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Collagen fractionation: Separation of native types I, II and III by differential prectipitation
ANALYTICAL
Collagen
BIOCHEMISTRY
71, 114- 118 (1976)
Fractionation: Separation of Native Types I, II and III by Differential Precipitation’
ROBERT L. TRELSTAD~, VERONICA M. CATANESE AND DEBRA F. RUBIN Developmental Biology Laboratory, Departments Harvard Medical School, Massachusetts Boston, Massachusetts
of Medicine and Pathology, General Hospital, 02114
Received July 28. 1975; accepted October 21, 1975 Collagen types I, II and III can be purified in their native state from heterogeneous collagen solutions by fractional precipitation at neutral pH using ammonium sulfate, sodium chloride and ethanol as precipitants. This method of collagen separation is useful as a preparative procedure and should also serve as an analytical tool for identification of unknown radioactively labeled collagens.
It has recently been shown that at least four different molecular species of collagen are present in mammalian tissues. (1). The native molecule in each contains three polypeptide chains or (Y chains wound in a right handed superhelix. The amino acid sequence and distribution of the polypeptide chains, however, is distinct for each of the different collagen types. Type I collagen, [al(I)],o2, is found predominantly in skin, bone, tendon, ligaments and cornea and contains two identical al(I) chains and one dissimilar a2 chain (1,2). Type II collagen, [(ul(II)],, is found principally in cartilage and consists of three identical (Al chains (3). Type III collagen, [(ul(III)],, has been found in fetal skin, in internal organs and in the aorta and consists of three identical al(II1) chains (4-6). Type IV collagen, [czl(IV)]~, has been isolated from the basement membranes of the lens capsule and the renal glomerulus (7) and has also been isolated from the aorta (6); it consists of three identical al(IV) chains. Since the native collagen molecules are long relatively rigid rods with a tendency to interact and form aggregates they cannot be separated using chromatographic procedures under native conditions. In a previous study of type I and type II collagens we noted that these two molecules had significant differences in their solubilities at neutral pH and that a separation based on salt fractionation could be effected (8). Recently, studies on i This is publication No. 678 of the Robert W. Diseases Causing Deformities. This investigation (AM 3564) and from the American Cancer 1441-C-I). 2 Recipient Faculty Research Award, American 114 Copyright All rights
0 1976 by Academic Press. Inc. of reproduction in anv form reserved
Lovett Memorial Group for the Study of was supported by a grant from the NIH Society, Massachusetts Division (No. Cancer Society (PR 107).
COLLAGEN
FRACTlONATlON
115
type III collagen have indicated that it too can be separated from type I collagen by salt fractionation (45). The present study reports the relative precipitability of collagen types I, II, and III in a neutral phosphate buffer using the three different precipitants, ammonium sulfate, sodium chloride and ethanol. MATERIALS
AND METHODS
Collagens from human skin, cartilage and uterine leiomyomas were isolated using limited proteolytic digestion with pepsin as described previously (4,6). In brief, tissues were homogenized in 0.1 M acetic acid, the pH was adjusted to 2.5 with HCl and the preparation was digested with Worthington 2x Pepsin (20 mg pepsin/g wet weight tissue) for 3 days at 4°C. The extracts were neutralized with NaOH to inactivate peptic activity and the solubilized collagens were salted out by addition of solid sodium chloride to 20%. Prior to pepsinization, the cartilage was extracted with 2.0 M magnesium chloride, 0.05 M Tris (pH 7.5) at 4°C overnight to remove proteoglycans. The cartilage preparation consisted of over 90% type II collagen with a small contaminant of type I collagen presumably from the perichondrium. The leiomyoma digest consisted of approximately an equal mixture of types I and III and the skin approximately 85% type I and the remainder type III. Pure types I and III were obtained by salt fractionation using sodium chloride following the methods described below before use. The purity of each preparation was established by chromatography on CM-cellulose and amino acid analysis of the native collagens and their isolated chains using previously described procedures (3,8). The purified collagen preparations of all three types were solubilized at 4°C in 0.4 ionic strength (0.16 M) phosphate buffer (pH 7.6), at aconcentration of 1 mg/ml, centrifuged after solubilization at 34,000 rpm for 2 hr and then subjected to fractionation. Precipitation with ammonium sulfate was accomplished by slowly adding a cold solution of saturated ammonium sulfate to the collagen solution with stirring; with sodium chloride by adding a cold solution of 4.4 M NaCl to the collagen solution slowly with stirring and with alcohol by adding cold 100% ethanol to the solution with stirring. To analyze the degree of precipitation, aliquots from the supernatant after centrifugation at 16,000 t-pm for 45 min in a Sorvall RC-2, SS-34 rotor, were removed, hydrolyzed under N2 in 6 N HCl at 110°C for 24 hr and analyzed for hydroxyproline on a Jeolco 5AH amino acid analyzer. Two separate series of precipitations were performed using the same three sets of precipitants. In one series, the precipitant was added and the preparation was centrifuged within 30 min following its addition: this set is hereafter referred to as rapid precipitation. In the second series
