- Email: [email protected]
[47] Vertebrate collagenases
420
AMINO ACIDS AND PEPTIDES
[47]
suspension. The ethanol concentration is increased to about 35% in order to maintain the inhibitor in solution, and the coupling is allowed to proceed at 5 ° for 16 hours. The resultant Sepharose-4-phenylbutylamine is washed extensively with the bicarbonate buffer containing 30% ethanol and stored in 50 mM Bicine buffer, pH 8.0; 50 mM Tris chloride may be used in place of Bicine. Illustrative Procedure: Purification of Commercial a-Chymotrypsin A column of the given affinity gel is equilibrated with 50 mM Tris chloride at pH 8.0 and room temperature. The enzyme sample is applied to the column in a minimum volume of the same buffer, and the equilibrating buffer is passed at a flow rate of 30-60 ml per hour until no more material absorbing at 280 nm is eluted. Typically, the eluted material amounts to 5 - 1 0 % of the initial protein applied to the column and is free of chymotryptic activity. The buffer is then changed to 0.1 M acetic acid at pH 3.0, and the remaining protein is rapidly eluted. In order to minimize autolysis, the amount of pH 8.0 buffer should be the smallest amount practically possible. Miller et al." have reported improved recovery of activity if the enzyme is applied to the column in a small volume of 1 mM HC1, the starting buffer contains 50 mM CaC12, and the column is run at between 2 and 6 °.
[47]
Vertebrate
Collagenases
B y ARTHUR Z. EISEN, EUGENE A. BAUER, GEORGE P. STRICKLIN,
and JOHN J. JEFFREY Gallop and associates 1 were the first to recognize the high affinity of a collagenase for its substrate and to take advantage of this property in the purification of the collagenase from Clostridium histolyticum. The enzyme was allowed to bind to native collagen fibrils and was subsequently recovered after digestion of the substrate and release of the enzyme. Similar use of collagen fibrils for purification of vertebrate tissue collagenase is much less effective, since the limited cleavage of collagen catalyzed by these enzymes 2 makes recovery of the enzyme from the reaction mixture difficult. To overcome this problem, collagen in solution has ~P. M. Gallop, S. Seifter, and E. Meilman, 1. Biol. Chem. 227, 891 (1957). 2A. Z. Eisen, E. A. Bauer, and J. J. Jeffrey, 1. Invest. Dermatol. 55, 359 (1970).
[47]
VERTEBRATE COLLAGENASES
421
been employed for coupling to a solid matrix (agarose) for use in affinity chromatography? Sources of Collagenase. Vertebrate collagenases, including those from human and animal sources, can be obtained from tissue culture using well-established techniques. 2 Collagenase activity is measured throughout all stages of purification by the enzymatic release of soluble [l'C]glycinecontaining peptides from native, reconstituted guinea pig skin collagen fibrils.' Affinity Chromatography. Collagen was coupled to agarose (Sepharose 4B) according to the method described for other ligands. ~ In a typical preparation, approximately 25 ml of cyanogen bromide-activated Sepharose 4B was suspended in 50 ml of 0.2 M NaHCO3 at pH 9.0 and 75 mg of highly purified native guinea pig skin collagen, prepared by the method of Gross G in 25 ml of 0.4 M NaCI, was added immediately. The mixture was stirred gently for 18 hours at 4 ° , filtered, washed with water, and equilibrated with 50 mM Tris chloride at p H 7.5 containing 5 mM CaC12. The efficiency with which native guinea pig skin collagen is coupled to Sepharose was determined by measuring the hydroxyproline content of the collagen Sepharose beads at the end of a reaction. In every case, 7 0 - 9 5 % of the collagen should be coupled to Sepharose under the above conditions, producing a slurry which contains approximately 1.8 mg collagen per milliliter of packed Sepharose. Affinity chromatography was carried out at 4 ° by applying 5 - 1 0 mg of partially purified enzyme protein to a column (1.2 × 3 cm) containing collagen-Sepharose which had been equilibrated with 50 mM Tris chloride-5 mM CaCI2, pH 7.5. For greater amounts of enzyme protein, larger columns were used; adequate flow rates were maintained by increasing the column diameter. The starting buffer was passed through the column until the absorbance at 280 nm returned to the base line. Elution was accomplished using the same buffer containing 1 M NaC1. Harsher methods of elution, such as lowering the pH, have been used with other enzymes, but the lability of most human and animal collagenases at acid pH makes such an approach hazardous. Unbound enzyme protein can be subjected to rechromatography on collagen-Sepharose until no further enzyme activity remains. This is usually done after the first peak is lyophilized and reconstituted in starting buffer, but this step E. A. Bauer, J. J. Jeffrey, and A. Z. Eisen, Biochem. Biophys. Res. Commun. 44, 813 (1971). ' Y. Nagai, C. M. Lapiere, and J. Gross, Biochemistry 5, 3123 (1966). P. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, Proc. Nat. ,4cad. Sci. U.S. 61, 636 (1968). ~J. Gross, J. Exp. Med. 107, 247 (1958).
