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Residual Proteins of the Vitreous*
RESIDUAL P R O T E I N S O F T H E VITREOUS* ANITA A.
SURAN, M.S.,
AND W.
K.
MCEWEN,
PH.D.
San Francisco, California
Most of the work demonstrating different components of the residual protein of the vitreous has been either microscopic or elec tron micrographic delineation of different types of fibers in the vitreous body.1"5 An alytic differentiation of residual protein com ponents has been done by several investiga tors. Balazs6 showed that the residual pro tein is not distributed uniformly throughout the vitreous. Woodin and Boruchoff7 and Gross, Matolsty, and Cohen8 reported analy tic data for various fractions of the residual protein. Dische and Zelmanes9 analyzed the carbohydrate components of acid extracts of the residual proteins. Proteolytic enzyme analysis has been used by Bembridge, Craw ford, and Pirie 4 and by Balazs and Varga.10 This paper details some experimental work on the analysis of the residual protein of the vitreous. There are two points of inter est in addition to the partial analysis of the residual protein of the vitreous. One is the strong hygroscopicity of the residual pro tein, the other is an interesting mechanism of action of lytic enzymes on collagen.
distilled water with toluene and carbon tetrachloride as protection against bacterial con tamination, (2) filtration of this washed ma terial through loose nylon stocking (60 mesh, 15 denier), and (3) bringing to constant weight. This constancy is with respect both to drying and to hyaluronidase. The centrifugation method is by high speed (^100,000 X G) centrifugation to spin down the re sidual protein and is followed by several washings and finally drying to constant weight. The same weight of residual protein is obtained with or without hyaluronidase treatment. Both methods yield an average of 10.5 mg. percent for the residual protein. This ma terial, like many proteins, is hygroscopic. Because it is finally divided, its rate of watet absorption'is rapid. It will rapidly increase to 113 percent of its weight in 20 minutes when exposed to laboratory air and will reach equilibrium at about 120 percent of its dry weight. It will be noted that kangaroo tail tendon (a typical collagen) is also hygro scopic and will slowly increase in weight to 129 percent of its dry weight when exposed to air over a 16-hour period.
METHODS AND MATERIAL I. PREPARATION OF RESIDUAL PROTEIN
The vitreous were prepared by careful dissection and, after rinsing thoroughly and removing any extraneous tissues, the portion two to four mm. posterior to the lens was used. This excluded the zonular fibers. The residual protein of the vitreous was isolated by either of two methods, filtration or centrifugation. The filtration method consisted of (1) washing the vitreous for about a three-week period in frequent changes of * From the Francis I. Proctor Foundation for Research in Ophthalmology, University of Cali fornia Medical Center. This investigation was sup ported in part by Research Grant B-180 from the National Institute of Neurological Diseases and Blindness of the National Institutes of Health, De partment of Health, Education, and Welfare.
II. ENZYMATIC HYDROLYSIS
Wyeth's hyaluronidase* was used in a concentration of 10,000 TRU of hyaluroni dase per 100 ml. of a 0.1 M acetate buffer (pH 5.4) in 0.15 M NaCl. One ml. of this buffer was used with each mg. of material to be hydrolyzed. Tryptar, a brand of trypsin, was used as a 100 mg. percent solution in 0.2 M borate buffer (pH 7.8). One mg. of enzyme was used to hydrolyze from two to three mg. of material. Collagenase* from Clostridium welchii was
338
t Courtesy of Joseph Seifter, Wyeth, Incor porated. * Courtesy of Wellcome Research Laboratories, Kent, England.
RESIDUAL PROTEINS OF VITREOUS used as a 100 mg. percent solution in the same buffer as was used for the trypsin. One mg. of enzyme was used with one mg. of sub strate. Most hydrolyses were carried out at 37°C. for 16 hours. III.