116
TRELSTAD,
CATANESE
AND RUBIN
the preparations were gently agitated overnight and then centrifuged 24 hr following addition of the precipitant; this set is referred to as slow precipitation. RESULTS
AND DISCUSSION
Different degrees of precipitation of collagen types I, II and III can readily be effected at neutral pH as indicated by the precipitation curves in Fig. 1. In general Type III is the least soluble of the three collagens under all conditions of precipitation and is most easily separated by rapid precipitation with 14% saturated ammonium sulfate or by slow precipitation with 1.5 M NaCl. The precipitates formed from all collagen types in both ammonium sulfate and ethanol are translucent and readily redissolved after centrifugation, whereas those formed after sodium chloride precipitation tend to compact upon centrifugation and are less readily redissolved. The decrease in solubility of the type II collagen seen in all three precipitants at the lower concentrations was shown to represent an apSLOW
PRECIPITA
Tl ION i
PI
FIG. 1. Precipitation curves for collagen types I, II and 111 from neutral solutions (pH 7.6; ionic strength 0.4) at initial concentrations of 1 mg/l ml. Slow precipitations: 24 hr between addition of precipitant and centrifugation. Rapid precipitation: 30 min between addition of precipitant and centrifugation.
COLLAGEN
FRACTIONATION
117
proximate 5- 10% contamination of this preparation with type I collagen as judged by amino acid analysis of the precipitate. The cartilage preparation had not initially been salt fractionated to obtain a pure type II preparation as was necessary for the types I and III collagens. The data derived from the precipitation of the purified collagen solutions at a fixed concentration were directly applicable to mixtures of collagens in tissue extracts in which neither the ratio of collagen species nor collagen concentration was known. In general the wt/vol ratio of tissue to solvent during collagen extraction was 10 and after the initial 20% sodium chloride precipitation the collagen was resolubilized at an estimated concentration of between 0.5 and 2.0 mg/ml. Reproducible salt fractionation was routinely obtained in such preparations. The most effective separation procedure was the overnight precipitation with sodium chloride. The slower precipitation presumably reflects more nearly the equilibrium solubility state of the collagen and is also preferred in analytical procedures where identification of unknown radioactively labeled collagen is being attempted. During the preparative procedures, however, for type III collagen rapid precipitation with 14% ammonium sulfate is preferred both because of speed and ease of resolubilization of the precipitate. Adequate fractionation of an extract for preparative purposes generally required at least three precipitations. Neutral solutions of collagen in buffers other than phosphate can also be successfully fractionated in a manner similar to that described here. Collagen in acidic solution such as 0.1 M acetic acid precipitates at lower concentrations of all three precipitants and such solution conditions are not suitable for fractional precipitation. The use of concentrated solutions of sodium chloride and ammonium sulfate is important for reproducible fractionation. Addition of these precipitants in the crystalline state to the solutions causes more extensive precipitation of the collagen than when the solubilized forms of the salts are employed. The basis for the differences in solubility in these three collagenous proteins is unknown. The possibility that the high hexose content (4-5%) of the type II collagen renders it more soluble was suggested on the basis of the generalization that carbohydrate rich glycoproteins are more soluble than carbohydrate poor ones (9). The hexose content of types I and III collagens however are very similar and the differences in the solubility of these two species is thus apparently not based on carbohydrate content. Furthermore, preliminary observations on the solubility of type IV collagen from the aorta and spleen in which the molecule contains nearly 10% hexose by weight (6) indicate a precipitation profile more similar to type III than type II, suggesting that the hexose content of the collagens may not be a major determinant of solubility properties. The identification of unknown collagens studied by means of radioactive tracers requires criteria for identification by several different methodologies. At present chromatographic profiles on carboxymethyl