422
[47]
AMINO ACIDS AND PEPTIDES
can be performed without reducing the volume of the unbound enzyme peak. Although collagenase can be obtained by passing crude enzyme preparations through collagen-Sepharose, a number of contaminating proteins also bind to the matrix. Thus, the use of affinity chromatography to obtain pure collagenase must be accomplished by initial partial purification of these enzymes to reduce the likelihood of nonspecific adsorption of components present in the crude mixtures. Usually, ammonium sulfate fractionation, gel filtration on Sephadex G-150, and DEAE chromatography have been employed as preliminary steps? With appropriate starting solutions, the enzymatically active eluent fraction usually appears as a single band on polyacrylamide gel electrophoresis although minor contaminating protein bands may be present occasionally. In most instances these can be removed by rechromatography on freshly prepared collagen-Sepharose. Unfortunately, the yields of homogeneous enzyme using affinity chromatography are low. Starting with 10 mg of partially purified collagenase, a single chromatographic purification may yield no more than 0.5 mg of the desired enzyme. In addition, there is a considerable loss of enzyme activity from the highly purified preparations which are rerun on coIlagenSepharose, and usually only about 2% of the material applied to the column is recovered. In spite of some of the difficulties encountered, this approach has permitted sufficient electrophoretically pure collagenase to be obtained from human skin, rheumatoid synovium, and tadpole skin to produce functionally monospecific antisera in rabbitsJ -9 A major limitation of this method of purification is the rather small number of rigid collagen molecules which can be spatially accommodated on an agarose bead. A yield of approximately 2 mg of collagen per packed milliliter of Sepharose appears to be about the maximum amount of this protein that can be coupled to agarose. In addition, attempts to make use of side arms to increase the extent of binding of the collagen to the matrix have been unsuccessful. Several modifications of the method have been used in an effort to improve the yield of vertebrate collagenases. These include: (1) the use of purified alpha chains from denatured collagen, in the hope that greater molar coupling could be achieved by eliminating the restrictions imposed by the rodlike structure of native collagen. Indeed, when purified alpha1 ~E. A. Bauer, A. Z. Eisen, and J. J. Jeffrey, 1. CIhL Invest. 50, 2056 (1971). 8A. Z. Eisen, E. A. Bauer, and J. J. Jeffrey, Proc. Nat. Acad. Sci. U.S. 68, 248
(1971). E. A. Bauer, A. Z. Eisen, and J. J. Jeffrey, J.
Biol. Chem.
247, 6679 (1972).
[47]
VERTEBRATE COLLAGENASES
423
chains from guinea pig skin collagen were employed as a ligand, yields of 4-5 mg protein per milligram of packed Sepharose were obtained. (2) The use of the cyanogen bromide peptide alphal-CB-7 obtained from the purified alpha1 chain of rat skin collagen. This peptide was chosen as a ligand for coupling to Sepharose since it contains the site at which most vertebrate collagenases initiate their cleavage of the collagen molecule. Here too, it was reasoned that this property might enhance the specific binding of collagenase to its substrate and increase the amount of enzyme bound to the Sepharose. Partially purified human skin collagenase, when subjected to affinity chromatography on either alpha~-Sepharose or alphal-CB7-Sepharose under the same conditions as described above results in preparations purified some 3- to 4-fold with a yield of approximately 20%. However, the eluted enzyme peak still contains numerous contaminating bands when examined on polyacrylamide gel electrophoresis. Undoubtedly the failure of alphal-Sepharose or alphal-CB-7-Sepharose to yield pure collagenase preparations is, in part, due to the fact that neither ligand possesses the native triple helical collagen structure. Thus, both are potential substrates for other, less specific proteases. Nevertheless, these ligands may be useful as an initial purification step in which yield is more important than specificity. (3) The entrapment of collagen in a polyacrylamide matrix. This method ~° has been used for the purification of tadpole collagenase. Purified collagen was treated with Proctase ~ to remove the nonhelical (telopeptide) amino-terminal region of the collagen molecule in an effort to reduce the binding on noncollagenolytic proteases to the collagen molecules entrapped in polyacrylamide. In these studies crude tadpole collagenase preparations were pretreated with hyaluronidase and ribonuclease to improve the purity of the final enzyme preparation. Affinity chromatography was performed using 5% polyacrylamide gels containing approximately 58 mg of entrapped collagen. The collagenase was eluted with sodium acetate at pH 5.2 containing 0.2 M NaC1 and 5 mM CaC12. This technique resulted in an approximate 300-fold purification of the tadpole collagenase with a yield of 69%.1° In our experience the use of pH 5.2 acetate buffer, rather than a neutral pH buffer, to elute human collagenases results in a loss of approximately 80% of the enzyme activity. In addition, regardless of the mode ~0y. Nagai and H. Hori, Biochim. Biophys..4cta 263, 564 (1972). l~Proctase is an acid proteinase obtained from the broth filtrate of cultures of Aspergillus niger var. macrosporus. Presumably, the nonhelical amino terminus of the collagen molecule could be removed just as well by using pepsin.