TABLE 1 P E R C E N T REMAINING AFTER TRYPSIN DEGRADATIONS (COMPOSITE DATA)
Number of Trypsin Attacks
ANALYSES
Hydroxyproline was determined by a slight modification of the method of Martin and Axelrod, 11 hexosamine by the method of Boas,12 and nitrogen following the method of Grunbaum, Shaffer, and Kirk. 13 Both cir cular and two dimensional chromatograms were used. The solvent for circular chromatography and for the first dimension was n-propanol, H 2 0 , NH 4 OH in proportion of 67:32:1. The second dimension was chromatographed with a solvent developed during the course of this work and is a mixture of benzene, ethyl alcohol (95 percent), and onepercent acetic acid in the proportions 20:85:30. This solvent seemed more applicable than the usual phenol, collidine, leutidine types of solvents under conditions where no special ventilation was available. Good resolutions were obtained except in the case of methionine and valine which give one spot and a procedure is available for separating these two amino acids. The material on the chromatogram was visualized by the usual sprays: (a) Ninhydrin for amino acids (fol lowed by Cu ( N 0 3 ) 2 for preservation, (b) Aniline phthalate for reducing sugars, and (c) Isatin chiefly for proline and hydroxy proline. RESULTS AND DISCUSSION
It is generally believed that trypsin will attack many denatured proteins. It has only weak action toward undenatured proteins.1* The slow attrition of collagen by trypsin is shown in Table 1. It may be seen that when kangaroo tail tendon is subjected to repeated attacks of trypsin, with drying to constant weight between attacks, there is a slow, gradual loss of substrate.
339
1 2 3 4
Dried Intermittently Collagen (Kangaroo tail tendon) 93 91 89
■
Kept Wet
Residual Protein
Residual Protein
65 51 44 40
58 64 64
When residual protein is subjected simi larly to trypsin attack there is an early, large weight loss, quite different from the reaction of collagen. After this initial drop in weight there is a continual weight loss that is similar to, but larger in amount than, when kangaroo tendon collagen is used as a substrate. It will also be noted that when the residual protein is kept wet there is little of this continual loss. The interpretation of these results is that there is a component of the residual protein which is more susceptible to trypsin attack than is the remainder of the residual protein. The re mainder has slightly greater sensitivity to continuing trypsin attack than kangaroo tail tendon, probably by reason of its greater area. This difference in susceptibility to tryp sin may be a rate phenomenon rather than a qualitative difference between the two sub strates. Elimination of intermittent drying to constant weight appears to eliminate most of the gradual loss in weight due to trypsin activity with both substrates, which suggests that desiccation causes partial denaturation. The material which is easily hydrolyzed by trypsin is called the trypsin attackable fraction and the remainder from this initial treatment is called the trypsin resistant frac tion. The chemical analyses of these unpurified fractions are useful in showing trends and general characteristics but are not to be interpreted precisely. Table 2 gives the analyses of the whole residual protein and the trypsin resistant material. Our residual
340
A N I T A A. S U R A N A N D W. K. M c E W E N TABLE 2 ANALYSIS OF FRACTIONS OF RESIDUAL PROTEIN
Residual Protein Preparation Trypsin Resistant Material
Amount (mg. %)
%N
% Hydroxyproline
Hexosamine*
10.5 6.4
11.5 14.7
11.3 12.5
4.0 0.3
Sugarst + + + + + +
* Analysis courtesy Mr. Henry Dix, Department of Pathology. t Qualitative data from chromatography.
