHORESIS BOUNDARY DIAGRAMS method material % protein in complex sedimentation mucin redissolved 27 to 35 32 to 36 diffusion much redissolved 28 to 33 electrophoresis mucin redissolved 25 synovial fluid 27 hyaluronic acid, by filtration TABLE 3.--COMPOSITION OF HYALURONIC ACID COMPLEX g/ 100 g of complex glucosamine, as acetyl glucosamine 30-6 ylucuronic acid 35.9 protein 25-7 ash 6.8 99.0 2.5 3.3 93.2 - less 1 mole HzO/mole glucosamine less 1 mole HzO/mole glucuronic acid - - - MOLECULAR FORM.-A model of the molecule must be used in interpreting the results of dynamic measurements and three quantities have then to be determined : molecular weight, molecular volume (depending on degree of solvation), shape (this being expressed as axial ratio for an ellipsoidal model).Three independent measurements are therefore needed, unless the common assumption is made that solvation is unimportant. Various combinations of methods have been used.In the present case we have available the following data: (i) sedimentation rate extrapolated to zero concentration, (ii) variation of sedimentation rate with concentration (which can be interpreted by the “plug” theory of Fessler and Ogston I*), (iii) diffusion, (iv) intrinsic viscosity, (v) variation of intrinsic viscosity with velocity gradient, (vi) streaming birefringence. Of these, (i), (iv) and (v) were chosen 2 as the three independent measurements; it was decided not to use the diffusion data, because the diffusion coefficient varies both with the chemical potential and the frictional coefficient, making extrapolation to zero concentration uncertain. The non-dependence of the specific viscosity increment (vre1-1/$) on velocity gradient at zero concentration (v) (fig.2) showed that the non-Newtonian viscosity at higher concentration cannot be due to the particles being very elongated. A limit- ing value of 3 was estimated for the axial rati0.13~14 Use of (i) and (iv) then gave values of 1 -2 x lo7 for the molecular weight and 1.6 x 103 for the effective volume per g of dry material.3 The conclusion reached on this basis is sufficiently confirmed by considering the other data (table 4). Birefringence indicates the highest value of 14 for the axial ratio at a concentration 0.041 g/100 ml, but this estimated value is falling rapidly with concentration. The enormous value for the solvation (effective specific volume), which must be assumed if the axial ratio is small, is quite consistent with the view that the particle is a flexible chain.Current theories 15, 16 predict that a random chain of thisA . G . OGSTON AND J . E. STANIER 279 magnitude will have approximately the dimensions deduced above and that solvent entrained within it will be largely carried with the particle in dynamic measurements, so that the solid ellipsoidal model used is justified. TABLE 4.-ESTIMATES OF AXIAL RATIO OF HYALURONIC ACID COMPLEX method variation of intrinsic viscosity with velocity gradient << 3 variation of sedimentation with concentration - 4 angle of isocline < 5 birefringence < 14 VISCOSITY AND ELASTICITY.-The non-Newtonian viscosity observed at finite concentration must now be ascribed to an intertanglement of the particles.This would predict that solutions of hyaluronic acid should exhibit elasticity. Ogston et al.17 showed that this is the case, and later measurements (unpublished) have fully confirmed this finding (fig. 3). DEFusIoN.-The only data apparently inconsistent with the conclusions reached are those on diffusion. A single measurement 2 has indicated that solutions of hyaluronic acid have a highly abnormal osmotic pressure, which agrees approxi- mately with the predictions of Huggins’ 18 theory for the entropy of dilution. This FIG. 3.