118
TRELSTAD,
CATANESE
AND RUBIN
cellulose of intact OLchains and peptides produced by cyanogen bromide cleavage are the principal analytical tools available (10). Techniques for the identification of native molecules rely exclusively at present on electron microscopic methods (II- 13). Separation of native molecules by salt fractionation would appear to be another potentially useful technique since the precipitation profile of a radioactively labeled collagen can readily be determined without significant loss of material and the collagen then analyzed by chromatographic methods of the (Y chains and their peptides using standard techniques (unpublished data). In addition to precipitation from solution the relative insolubility of type III collagen can also be used for its selective isolation by employing extractants of relatively high ionic strength which solubilize the type I and II collagens and leave type III in a precipitated state for subsequent solubilization with a lower ionic strength buffer (14). REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Trelstad, R. L. (1973)J. Histochem. Cytochem. 21, 521-528. Miller, E. J., and Matukas, V. J. (1969) Proc. Nut Acad. Sci. USA 64, 1264-1268. Miller, E. J. (1971) Biochemistry 10, 1652-1659. Chung, E., and Miller, E. J. (1974) Science 183, 1200-1201. Epstein, E. H. (1974) J. Biol. Chem. 249, 3225-3231. Trelstad, R. L. (1974) Biochem. Biophys. Res. Commun. 57, 717-725. Kefalides, N. A. (1971) Biochem. Biophys. Res. Commun. 45, 226-234. Trelstad, R. L., Kang, A. H., Toole, B. P., and Gross, J. (1972) J. Biol. Chem. 247, 6469-6473. 9. Haurowitz, F. (1963) The Chemistry and Function of Proteins, Academic Press, New York. 10. Bradley, K., McConnell-Breul, S., and Crystal, R. G. (1974) Proc. Nat. Acad. Sci. USA 71, 2828-2832. 11. Trelstad, R. L., Kang, A. H., Igarashi, S., and Gross, J. (1970) Biochemistry 9, 4993-4998.
12. Bruns, R. R., Trelstad, R. L., and Gross, J. (1973) Science 181, 269-271. 13. Wiedemann, H., Chung, E., Fujii, T., Miller, E. J., and Kuhn, K. (1975)Eur. J. Biochem. 51, 363-368. 14. Byers, P. H., McKenney, K. H., Lichtenstein, J. R.. and Martin, G. R. (1974) Biochemistry 13, 5243-5248.
Collagen
BIOCHEMISTRY
71, 114- 118 (1976)
Fractionation: Separation of Native Types I, II and III by Differential Precipitation’
ROBERT L. TRELSTAD~, VERONICA M. CATANESE AND DEBRA F. RUBIN Developmental Biology Laboratory, Departments Harvard Medical School, Massachusetts Boston, Massachusetts
of Medicine and Pathology, General Hospital, 02114
Received July 28. 1975; accepted October 21, 1975 Collagen types I, II and III can be purified in their native state from heterogeneous collagen solutions by fractional precipitation at neutral pH using ammonium sulfate, sodium chloride and ethanol as precipitants. This method of collagen separation is useful as a preparative procedure and should also serve as an analytical tool for identification of unknown radioactively labeled collagens.
It has recently been shown that at least four different molecular species of collagen are present in mammalian tissues. (1). The native molecule in each contains three polypeptide chains or (Y chains wound in a right handed superhelix. The amino acid sequence and distribution of the polypeptide chains, however, is distinct for each of the different collagen types. Type I collagen, [al(I)],o2, is found predominantly in skin, bone, tendon, ligaments and cornea and contains two identical al(I) chains and one dissimilar a2 chain (1,2). Type II collagen, [(ul(II)],, is found principally in cartilage and consists of three identical (Al chains (3). Type III collagen, [(ul(III)],, has been found in fetal skin, in internal organs and in the aorta and consists of three identical al(II1) chains (4-6). Type IV collagen, [czl(IV)]~, has been isolated from the basement membranes of the lens capsule and the renal glomerulus (7) and has also been isolated from the aorta (6); it consists of three identical al(IV) chains. Since the native collagen molecules are long relatively rigid rods with a tendency to interact and form aggregates they cannot be separated using chromatographic procedures under native conditions. In a previous study of type I and type II collagens we noted that these two molecules had significant differences in their solubilities at neutral pH and that a separation based on salt fractionation could be effected (8). Recently, studies on i This is publication No. 678 of the Robert W. Diseases Causing Deformities. This investigation (AM 3564) and from the American Cancer 1441-C-I). 2 Recipient Faculty Research Award, American 114 Copyright All rights
0 1976 by Academic Press. Inc. of reproduction in anv form reserved
Lovett Memorial Group for the Study of was supported by a grant from the NIH Society, Massachusetts Division (No. Cancer Society (PR 107).