protein results are close to those of Woodin.7 We show a slightly higher hydroxyproline content. The analysis of the trypsin resistant fraction is similar both to Pirie's 15 precipi tated protein and to Matolsty's Vitrosin.8 It appears that the hexosamine has been largely hydrolyzed but that other carbohydrate re mains with this fraction. Although this re sistant fraction appears analytically to be more collagenlike than the initial residual protein, it still differs considerably from the typical collagen analysis. From these data it is apparent that the material hydrolyzed by trypsin contains the hexosamine, considerable hydroxyproline, and accounts for some 40 percent of the material. The trypsin resistant fraction of the re sidual protein may be further degraded by a combination of repeated drying, trypsin, and hyaluronidase treatments or by collage nase. The hydrolysis is not complete as there is a small residue of 1.0 to 2.5 mg. percent. It is of interest to note that the hydrolysis of both collagen and the trypsin resistant fraction by drying and trypsin may be in creased by treatment with hyaluronidase. This is shown in Table 3. The increased breakdown of both proteins after hyaluroni dase treatments is another example of the importance of sugar in the collagen molecule as previously shown by Banga16 and Jack son.17 The residue from collagenase treatments was hydrolyzed with acid and chromatographed. There was no proline or hydroxy-
TABLE 3 E F F E C T OF HYALURONIDASE ON SUCCESSIVE TRYPSIN DEGRADATIONS
% Remaining Treatment
Trypsin 1 Trypsin 2 Trypsin 3 Hyaluronidase 1 Trypsin 4 Trypsin 5 Trypsin 6
Residual Protein
Collagen (Kangaroo tail tendon)
Dried Inter mittently
Dried Inter mittently
94 90
93 91 89 81 77 72 62
60 36
Kept Wet
85
proline although the other amino acids were present in this fraction. SUMMARY
1. The residual protein of bovine vitre ous may be differentiated enzymatically into three components: a. A trypsin hydrolyzable fraction which accounts for approximately 35 to 40 per cent of the residual protein. It contains hy droxyproline and hexosamine. b. A trypsin resistant fraction which is rapidly hydrolyzed by collagenase and may be slowly attacked by repeated trypsin, hy aluronidase, and drying treatments. This fraction is suggestive of collagen. c. A residue resistant to the above enzyme treatments. It contains amino acids but no proline or hydroxyproline. University of California Medical Center (22).
RESIDUAL PROTEINS OF VITREOUS
341
REFERENCES
1. Matolsty, A. G., Gross, J., and Grignolo, A.: A study of the fibrous components of the vitreous body with the electron microscope. Proc. Soc. Exper. Biol. and Med., 76:857, 1951. 2. Matolsty, A. G.: A study of the structural protein of the vitreous body. J. Gen. Physiol., 36:39, 1952. 3. Grignolo, A.: Fibrous components of the vitreous body. Arch. Ophth., 47 :760, 1952. 4. Bembridge, B. A., Crawford, G. N. C, and Pirie, A.: Phase contrast microscopy of the animal vitreous body. Brit. J. Ophth., 36:131, 1952. 5. Cagianut, B., and Wunderly, C.: Protein studies in the human vi'reous body. Brit. J. Ophth., 37: 229, 1953. 6. Balazs, Endre A.: Structure of the vitreous gel. A d a XVII Intemat. Cong. Ophth., pp. 1019-1024, 1954. 7. Woodin, A. M., and Boruchoff, S. A.: Particle interaction in solutions derived from ox vitreous humor. J. Biophysic. Biochem. & Cytol., 1:489, 1955. 8. Gross, J., Matolsty, A. G., and Cohen, C.: Vitrosin, a member of the collagen class. J. Biophysic. Biochem. & Cytol., 1:215, 1955. 9. Dische, Z., and Zelmanes, G.: Polysaccharides of the vitreous fibers. Arch. Ophth., 54:528, 1955. 10. Varga, L., and Balazs, E. A.: Studies on the structure of the vitreous body: II. Electrophoretic and sedimentation properties. Am. J. Ophth., 38 :29, 1954. 11. Martin, C. J., and Axelrod, A. E.: A modified method for the determination of hydroxyproline. Proc. Soc. Exper. Biol. & Med., 83:461, 1953. 12. Boas, N. F . : Method for the determination of hexosamine in tissues. J. Biol. Chem., 204:553, 1953. 13. Grunbaum, B. W., Shaffer, F. L., and Kirk, P. L.: Kjeldahl determination of nitrogen with sealed tube digestion. Anal. Chem., 24:1487, 1952. 14. The Enzymes, (eds. Sumner, J. B., and Myrback, Karl) : New York, Acad. Press, 1951, p. 847. 15. Pirie, A., Schmidt, G., and Waters, J. W.: Ox vitreous humor: I. The residual protein. Brit. J. Ophth., 32:321, 1948. 16. Banga, I., and Balo, J.: Studies on the elastolysis of elastin and collagen. Acta Physiol. Acad. Sci. Hung., 6:235, 1954. Cited in Chem. Abts., 49 :8337, 1955. 17. Jackson, D. S.: Chondroitin sulfate as a factor in the stability of tendon. A chapter in Nature and Structure of Collagen, (ed. Randall, J. T.) New York, Acad. Press, Inc., 1953.