-Amplitude ratio against frequency curves obtained in a coaxial cylinder elasto- matter is being investigated further. Attempts have been made to extrapolate the diffusion to zero concentration, using Huggins’ theory and either the sedimentation rate or the viscosity (at zero velocity gradient) to measure the variation of frictional coefficient (fig.4). The extra- polation using the sedimentation rate gives an extrapolated value for D in approxi- mate agreement with the other data. It seems that the diffusion coefficient of material of this sort is difficult to interpret. BIOLOGICAL IMPLIcAnoNs.-It is known that material which resembles the hyaluronic acid of synovial fluid is widely distributed in the intercellular spaces of higher animals. Little is known of its concentration and physicochemical state in these sites. It is not necessary to suppose that the same properties are of im- portance wherever it occurs. The three striking properties which may be of biological significance are : (i) OSMOTIC PwssuRE.-This is large, considering its large molecular weight.Moreover, it may perhaps increase the osmotic contribution of free protein present in solution ; it is difficult otherwise to explain the high osmotic pressure of synovial fluids obtained by Ropes et al.19 This property might be important in determining the distribution of water in intercellular spaces. viscometer. A, synovial fluid ; B, buffer. These alternatives give widely different results.280 HYALURONIC ACID COMPLEX (ii) VISCOSITY.-A fluid containing a low concentration of a chain polymer will have a relatively high viscosity. MacConaill20 has pointed out the structural resemblance of joints to the Michel (" slipper ") type of thrust bearing3 The non-Newtonian viscosity would endow such a bearing with the property of with- FIG.4.-Variation of the dif- fusion coefficient with volume fraction of hyaluronic acid complex : 0 experimental values of Ogston and Stanier ;1 0 calculated from viscosity ; x calculated from sedimenta- tion constant. standing a pressure load nearly independent of its rate of movement over a con- siderable range of rate. (iii) ELASTICITY.-It seems unlikely that the elastic forces generated in joint fluid would be large enough to protect its bearing surfaces. In other sites, at possibly higher concentrations, the elasticity of hyaluronic acid might have some significance. The elasticity may, however, be merely a consequence of the physico- chemical structure which underlies the other properties mentioned. 1 Ogston and Stanier, Biochem. J., 1950,46, 364. 2 Ogston and Stanier, ibid., 1951, 49, 585. 3 Ogston and Stanier, ibid., 1952 (in press). 4 Ropes, Robertson, Rossemisl, Peabody and Bauer, Acta med. Scand., 1947, Suppl. 5 Johnston, Ogston and Stanier, Analyst, 1951, 76,88. 6 Ogston and Stanier, Biochem. J., 1951,49,491. 7 Cecil and Ogston, J. Sci. Insfr., 1951, 28, 253. 8 Johnston and Ogston, Trans. Faraduy Soc., 1946,42,789. 9 Cecil and Ogston, Biochem. J., 1948,43,592. 10 Coulson, Cox, Ogston and Philpot, Proc. Roy. SOC. A, 1948, 192, 382. 11 Meyer, Smyth and Dawson, J. Biol. Chem., 1939, 128, 319. 12 Fessler and Ogston, Trans. Farahy SOC., 1951,47, 667. 13 Mehl, Oncley and Simha, Science, 1940, 92, 132. 14Burgers, Verk. Akad. Wet. Amsf, 1938, 16a, no. 4. 15 Kuhn and Kuhn, Helv. chim. Acta, 1947,30, 1233. 16 Kirkwood and Riseman, J. Chem. Physics, 1948, 16, 565. 17 Ogston, Stanier, Toms and Strawbridge, Nature, 1950, 165, 571. 18 Huggins, J. Physic. Chern., 1942,46, 151. 19 Ropes, Bennett and Bauer, J. d i n . Invest., 1939, 18, 351. 20 MacConaill, J. Anat., 1931-32, 66, 210. 21 see, for example, Dodge and Thompson, Fluid Mechanics. 196,700.