COLLAGEN
FRACTlONATlON
115
type III collagen have indicated that it too can be separated from type I collagen by salt fractionation (45). The present study reports the relative precipitability of collagen types I, II, and III in a neutral phosphate buffer using the three different precipitants, ammonium sulfate, sodium chloride and ethanol. MATERIALS
AND METHODS
Collagens from human skin, cartilage and uterine leiomyomas were isolated using limited proteolytic digestion with pepsin as described previously (4,6). In brief, tissues were homogenized in 0.1 M acetic acid, the pH was adjusted to 2.5 with HCl and the preparation was digested with Worthington 2x Pepsin (20 mg pepsin/g wet weight tissue) for 3 days at 4°C. The extracts were neutralized with NaOH to inactivate peptic activity and the solubilized collagens were salted out by addition of solid sodium chloride to 20%. Prior to pepsinization, the cartilage was extracted with 2.0 M magnesium chloride, 0.05 M Tris (pH 7.5) at 4°C overnight to remove proteoglycans. The cartilage preparation consisted of over 90% type II collagen with a small contaminant of type I collagen presumably from the perichondrium. The leiomyoma digest consisted of approximately an equal mixture of types I and III and the skin approximately 85% type I and the remainder type III. Pure types I and III were obtained by salt fractionation using sodium chloride following the methods described below before use. The purity of each preparation was established by chromatography on CM-cellulose and amino acid analysis of the native collagens and their isolated chains using previously described procedures (3,8). The purified collagen preparations of all three types were solubilized at 4°C in 0.4 ionic strength (0.16 M) phosphate buffer (pH 7.6), at aconcentration of 1 mg/ml, centrifuged after solubilization at 34,000 rpm for 2 hr and then subjected to fractionation. Precipitation with ammonium sulfate was accomplished by slowly adding a cold solution of saturated ammonium sulfate to the collagen solution with stirring; with sodium chloride by adding a cold solution of 4.4 M NaCl to the collagen solution slowly with stirring and with alcohol by adding cold 100% ethanol to the solution with stirring. To analyze the degree of precipitation, aliquots from the supernatant after centrifugation at 16,000 t-pm for 45 min in a Sorvall RC-2, SS-34 rotor, were removed, hydrolyzed under N2 in 6 N HCl at 110°C for 24 hr and analyzed for hydroxyproline on a Jeolco 5AH amino acid analyzer. Two separate series of precipitations were performed using the same three sets of precipitants. In one series, the precipitant was added and the preparation was centrifuged within 30 min following its addition: this set is hereafter referred to as rapid precipitation. In the second series
116
TRELSTAD,
CATANESE
AND RUBIN
the preparations were gently agitated overnight and then centrifuged 24 hr following addition of the precipitant; this set is referred to as slow precipitation. RESULTS
AND DISCUSSION
Different degrees of precipitation of collagen types I, II and III can readily be effected at neutral pH as indicated by the precipitation curves in Fig. 1. In general Type III is the least soluble of the three collagens under all conditions of precipitation and is most easily separated by rapid precipitation with 14% saturated ammonium sulfate or by slow precipitation with 1.5 M NaCl. The precipitates formed from all collagen types in both ammonium sulfate and ethanol are translucent and readily redissolved after centrifugation, whereas those formed after sodium chloride precipitation tend to compact upon centrifugation and are less readily redissolved. The decrease in solubility of the type II collagen seen in all three precipitants at the lower concentrations was shown to represent an apSLOW
PRECIPITA
Tl ION i
PI
FIG. 1. Precipitation curves for collagen types I, II and 111 from neutral solutions (pH 7.6; ionic strength 0.4) at initial concentrations of 1 mg/l ml. Slow precipitations: 24 hr between addition of precipitant and centrifugation. Rapid precipitation: 30 min between addition of precipitant and centrifugation.