424
AMINO ACIDS AND PEPTIDES
[48]
of elution the enzyme preparations obtained contain multiple bands on polyacrylamide gel electrophoresis. Although this method may provide an excellent initial purification step, it must be followed by chromatography on collagen-Sepharose or another chromatographic procedure to obtain highly purified collagenase. Acknowledgment This work was supported by U.S. Public Health Service Research Grants AM 12129, AM 05611, and HD 05291.
[48] Plasminogen B y B. A. K. CHIBBER, D. G. DEUTSCH, and E. T. MERTZ
Plasminogen is the zymogen of the proteolytic enzyme plasmin (EC 3.4.4.14), the enzyme responsible for the dissolution of fibrin clots in blood. In a continuing effort to resolve problems of homogeneity, yield, stability, reproducibility, solubility at physiological pH, and isolation of the native form of the zymogen, various methods have evolved over the three decades following the original euglobulin precipitation procedure for concentrating plasminogen from p l a s m a ? These efforts culminated in combining gel filtration and ion-exchange chromatography techniques as the procedures of choice. 2,3 The development of affinity chromatography in recent years' provided an attractive alternative to conventional multistep purification procedures as a gentle and effective technique for the purification of plasminogen. The application of affinity methods to the purification of plasminogen was based upon the ability of this zymogen to bind various aliphatic ,0-amino acids. ~-9 These amino acids inhibit the activation of the zymoIH. A. Milstone, 1. Immunol. 42, 109 (1941). 2K. C. Robbins, L. Summaria, D. Elwyn, and G. H. Barlow, J. Biol. Chem. 240, 541 (1965). K. C. Robbins and L. Summaria, this series, Vol. 19, p. 184. ' P. Cuatrecasas and C. C. Anfinsen, this series, Vol. 22, p. 345. aN. Alkjaersig, A. P. Fletcher, and S. Sherry, J. Biol. Chem. 234, 832 (1959). 6 F. B. Ablondi, J. J. Hagan, M. Phillips, and E. C. DeRenzo, Arch. Biochem. Biophys. 82, 153 (1959). F. Markwardt, H. Landmann, and A. Hoffman, Hoppe-Seyler's Z. Physiol. Chem. 340, 174 (1965). AM. Maki and F. K. Ellery, Thromb. Diath. Haemorrh. 16, 668 (1966). 0y. Abiko, M. Iwamoto, and M. Tomikawa, Biochim. Biophys. Acta 185, 424 (1969).
AMINO ACIDS AND PEPTIDES
[47]
suspension. The ethanol concentration is increased to about 35% in order to maintain the inhibitor in solution, and the coupling is allowed to proceed at 5 ° for 16 hours. The resultant Sepharose-4-phenylbutylamine is washed extensively with the bicarbonate buffer containing 30% ethanol and stored in 50 mM Bicine buffer, pH 8.0; 50 mM Tris chloride may be used in place of Bicine. Illustrative Procedure: Purification of Commercial a-Chymotrypsin A column of the given affinity gel is equilibrated with 50 mM Tris chloride at pH 8.0 and room temperature. The enzyme sample is applied to the column in a minimum volume of the same buffer, and the equilibrating buffer is passed at a flow rate of 30-60 ml per hour until no more material absorbing at 280 nm is eluted. Typically, the eluted material amounts to 5 - 1 0 % of the initial protein applied to the column and is free of chymotryptic activity. The buffer is then changed to 0.1 M acetic acid at pH 3.0, and the remaining protein is rapidly eluted. In order to minimize autolysis, the amount of pH 8.0 buffer should be the smallest amount practically possible. Miller et al." have reported improved recovery of activity if the enzyme is applied to the column in a small volume of 1 mM HC1, the starting buffer contains 50 mM CaC12, and the column is run at between 2 and 6 °.