ISSN:0366-9033    

DOI:10.1039/DF9531300275

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

GENERAL DISCUSSION 28 1 GENERAL DISCUSSION Mr. A. M. Woodin (Inst. of Ophthalmology, London) said: The properties of the mucoid from cornea may be relevant to the problem of the mucopolysaccharide- protein linkage in the particularly stable mucoids formed in connective tissues from mucopolysaccharides which contain ester sulphur. The mucoid from cornea (the phenol-insoluble mucoid described by Woodin 1) contains about 40 % protein. There is 2.2 % tyrosine, 0.9 % tryptophane and 0.5 % hydroxypyroline in the mucoid and it is not hydrolyzed by CI. welchii collagenase; from this, it can be concluded that the protein component is not a collagen derivative. The mucopolysaccharide component contains ester sulphur, galactose and hexosamine. The substance is electrophoretically homogeneous in the range pH 1.4-1 1.3.Extrapolation of the pH against mobility curve gives an isoelectric point at pH 1.0. The stability in this range excludes the possibility that carboxyl or a-amino groups are responsible for an electrostatic linkage. At pH 12 the mucoid dissociates nto two components and so some protein may be combined through its lysine side-chains. However, as the slow component at pH 12 is responsible for only 15 % of the total refraction, some protein is still combined above pH 12. If this protein is combined electrostatically, it must be by means of an interaction between the arginine and the bisulphate groups. Alternatively, it can be bound covalently. It will be difficult to distinguish an arginine-bisulphate interaction from a covalent bond since it will only be suppressed at a pH where labile covalent bonds will be hydrolyzed.Dissociationalso occurs on electrophoresis in the presence of 0.5 M guanidine hy- drochloride at pH 6. This reagent also reduces the mobility of the mucopoly sac- charide to half its normal value, indicating a strong guanidine-bisulphate interaction. Confirmation of the dissociating action of strong alkali and the amidinium ion is provided by osmotic pressure measurements. In salt solutions (NaCI 0-5 M, phosphate 0.05 M, pH 7) the mucoid has an average molecular weight of about 220,000. At pH 12 this is reduced to about 11O,OOO, while in the presence of 5 M urea the average molecular weight is about 80,000. The effects of alkali and urea are partly reversible. It is possible that analysis of the action of the amidinium ion will enable a distinction to be made between a covalent bond and an electrostatic arginine- bisulphate bond in the molecule.Interpretation of the action of urea and guanidine must await identification of the products of dissociation, but as a working hypoth- esis it can be suggested that, if there is an arginine-bisulphate linkage in the mole- cule, then the amidinium ion in high concentration would induce dissociation by competing with the guanidyl groups of the arginine. Such an action of urea has already been suggested by Tsao, Bailey and Adairz to account for the de- polymerizition of tropomyosin. It may also be noted that an arginine-bisulphate interaction would be a very stable linkage. Walker 3 has indicated that interactions involving amidinium ions and anions with alternative positions for the negative charge would be particularly stable through formation of a doublet ion pair.Such ion pair formation could well account for the stability of these mucoids. Mr. J. H. Verhoog (Leeuwarden, Netherlands) said: I should like to make some comment on the last part of Prof. Stacey’s paper, namely that in which he speaks of the so-called “ artificial ” mucoprotein from molasses. Prof. Stacey remarks that the complex is evidently some type of polymer formed by interaction of ducing groups of the sugar with the amino groups of the amino acids, but unlike 1 Woodin, Biochem. J., 1952, 51, 319. 2 Tsao, Bailey and Adair, Biochem. J., 1951, 49, 27. Walker, J. Chem. SOC., 1949, p.1996.282 GENERAL DISCUSSION other complexes of this type, he says, it can easily bc dissociated hy ucids. Now we are interested in the so-called Maillard reaction, that is the browning reaction which occurs when reducing sugars are heated with amino acids or proteins. We carried out a considerable number of experiments with amino acids or proteins and reducing sugars, but we never succeeded in recovering the sugars from the brown complexes formed by acid nor by alkaline hydrolysis. On the other hand, however, these complexes show reducing properties, e.g. against Luff's reagent, which is a specific sugar reagent. So I should like to ask Prof. Stacy if this " artificial " mucoprotein has any reducing properties which are specific for sugars. Moreover, is it not possible that this compound is a real mucoprotein or a decomposition product of a real mucoprotein rather than an " artificial " mucoprotein ? Prof.M. Stacey (Birmingham) said : In my experience all