DISCUSSION DR. ZACHARIAS DISCHE (New York) : I would
like to say a few words concerning Dr. McEwen's very interesting paper, because I think the criticism by Dr. Balazs, while it is completely justified as far as the loose use of the words "residual protein" is concerned, may do a certain injustice to the paper and the results Dr. McEwen has shown. It is perfectly true that the residual protein is a mixture which could be brought about by contami nation, but in addition I think we must not dismiss the idea that it is a mixture by structural co-ordina tion of various things. It is difficult to explain Dr. McEwen's results only by simple combination, be cause almost 40 percent of the total protein goes off on the trypsin digestion. One possibility to reconcile that with our con cepts of the chemical nature of collagen is that in
this residual protein there is a structural co-ordina tion of certain small collagen fibers by very small amounts of highly active high polymers of the kind of hyaluronic acid. As soon as the latter are re moved, the structure disintegrates and perhaps goes simply into solution. I am not quite sure how Dr. McEwen determined the tryptic effects on his ma terial. Perhaps this would be a crucial thing to answer. We found in our residual protein, which was taken from the center of the vitreous, that we can not remove certain traces of hyaluronic acid even by the most exhaustive washing and combined homogenization and treatment with hyaluronidase. We had the impression that there may be a structural combination between certain collagen fibers and hyaluronic acid which may be of major significance.
SURAN, M.S.,
AND W.
K.
MCEWEN,
PH.D.
San Francisco, California
Most of the work demonstrating different components of the residual protein of the vitreous has been either microscopic or elec tron micrographic delineation of different types of fibers in the vitreous body.1"5 An alytic differentiation of residual protein com ponents has been done by several investiga tors. Balazs6 showed that the residual pro tein is not distributed uniformly throughout the vitreous. Woodin and Boruchoff7 and Gross, Matolsty, and Cohen8 reported analy tic data for various fractions of the residual protein. Dische and Zelmanes9 analyzed the carbohydrate components of acid extracts of the residual proteins. Proteolytic enzyme analysis has been used by Bembridge, Craw ford, and Pirie 4 and by Balazs and Varga.10 This paper details some experimental work on the analysis of the residual protein of the vitreous. There are two points of inter est in addition to the partial analysis of the residual protein of the vitreous. One is the strong hygroscopicity of the residual pro tein, the other is an interesting mechanism of action of lytic enzymes on collagen.
distilled water with toluene and carbon tetrachloride as protection against bacterial con tamination, (2) filtration of this washed ma terial through loose nylon stocking (60 mesh, 15 denier), and (3) bringing to constant weight. This constancy is with respect both to drying and to hyaluronidase. The centrifugation method is by high speed (^100,000 X G) centrifugation to spin down the re sidual protein and is followed by several washings and finally drying to constant weight. The same weight of residual protein is obtained with or without hyaluronidase treatment. Both methods yield an average of 10.5 mg. percent for the residual protein. This ma terial, like many proteins, is hygroscopic. Because it is finally divided, its rate of watet absorption'is rapid. It will rapidly increase to 113 percent of its weight in 20 minutes when exposed to laboratory air and will reach equilibrium at about 120 percent of its dry weight. It will be noted that kangaroo tail tendon (a typical collagen) is also hygro scopic and will slowly increase in weight to 129 percent of its dry weight when exposed to air over a 16-hour period.