COLLAGEN
FRACTIONATION
117
proximate 5- 10% contamination of this preparation with type I collagen as judged by amino acid analysis of the precipitate. The cartilage preparation had not initially been salt fractionated to obtain a pure type II preparation as was necessary for the types I and III collagens. The data derived from the precipitation of the purified collagen solutions at a fixed concentration were directly applicable to mixtures of collagens in tissue extracts in which neither the ratio of collagen species nor collagen concentration was known. In general the wt/vol ratio of tissue to solvent during collagen extraction was 10 and after the initial 20% sodium chloride precipitation the collagen was resolubilized at an estimated concentration of between 0.5 and 2.0 mg/ml. Reproducible salt fractionation was routinely obtained in such preparations. The most effective separation procedure was the overnight precipitation with sodium chloride. The slower precipitation presumably reflects more nearly the equilibrium solubility state of the collagen and is also preferred in analytical procedures where identification of unknown radioactively labeled collagen is being attempted. During the preparative procedures, however, for type III collagen rapid precipitation with 14% ammonium sulfate is preferred both because of speed and ease of resolubilization of the precipitate. Adequate fractionation of an extract for preparative purposes generally required at least three precipitations. Neutral solutions of collagen in buffers other than phosphate can also be successfully fractionated in a manner similar to that described here. Collagen in acidic solution such as 0.1 M acetic acid precipitates at lower concentrations of all three precipitants and such solution conditions are not suitable for fractional precipitation. The use of concentrated solutions of sodium chloride and ammonium sulfate is important for reproducible fractionation. Addition of these precipitants in the crystalline state to the solutions causes more extensive precipitation of the collagen than when the solubilized forms of the salts are employed. The basis for the differences in solubility in these three collagenous proteins is unknown. The possibility that the high hexose content (4-5%) of the type II collagen renders it more soluble was suggested on the basis of the generalization that carbohydrate rich glycoproteins are more soluble than carbohydrate poor ones (9). The hexose content of types I and III collagens however are very similar and the differences in the solubility of these two species is thus apparently not based on carbohydrate content. Furthermore, preliminary observations on the solubility of type IV collagen from the aorta and spleen in which the molecule contains nearly 10% hexose by weight (6) indicate a precipitation profile more similar to type III than type II, suggesting that the hexose content of the collagens may not be a major determinant of solubility properties. The identification of unknown collagens studied by means of radioactive tracers requires criteria for identification by several different methodologies. At present chromatographic profiles on carboxymethyl
118
TRELSTAD,
CATANESE
AND RUBIN
cellulose of intact OLchains and peptides produced by cyanogen bromide cleavage are the principal analytical tools available (10). Techniques for the identification of native molecules rely exclusively at present on electron microscopic methods (II- 13). Separation of native molecules by salt fractionation would appear to be another potentially useful technique since the precipitation profile of a radioactively labeled collagen can readily be determined without significant loss of material and the collagen then analyzed by chromatographic methods of the (Y chains and their peptides using standard techniques (unpublished data). In addition to precipitation from solution the relative insolubility of type III collagen can also be used for its selective isolation by employing extractants of relatively high ionic strength which solubilize the type I and II collagens and leave type III in a precipitated state for subsequent solubilization with a lower ionic strength buffer (14). REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Trelstad, R. L. (1973)J. Histochem. Cytochem. 21, 521-528. Miller, E. J., and Matukas, V. J. (1969) Proc. Nut Acad. Sci. USA 64, 1264-1268. Miller, E. J. (1971) Biochemistry 10, 1652-1659. Chung, E., and Miller, E. J. (1974) Science 183, 1200-1201. Epstein, E. H. (1974) J. Biol. Chem. 249, 3225-3231. Trelstad, R. L. (1974) Biochem. Biophys. Res. Commun. 57, 717-725. Kefalides, N. A. (1971) Biochem. Biophys. Res. Commun. 45, 226-234. Trelstad, R. L., Kang, A. H., Toole, B. P., and Gross, J. (1972) J. Biol. Chem. 247, 6469-6473. 9. Haurowitz, F. (1963) The Chemistry and Function of Proteins, Academic Press, New York. 10. Bradley, K., McConnell-Breul, S., and Crystal, R. G. (1974) Proc. Nat. Acad. Sci. USA 71, 2828-2832. 11. Trelstad, R. L., Kang, A. H., Igarashi, S., and Gross, J. (1970) Biochemistry 9, 4993-4998.
12. Bruns, R. R., Trelstad, R. L., and Gross, J. (1973) Science 181, 269-271. 13. Wiedemann, H., Chung, E., Fujii, T., Miller, E. J., and Kuhn, K. (1975)Eur. J. Biochem. 51, 363-368. 14. Byers, P. H., McKenney, K. H., Lichtenstein, J. R.. and Martin, G. R. (1974) Biochemistry 13, 5243-5248.