complexes formed by the Maillard reaction cannot be regenerated because the sugar portion has been transformed into combined furfural derivatives. It is to be expected that they will still show colour reactions for sugars since many of these reactions are dependent upon furfural derivative formation. It gives colour reactions for glucose, galactose, arabinose and xylose and for seven common amino acids and there are approximately three sugar residues to one amino acid. There is only one free hydroxyl group per sugar molecule. Nothing of this type has yet been described and no mucoprotein has yet been isolated from sugar juice.We are inclined to the view that it is a condensation product of an unusual kind. Simple N-glycosides can sometimes be regenerated and so can acylated sugars. Dr. R. Consden (Canadian Red Cross Memorial Hospital, Maidenhead) said : Fibrin was prepared by three methods. In the first preparation, citrated plasma from a healthy human subject was treated with thrombin and the clot worked up by dissolving in 1 % chloracetic acid and precipitating with trichloracetic acid according to the method of Lorand.1 Solution and precipitation were repeated a number of times, and samples at each stage removed for analysis. Following Lorand's second method,z the fibrin clot was dissolved in strong urea solution and reprecipitated by pouring into a large volume of 0.8 % saline.In the third method, fibrin was obtained by treatment of fibrinogen with thrombin. The fibrinogen was prepared from human citrated plasma by ether precipitation according to the method of Kekwick, Mackay and Record.3 After dissolving in saline and re- precipitating with ether twice more, it was over 90 % clottable by thrombin. All the above preparations liberated significant quantities of reducing sugars after hydrolysis. Reducing sugar estimations, after hydrolysis with N HCl at 100-110" for 3 h were carried out by the copper reduction method of Schaffer and Somogyi,4 and hexosamine was determined after hydrolysis with 4 N HCl at 100"-110" for 4 h, by the method of Elson and Morgan.5 The results are recorded in table 1 and all figures are based on material dried to constant weight at 100"-110".The molasses complex is strongly reducing to Fehlings solution. TABLE 1 .-ANALYSES OF HUMAN FIBRIN AND FIBRINOGEN fibrin * fibrinogen I 11 111 reducing sugar (as glucose, %) 3-8 4-8 4-5 4.6 - - 17.1 16.9 fibrin IT1 : from fibrinogen. 2 Lorand, Nature, 1950, 166, 694. hexosamine (%) 0.8 1.2 1.1 1.1 N (%I * fibrin I : chloracetic-trichloracetic acid preparation ; four times dissolved and precipitated. fibrin I1 : urea preparation. 1 Lorand, Nature, 1951, 167, 992. 3 Kekwick, Mackay and Record, Nature, 1946, 157, 629. 4 Shaffer and Somogyi, J. Biol. Chem., 1933, 100, 695. 5 Elson and Morgan, Biuchem. J., 1933, 27, 1824.GENERAL DISCUSSION 283 Crude fibrin as prepared from treatment of plasma with thrombin, gave very similar figures to those of the four times chloracetic-trichloracetic acid treated material (fibrin I), and none of the intermediate materials showed any significant difference.The lower reducing sugar and hexosamine figures of fibrin I compared with those of the other preparations are not unexpected since any occluded pro- tein would be reprecipitated with the fibrin by irichloracetic acid. Nevertheless the failure to lower these values by repeated treatments with chloracetic and trichloracetic acids suggests that there is a polysaccharide present, which is firmly bound to the protein. Fibrin I1 and 111, which are expected to be purer products than fibrin I have similar carbohydrate contents and these are both higher than that of fibrin 1.The polysaccharide of fibrin appears to be derived from the original fibrinogen, since little, if any, change occurs in carbohydrate content after the conversion. Ionophoresis on paper in borate buffer at pH 8.6 of the fibrin and fibrinogen hydrolysates as described by Consden and Stanier 1 revealed three sugar components having mobilities of hexosamine, mannose and galactose each being present in similar amounts. These sugars were confirmed by two-dimensional separations (chromatography at right-angles to the direction of ionophoresis), and subse- quent experiments have identified the hexosamine as glucosamine. Fibrin and fibrinogen therefore appear to be mucoproteins containing several percent of bound polysaccharide. In common with some other blood muco- proteins, the constituents of the polysaccharide are glucosmine, mannose and galactose.Thanks are due to Dr. L. Lorand for helpful advice. Prof. M. Stacey (Birmingham) said : Dr. Consden’s valuable contribution is welcomed and it is anticipated that the prosthetic groups in most serum proteins would have a very similar carbohydrate prosthetic group. These carbohydrates are of relatively low molecular weight and are of a highly-branched type. Prof. F. Haurowitz (Indiana Universify, U.S.A.) said: Since the blood group substances have the properties of carbohydrates and not of proteins, one has to assume that the surface of their macromolecules is formed by carbohydrate residues and not by amino acids. Prof. M. Stacey (Birmingham) said: The serological reactions of the blood group substances are dominated by the carbohydrate structures while the amino acid components are arranged in such a way that their presence can only be revealed by chemical methods after acid hydrolysis. The removal of L-fucose residues by acids from the ends of the carbohydrate chains profoundly alters the specific properties of the factors.Prof. W. T. Astbury and Dr. N. N. Saha (Leeds University) (communicated): At the suggestion of Dr. W. T. J. Morgan, who also kindly supplied the material, we have tried to find out something about the structure of hog gastric mucin A-substance (which is closely related cerologically to the human blood group A-factor) by the methods of X-ray and infra-red analysis. The substance gave only a very ill-defined X-ray diagram, with vague haloes round about 9-10A and 4-5A recalling protein side-chain and backbone spacings, and all attempts to improve it by inducing orientation were fruitless-except as regards another reflection at about 4.1 A, presumably arising from a fatty contaminant.This fatty component was so tightly held that it took prolonged boiling in benzene to remove it, and then, of course, the structure of the original A-substance was ruined. The interesting point, though, was that the diagram had changed into one of the type of denatured protein, indicating that peptide chains must have been present, not merely single amino acids. This inference was further strength- ened by the infra-red absorption spectrum of the original A-substance, which showed among other features strong bands at 1650 and 1550 cm-1 characteristic of peptide groups.1 Consden and Stanier, Nature, 1952, 169, 783.284 GENERAL DISCUSSION Dr. R. D. B. Fraser (King's College, London) said: From observations of the infra-red dichroism exhibited by oriented sheets of dried nucleoprotein Ambrose and Butler have concluded that the nucleic acid component has a configuration similar to that determined by us for isolated nucleic acid,l and that the protein component exists in the P-configuration with the chain axes lying parallel to those of the nucleic acid. We have also been interested in the protein configuration, but have preferred to work with material as near as possible to that found in the living cell. The head of the squid sperm contains highly oriented deoxyribose nucleoprotein, and Dr.Wilkins has succeeded in separating these heads from the tails and lining them up on silver chloride plates. The infra-red dichroism exhibited by these sperm heads was measured with an infra-red microscope 2 and the results are shown in the figure. The dishroisms 1000 1200 1400 1600 WAVE NUMBER cm.-' FIG. 1.-The infra-red dichroism exhibited by oriented sperm heads of Laligo. The dashed curve is measured with the electric vector vibrating parallel to the length of the sperm head and the full curve perpendicular. A is the observed absorption, B, the estimated contribution of nucleic acid, and C, the protein. of the absorption bands at 967, 1052 and 1087cm-1 confirm the suggestion of Ambrose and Butler that the nucleic acid component has the same configuration as that existing in extracted nucleic acid. As regards the protein configuration we have attempted to correct the di- chroism of the NH deformation band for the nucleic acid dichroism and find, as did Ambrose and Butler, a small parallel dichroism for this absorption band. N7e do not feel, however, that this provides reasonably conclusive evidence for the P-configuration as they suggest.Firstly, because the frequency of 1540 cm-1 is that which Elliott and Ambrose have shown to be characteristic of the ct- configuration,3 and secondly because we have recently observed an absorption band in tobacco mosaic virus of this frequency and dichroism where the inter- pretation was completely different,4 showing that observation of this band alone is not a conclusive test of configuration and chain direction.* Fraser, M. J. and R. D. B., Nature, 1951, 167, 759. 2 Fraser, R. D. B., Faraday Soc. Discussion, 1950, 9, 378. 