METHODS AND MATERIAL I. PREPARATION OF RESIDUAL PROTEIN
The vitreous were prepared by careful dissection and, after rinsing thoroughly and removing any extraneous tissues, the portion two to four mm. posterior to the lens was used. This excluded the zonular fibers. The residual protein of the vitreous was isolated by either of two methods, filtration or centrifugation. The filtration method consisted of (1) washing the vitreous for about a three-week period in frequent changes of * From the Francis I. Proctor Foundation for Research in Ophthalmology, University of Cali fornia Medical Center. This investigation was sup ported in part by Research Grant B-180 from the National Institute of Neurological Diseases and Blindness of the National Institutes of Health, De partment of Health, Education, and Welfare.
II. ENZYMATIC HYDROLYSIS
Wyeth's hyaluronidase* was used in a concentration of 10,000 TRU of hyaluroni dase per 100 ml. of a 0.1 M acetate buffer (pH 5.4) in 0.15 M NaCl. One ml. of this buffer was used with each mg. of material to be hydrolyzed. Tryptar, a brand of trypsin, was used as a 100 mg. percent solution in 0.2 M borate buffer (pH 7.8). One mg. of enzyme was used to hydrolyze from two to three mg. of material. Collagenase* from Clostridium welchii was
338
t Courtesy of Joseph Seifter, Wyeth, Incor porated. * Courtesy of Wellcome Research Laboratories, Kent, England.
RESIDUAL PROTEINS OF VITREOUS used as a 100 mg. percent solution in the same buffer as was used for the trypsin. One mg. of enzyme was used with one mg. of sub strate. Most hydrolyses were carried out at 37°C. for 16 hours. III.
TABLE 1 P E R C E N T REMAINING AFTER TRYPSIN DEGRADATIONS (COMPOSITE DATA)
Number of Trypsin Attacks
ANALYSES
Hydroxyproline was determined by a slight modification of the method of Martin and Axelrod, 11 hexosamine by the method of Boas,12 and nitrogen following the method of Grunbaum, Shaffer, and Kirk. 13 Both cir cular and two dimensional chromatograms were used. The solvent for circular chromatography and for the first dimension was n-propanol, H 2 0 , NH 4 OH in proportion of 67:32:1. The second dimension was chromatographed with a solvent developed during the course of this work and is a mixture of benzene, ethyl alcohol (95 percent), and onepercent acetic acid in the proportions 20:85:30. This solvent seemed more applicable than the usual phenol, collidine, leutidine types of solvents under conditions where no special ventilation was available. Good resolutions were obtained except in the case of methionine and valine which give one spot and a procedure is available for separating these two amino acids. The material on the chromatogram was visualized by the usual sprays: (a) Ninhydrin for amino acids (fol lowed by Cu ( N 0 3 ) 2 for preservation, (b) Aniline phthalate for reducing sugars, and (c) Isatin chiefly for proline and hydroxy proline. RESULTS AND DISCUSSION
It is generally believed that trypsin will attack many denatured proteins. It has only weak action toward undenatured proteins.1* The slow attrition of collagen by trypsin is shown in Table 1. It may be seen that when kangaroo tail tendon is subjected to repeated attacks of trypsin, with drying to constant weight between attacks, there is a slow, gradual loss of substrate.