[47]
Vertebrate
Collagenases
B y ARTHUR Z. EISEN, EUGENE A. BAUER, GEORGE P. STRICKLIN,
and JOHN J. JEFFREY Gallop and associates 1 were the first to recognize the high affinity of a collagenase for its substrate and to take advantage of this property in the purification of the collagenase from Clostridium histolyticum. The enzyme was allowed to bind to native collagen fibrils and was subsequently recovered after digestion of the substrate and release of the enzyme. Similar use of collagen fibrils for purification of vertebrate tissue collagenase is much less effective, since the limited cleavage of collagen catalyzed by these enzymes 2 makes recovery of the enzyme from the reaction mixture difficult. To overcome this problem, collagen in solution has ~P. M. Gallop, S. Seifter, and E. Meilman, 1. Biol. Chem. 227, 891 (1957). 2A. Z. Eisen, E. A. Bauer, and J. J. Jeffrey, 1. Invest. Dermatol. 55, 359 (1970).
[47]
VERTEBRATE COLLAGENASES
421
been employed for coupling to a solid matrix (agarose) for use in affinity chromatography? Sources of Collagenase. Vertebrate collagenases, including those from human and animal sources, can be obtained from tissue culture using well-established techniques. 2 Collagenase activity is measured throughout all stages of purification by the enzymatic release of soluble [l'C]glycinecontaining peptides from native, reconstituted guinea pig skin collagen fibrils.' Affinity Chromatography. Collagen was coupled to agarose (Sepharose 4B) according to the method described for other ligands. ~ In a typical preparation, approximately 25 ml of cyanogen bromide-activated Sepharose 4B was suspended in 50 ml of 0.2 M NaHCO3 at pH 9.0 and 75 mg of highly purified native guinea pig skin collagen, prepared by the method of Gross G in 25 ml of 0.4 M NaCI, was added immediately. The mixture was stirred gently for 18 hours at 4 ° , filtered, washed with water, and equilibrated with 50 mM Tris chloride at p H 7.5 containing 5 mM CaC12. The efficiency with which native guinea pig skin collagen is coupled to Sepharose was determined by measuring the hydroxyproline content of the collagen Sepharose beads at the end of a reaction. In every case, 7 0 - 9 5 % of the collagen should be coupled to Sepharose under the above conditions, producing a slurry which contains approximately 1.8 mg collagen per milliliter of packed Sepharose. Affinity chromatography was carried out at 4 ° by applying 5 - 1 0 mg of partially purified enzyme protein to a column (1.2 × 3 cm) containing collagen-Sepharose which had been equilibrated with 50 mM Tris chloride-5 mM CaCI2, pH 7.5. For greater amounts of enzyme protein, larger columns were used; adequate flow rates were maintained by increasing the column diameter. The starting buffer was passed through the column until the absorbance at 280 nm returned to the base line. Elution was accomplished using the same buffer containing 1 M NaC1. Harsher methods of elution, such as lowering the pH, have been used with other enzymes, but the lability of most human and animal collagenases at acid pH makes such an approach hazardous. Unbound enzyme protein can be subjected to rechromatography on collagen-Sepharose until no further enzyme activity remains. This is usually done after the first peak is lyophilized and reconstituted in starting buffer, but this step E. A. Bauer, J. J. Jeffrey, and A. Z. Eisen, Biochem. Biophys. Res. Commun. 44, 813 (1971). ' Y. Nagai, C. M. Lapiere, and J. Gross, Biochemistry 5, 3123 (1966). P. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, Proc. Nat. ,4cad. Sci. U.S. 61, 636 (1968). ~J. Gross, J. Exp. Med. 107, 247 (1958).