3 Ambrose and Elliott, Proc. Roy. SOC. A, 195 1, 208, 75. Fraser, R. D. B., Nature (in press).GENERAL DISCUSSION 285 Dr. J. T. Edsall (Harvard University) said: I think that some comment is worth making on the unusual acid glycoprotein of human plasma which has been crystallized in our laboratory by K. Schmid. He has reported some of the findings in a preliminary note,l and a more extensive paper by Dr. Schmid is due to appear shortly. This protein is electrophoretically an or-globulin. It is extremely soluble in most solvents, and remains behind in the supernatant solution after the albumin, the y-globulin, the lipoproteins and all the major plasma constituents have been precipitated.The isoelectric point was found to be at pH 2.7 i 0.1 in phosphate buffers of ionic strength 0-1 ; the sedimentation constant is s20, W= 3.16s. The glycoprotein could be precipitated from alcoholic solutions in the cold in the presence of barium ions (0.02 M). It was crystallized as a lead salt ; the crystals, though small, were beautifully formed with very well-defined faces. The hexose content was estimated as 17 %, hexosamine as 12 %. To my know- ledge, this is the first glycoprotein of such high carbohydrate content that has been obtained in the crystalline state. Weimer, Mehl and Winzler2 have studied an electrophoretically homogeneous mucoprotein of extremely similar composition and isoelectric point.It is probable that their glycoprotein and Schmid’s are identical, but a few still unexplained discrepancies in composition remain between the two preparations. The investigators in both laboratories concluded that the concentration in plasma of this glycoprotein (or mucoprotein) amounted to about 0.7 % of the total protein. Prof. M. Stacey (Birmingham) said: A crystalline mucoprotein with high choline esterase activity had been isolated some years ago at Birmingham. It contained about 20 % of a carbohydrate residue. Mr. E. J. Ambrose (Chester Beatty Res. Inst., S.W.3) (communicated): In view of what Dr. Frazer had just told us, I would agree with him when he says that the infra-red dichroism of stretched sperm can be interpreted either as an arrangement of 19 chains, lying parallel to the direction of stretching, or of u chains lying perpendicular to this direction.I think that it is satisfactory that there is general experimental agreement about the small effects observed. I would suggest that for tobacco mosaic virus, which contains a small proportion of nucleic acid, the high frequency of the peptide C=O band may correspond to the a-form but that for sperm the low frequency of the peptide C=O band (164Ocm-1) rather suggests the presence of a /%form, which is stabilized by nucleic acid. Dr. J. E. Stanier (Oxford University) said: We agree that the viscosity of solutions of hyaluronic acid depends both on the method of preparaton and on the source of the material.For this reason the order of viscosities given by Dr. Meyer for different sources is not always the correct one. Fig. 2 shows that our preparation of hyaluronic acid complex from synovial fluid (equal in viscosity to the native fluid at equivalent concentration) has a viscosity similar to that of the native (i.e. untreated) pleural tumour fluid of Meyer and Chaffee.3 The umbilical cord preparation of Jeanloz and Forchielli 4 which contains little or no protein is less viscous at equivalent concentration, and that of Meyer 5 less viscous still. This suggests that the amount of treatment which the material receives, and especially the amount of protein which is removed, rather than the source of material, is the important factor in determining the viscosity.We have no definite suggestion about how the protein maintains the viscosity of our material. We can only say that removal of protein to less than about 25 % of the whole causes a fall in the viscosity, which is not restored by readdition 1 Schmid, J. Amer. Chem. SOC., 1950, 72, 2815 ; 1953, 75, 60. 2 Weimer, Mehl and Winzler, J. Biol. Chem., 1950, 185, 561. 3 Meyer and Chaffee, J. Biochem. Soc., 1940, 133, 83. 4 Jeanloz and Forchielli, J. Biochem. Soc., 1950, 186, 495. 5 Meyer, J. Biochem. Soc., 1948, 176, 993. Smith, Brown, Weimer and Winzler, J. Biol. Chem., 1950, 185, 569.286 GENERAL DISCUSSION of the protein. On the other hand we have made non-viscous preparations containing the full amount of protein. Dr. Meyer’s suggestion, that the high viscosity of our preparations could be due to interaction between hyaluronic acid particles rather than to any part played by the protein, cannot explain the high intrinsic viscosity, for there can be no interaction at zero concentration.Dr. A. G. Ogston (Oxford University) said: Divergence between our analytical results and Meyer’s are understandable, because different materials have been studied. Meyer’s products are obtained by more or less vigorous treatment and may represent a component or structure common to all forms of “ hyaluronic acid ”. These, as they occur in nature, are more likely to be a family of com- plexes than a single substance. We have been at pains to study one member of this family as nearly as possible in its native state.Electrophoretic mobilities alone, as has been shown in a number of systems, are not sufficient to identify chemical constitution. The neutral sugar and the high glucuronic acid contents of our samples cannot be due to the protein present in them. Neutral sugars have been shown by Consden still to be present in a sample whose protein content has been considerably reduced and the glucuronic acid/glucosamine