339
1 2 3 4
Dried Intermittently Collagen (Kangaroo tail tendon) 93 91 89
■
Kept Wet
Residual Protein
Residual Protein
65 51 44 40
58 64 64
When residual protein is subjected simi larly to trypsin attack there is an early, large weight loss, quite different from the reaction of collagen. After this initial drop in weight there is a continual weight loss that is similar to, but larger in amount than, when kangaroo tendon collagen is used as a substrate. It will also be noted that when the residual protein is kept wet there is little of this continual loss. The interpretation of these results is that there is a component of the residual protein which is more susceptible to trypsin attack than is the remainder of the residual protein. The re mainder has slightly greater sensitivity to continuing trypsin attack than kangaroo tail tendon, probably by reason of its greater area. This difference in susceptibility to tryp sin may be a rate phenomenon rather than a qualitative difference between the two sub strates. Elimination of intermittent drying to constant weight appears to eliminate most of the gradual loss in weight due to trypsin activity with both substrates, which suggests that desiccation causes partial denaturation. The material which is easily hydrolyzed by trypsin is called the trypsin attackable fraction and the remainder from this initial treatment is called the trypsin resistant frac tion. The chemical analyses of these unpurified fractions are useful in showing trends and general characteristics but are not to be interpreted precisely. Table 2 gives the analyses of the whole residual protein and the trypsin resistant material. Our residual
340
A N I T A A. S U R A N A N D W. K. M c E W E N TABLE 2 ANALYSIS OF FRACTIONS OF RESIDUAL PROTEIN
Residual Protein Preparation Trypsin Resistant Material
Amount (mg. %)
%N
% Hydroxyproline
Hexosamine*
10.5 6.4
11.5 14.7
11.3 12.5
4.0 0.3
Sugarst + + + + + +
* Analysis courtesy Mr. Henry Dix, Department of Pathology. t Qualitative data from chromatography.
protein results are close to those of Woodin.7 We show a slightly higher hydroxyproline content. The analysis of the trypsin resistant fraction is similar both to Pirie's 15 precipi tated protein and to Matolsty's Vitrosin.8 It appears that the hexosamine has been largely hydrolyzed but that other carbohydrate re mains with this fraction. Although this re sistant fraction appears analytically to be more collagenlike than the initial residual protein, it still differs considerably from the typical collagen analysis. From these data it is apparent that the material hydrolyzed by trypsin contains the hexosamine, considerable hydroxyproline, and accounts for some 40 percent of the material. The trypsin resistant fraction of the re sidual protein may be further degraded by a combination of repeated drying, trypsin, and hyaluronidase treatments or by collage nase. The hydrolysis is not complete as there is a small residue of 1.0 to 2.5 mg. percent. It is of interest to note that the hydrolysis of both collagen and the trypsin resistant fraction by drying and trypsin may be in creased by treatment with hyaluronidase. This is shown in Table 3. The increased breakdown of both proteins after hyaluroni dase treatments is another example of the importance of sugar in the collagen molecule as previously shown by Banga16 and Jack son.17 The residue from collagenase treatments was hydrolyzed with acid and chromatographed. There was no proline or hydroxy-
TABLE 3 E F F E C T OF HYALURONIDASE ON SUCCESSIVE TRYPSIN DEGRADATIONS
% Remaining Treatment
Trypsin 1 Trypsin 2 Trypsin 3 Hyaluronidase 1 Trypsin 4 Trypsin 5 Trypsin 6
Residual Protein
Collagen (Kangaroo tail tendon)
Dried Inter mittently
Dried Inter mittently
94 90
93 91 89 81 77 72 62
60 36
Kept Wet
85
proline although the other amino acids were present in this fraction. SUMMARY
1. The residual protein of bovine vitre ous may be differentiated enzymatically into three components: a. A trypsin hydrolyzable fraction which accounts for approximately 35 to 40 per cent of the residual protein. It contains hy droxyproline and hexosamine. b. A trypsin resistant fraction which is rapidly hydrolyzed by collagenase and may be slowly attacked by repeated trypsin, hy aluronidase, and drying treatments. This fraction is suggestive of collagen. c. A residue resistant to the above enzyme treatments. It contains amino acids but no proline or hydroxyproline. University of California Medical Center (22).