ratio tends to increase, if anything, as protein is removed. Dr. R. Consden (Canadian Red Cross Memorial Hospital, Maidenhead) said ; Various samples of hyaluronic acid, after mild hydrolysis, have been examined by ionophoresis on paper in borate buffer 1 and by paper chromatography. In addition to the expected hexosamine and uronic acid, all gave a polysaccharide, which was relatively resistant to the hydrolytic conditions employed, and some gave small but significant amounts of neutral hexoses.The ox synovial fluid hyaluronate prepared by ultraatration, described by Ogston and Stanier, gave galactose and mannose. These appeared to be present in similar amounts (galactose perhaps somewhat greater in quantity than mannose), * Consden and Stanier, Nature, 1952, 169, 783.GENERAL DISCUSSION 287 and each of the order of 3-5 % of the hyaluronate. These two sugars could thus account for the balance of about 7 % in Ogston and Stanier's analytical figures. A sample from which much of the protein had been eliminated by precipitating the ultrafilter residue with alcohol at - 20" from an alkaline solution 1 still con- tained mannose and galactose in similar amounts to those of the other preparations.Human umbilical cord hyaluronate preparations have also been found to give neutral sugars on hydrolysis, but in these cases they were galactose and glucose (several % of each), but little or no mannose. Some of these preparations were protein free, e.g. a sample (furnished by Dr. H. J. Rogers) prepared by the method of Hadidian and Pirie.2 None of these preparations had involved the use of proteolytic enzymes, and all had high relative viscosities. On the other hand, some samples of hyaluronate from various sources, which had received proteolytic enzyme treatment during their preparation and which were partly depolymerized, were found to contain much less or no neutral sugars. Thus, hyaluronate from mesothelioma fluid,3 supplied by Prof.B. Blix, contained only a trace of galactose. Papain had been used in this preparation. Human synovial fluid hyaluronate, which had received trypsin treatment during its preparation contained traces of galactose, but it should be pointed out that this hyaluronate was obtained from pooled rheumatoid fluid and may therefore differ from hyaluronate in normal fluid. The partial hydrolytic product found in hydrolysates of all hyaluronate samples gives with the aniline hydrogen phthalate reagent a characteristic yellow fluorescence in ultra-violet light, is positive to ninhydrin and to the Elson-Morgan reagent 4 for amino sugars. It is amphoteric and mobility experiments on paper suggest an isoelectric point of about 4 4 and pK,, about 7-5 for the basic group.Its maximum mobility towards the anode is about 0-8 that of leucylphenylalanine, suggesting a molecular weight somewhat higher than 278. A crystalline sub- stance having these properties was easily prepared by direct hydrolysis of hya- luronate (heating with N H2SO4 under reflux for 3 h and concentrating the hltrate after neutralization with baryta). Further hydrolysis of this substance liberates hexosamine and uronic acid, and it would therefore appear to be identical with the disaccharide, hyalobiuronic acid described by Rapport, Weissmann, Linker and Meyer.5 A similar substance has been observed in paper ionophoresis of a hydrolysate of chondroitin sulphate from human costal cartilage. In this case, of course, the amino-sugar residue would be galactosamine, not glucosamine. Galactose was also present in this hydrolysate. The occurrence of small amounts of neutral sugars in hydrolysates of hya- luronate might be due to contamination with other polysaccharides. For instance glucose in umbilical cord hyaluronate might conceivably arise from glycogen, and the galactose from blood group substances. Mannose and galactose in the ox synovial fluid hyaluronate could possibly be associated with the protein moiety of the protein + polysaccharide complex, but since these sugars are present in the same amounts after removal of much of the protein, they might indeed be part of the carbohydrate complex rather than that of the associated protein. The co-operation of Prof. Blix and Dr. Jean Stanier, Ogston and Rogers in these investigations is gratefully acknowledged. Prof. M. Stacey (Birmingham) said: We have obtained many protein-free preparations of hyaluronic acid, quite free also from sugars other than glucosamine and glucuronic acid. The last traces of protein were often tenaciously held though Dr. Consden's new observations concerned the blood group factors which can contaminate almost every type of tissue polysaccharide. 1 Ogston and Stanier, Biochern. J., 1952, 52, 149. 2 Hadidian and Pirie, Biochem. J., 1948, 42, 260. 3 Blix, Acta Soc. Med. Ups., 1951, 57, 47; Acta Chem. Scand., 1951, 5, 981. 4 Partridge, Biochem. J., 1948,42, 238. 5 Rapport, Weissmann, Linker and Meyer, Nature, 1951, 168, 996.