RESIDUAL PROTEINS OF VITREOUS
341
REFERENCES
1. Matolsty, A. G., Gross, J., and Grignolo, A.: A study of the fibrous components of the vitreous body with the electron microscope. Proc. Soc. Exper. Biol. and Med., 76:857, 1951. 2. Matolsty, A. G.: A study of the structural protein of the vitreous body. J. Gen. Physiol., 36:39, 1952. 3. Grignolo, A.: Fibrous components of the vitreous body. Arch. Ophth., 47 :760, 1952. 4. Bembridge, B. A., Crawford, G. N. C, and Pirie, A.: Phase contrast microscopy of the animal vitreous body. Brit. J. Ophth., 36:131, 1952. 5. Cagianut, B., and Wunderly, C.: Protein studies in the human vi'reous body. Brit. J. Ophth., 37: 229, 1953. 6. Balazs, Endre A.: Structure of the vitreous gel. A d a XVII Intemat. Cong. Ophth., pp. 1019-1024, 1954. 7. Woodin, A. M., and Boruchoff, S. A.: Particle interaction in solutions derived from ox vitreous humor. J. Biophysic. Biochem. & Cytol., 1:489, 1955. 8. Gross, J., Matolsty, A. G., and Cohen, C.: Vitrosin, a member of the collagen class. J. Biophysic. Biochem. & Cytol., 1:215, 1955. 9. Dische, Z., and Zelmanes, G.: Polysaccharides of the vitreous fibers. Arch. Ophth., 54:528, 1955. 10. Varga, L., and Balazs, E. A.: Studies on the structure of the vitreous body: II. Electrophoretic and sedimentation properties. Am. J. Ophth., 38 :29, 1954. 11. Martin, C. J., and Axelrod, A. E.: A modified method for the determination of hydroxyproline. Proc. Soc. Exper. Biol. & Med., 83:461, 1953. 12. Boas, N. F . : Method for the determination of hexosamine in tissues. J. Biol. Chem., 204:553, 1953. 13. Grunbaum, B. W., Shaffer, F. L., and Kirk, P. L.: Kjeldahl determination of nitrogen with sealed tube digestion. Anal. Chem., 24:1487, 1952. 14. The Enzymes, (eds. Sumner, J. B., and Myrback, Karl) : New York, Acad. Press, 1951, p. 847. 15. Pirie, A., Schmidt, G., and Waters, J. W.: Ox vitreous humor: I. The residual protein. Brit. J. Ophth., 32:321, 1948. 16. Banga, I., and Balo, J.: Studies on the elastolysis of elastin and collagen. Acta Physiol. Acad. Sci. Hung., 6:235, 1954. Cited in Chem. Abts., 49 :8337, 1955. 17. Jackson, D. S.: Chondroitin sulfate as a factor in the stability of tendon. A chapter in Nature and Structure of Collagen, (ed. Randall, J. T.) New York, Acad. Press, Inc., 1953.
DISCUSSION DR. ZACHARIAS DISCHE (New York) : I would
like to say a few words concerning Dr. McEwen's very interesting paper, because I think the criticism by Dr. Balazs, while it is completely justified as far as the loose use of the words "residual protein" is concerned, may do a certain injustice to the paper and the results Dr. McEwen has shown. It is perfectly true that the residual protein is a mixture which could be brought about by contami nation, but in addition I think we must not dismiss the idea that it is a mixture by structural co-ordina tion of various things. It is difficult to explain Dr. McEwen's results only by simple combination, be cause almost 40 percent of the total protein goes off on the trypsin digestion. One possibility to reconcile that with our con cepts of the chemical nature of collagen is that in
this residual protein there is a structural co-ordina tion of certain small collagen fibers by very small amounts of highly active high polymers of the kind of hyaluronic acid. As soon as the latter are re moved, the structure disintegrates and perhaps goes simply into solution. I am not quite sure how Dr. McEwen determined the tryptic effects on his ma terial. Perhaps this would be a crucial thing to answer. We found in our residual protein, which was taken from the center of the vitreous, that we can not remove certain traces of hyaluronic acid even by the most exhaustive washing and combined homogenization and treatment with hyaluronidase. We had the impression that there may be a structural combination between certain collagen fibers and hyaluronic acid which may be of major significance.