ISSN:0366-9033    

DOI:10.1039/DF9531300281

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

AUTHOR INDEX * Adair, G. S., 49. Alameri, Eva H., 176. Ambrose, E. J., 261, 285. Astbury, W. T., 95, 166, 283. Ayers, Janet, 189. Bailey, K., 145. Barbu, E., 48, 77, 95. Batchelor, W. H., 176. Botts, Jean., 125. Brand, B. P., 236. Butler, J. A. V., 231, 241, 261. Cohn, Edwin J., 176. Consden, R., 46, 282, 283. Cunnhgham, Jr., Leon W., 58. Doty, Paul, 51, 94. Durrum, E. L., 47. Edsall, John T., 9, 166, 231, 285. Falconer, J. S., 40. Fleming, Muriel, 217. Flodin, P., 46. Fraser, R. D. B., 284. Gilbert, G. A,, 159, 239. Goldberg, Richard J., 224, 244. Gorter, E., 205, 240, Green, N. Michael, 58. Gustavson, K. H., 161, 234. Hamer, D., 238. Hamoir, G., 116, 164, 165, 168. Harrap, B. S., 197. Haurowtiz, F., 95, 96, 243,283. Hill, Terrell L., 132. Horst, M. G. ter, 163. Hyde, A.J., 96. Iball, J., 242. Isliker, Henry C., 176. Jenden, D. J., 40. Johnson, P., 98, 161, 164. Joly, M., 77, 97. Jordan, D. O., 217. Klotz, Irving M., 189. McGavin, S., 242. Malstrom, Bo G., 234. Marrack, J. R., 243. Meyer, K., 240, 271. Morales, Manuel, 125, 167. Mould, D. L., 47. Murray Luck, J., 235. Myers, George E., 51. Naismith, W. E. F., 95, 98. Nanninga, L., 49, 95, 161, 205, 240. Neurath, Hans, 58, 93. Ogston, A. G., 48, 165, 275, 286. Pauling, Linus, 170. Pedersen, K. O., 49. Perlmann, Gertrude E., 67. Pethica, B. A., 237. Petri, Miss E. M., 151. Pope, C. G., 241. Robert, B., 49, 232, 236. Robert, L., 48, 232. Saha, N. N., 283. Schmid, Karl, 176. Schoberl, A., 231. Schulman, J. H., 197. Seibert, Florence B., 251. Shulman, Sidney, 109. Stacey, M., 245, 282, 283, 285, 281. Stanier, J. E., 276, 285. Staverman, A. J., 151, 163. Surgenor, Douglas M., 176. Taylor, D. B., 40, 50. Tietze, Frank, 58. Tiselius, Arne, 29. Tsao, T.-C., 145. Verhoog, H. J., 281. Walton, K., 237. Wassermann, A., 94, 164. Weber, G., 33. Williams, J. W., 224, 241, 244. Woodin, A. M., 281. * The references in heavy type indicate paper submitted for discussion. 288

ISSN:0366-9033    

DOI:10.1039/DF9531300288

出版商:RSC    

年代:1953

数据来源: RSC