422
[47]
AMINO ACIDS AND PEPTIDES
can be performed without reducing the volume of the unbound enzyme peak. Although collagenase can be obtained by passing crude enzyme preparations through collagen-Sepharose, a number of contaminating proteins also bind to the matrix. Thus, the use of affinity chromatography to obtain pure collagenase must be accomplished by initial partial purification of these enzymes to reduce the likelihood of nonspecific adsorption of components present in the crude mixtures. Usually, ammonium sulfate fractionation, gel filtration on Sephadex G-150, and DEAE chromatography have been employed as preliminary steps? With appropriate starting solutions, the enzymatically active eluent fraction usually appears as a single band on polyacrylamide gel electrophoresis although minor contaminating protein bands may be present occasionally. In most instances these can be removed by rechromatography on freshly prepared collagen-Sepharose. Unfortunately, the yields of homogeneous enzyme using affinity chromatography are low. Starting with 10 mg of partially purified collagenase, a single chromatographic purification may yield no more than 0.5 mg of the desired enzyme. In addition, there is a considerable loss of enzyme activity from the highly purified preparations which are rerun on coIlagenSepharose, and usually only about 2% of the material applied to the column is recovered. In spite of some of the difficulties encountered, this approach has permitted sufficient electrophoretically pure collagenase to be obtained from human skin, rheumatoid synovium, and tadpole skin to produce functionally monospecific antisera in rabbitsJ -9 A major limitation of this method of purification is the rather small number of rigid collagen molecules which can be spatially accommodated on an agarose bead. A yield of approximately 2 mg of collagen per packed milliliter of Sepharose appears to be about the maximum amount of this protein that can be coupled to agarose. In addition, attempts to make use of side arms to increase the extent of binding of the collagen to the matrix have been unsuccessful. Several modifications of the method have been used in an effort to improve the yield of vertebrate collagenases. These include: (1) the use of purified alpha chains from denatured collagen, in the hope that greater molar coupling could be achieved by eliminating the restrictions imposed by the rodlike structure of native collagen. Indeed, when purified alpha1 ~E. A. Bauer, A. Z. Eisen, and J. J. Jeffrey, 1. CIhL Invest. 50, 2056 (1971). 8A. Z. Eisen, E. A. Bauer, and J. J. Jeffrey, Proc. Nat. Acad. Sci. U.S. 68, 248
(1971). E. A. Bauer, A. Z. Eisen, and J. J. Jeffrey, J.
Biol. Chem.
247, 6679 (1972).
[47]
VERTEBRATE COLLAGENASES
423
chains from guinea pig skin collagen were employed as a ligand, yields of 4-5 mg protein per milligram of packed Sepharose were obtained. (2) The use of the cyanogen bromide peptide alphal-CB-7 obtained from the purified alpha1 chain of rat skin collagen. This peptide was chosen as a ligand for coupling to Sepharose since it contains the site at which most vertebrate collagenases initiate their cleavage of the collagen molecule. Here too, it was reasoned that this property might enhance the specific binding of collagenase to its substrate and increase the amount of enzyme bound to the Sepharose. Partially purified human skin collagenase, when subjected to affinity chromatography on either alpha~-Sepharose or alphal-CB7-Sepharose under the same conditions as described above results in preparations purified some 3- to 4-fold with a yield of approximately 20%. However, the eluted enzyme peak still contains numerous contaminating bands when examined on polyacrylamide gel electrophoresis. Undoubtedly the failure of alphal-Sepharose or alphal-CB-7-Sepharose to yield pure collagenase preparations is, in part, due to the fact that neither ligand possesses the native triple helical collagen structure. Thus, both are potential substrates for other, less specific proteases. Nevertheless, these ligands may be useful as an initial purification step in which yield is more important than specificity. (3) The entrapment of collagen in a polyacrylamide matrix. This method ~° has been used for the purification of tadpole collagenase. Purified collagen was treated with Proctase ~ to remove the nonhelical (telopeptide) amino-terminal region of the collagen molecule in an effort to reduce the binding on noncollagenolytic proteases to the collagen molecules entrapped in polyacrylamide. In these studies crude tadpole collagenase preparations were pretreated with hyaluronidase and ribonuclease to improve the purity of the final enzyme preparation. Affinity chromatography was performed using 5% polyacrylamide gels containing approximately 58 mg of entrapped collagen. The collagenase was eluted with sodium acetate at pH 5.2 containing 0.2 M NaC1 and 5 mM CaC12. This technique resulted in an approximate 300-fold purification of the tadpole collagenase with a yield of 69%.1° In our experience the use of pH 5.2 acetate buffer, rather than a neutral pH buffer, to elute human collagenases results in a loss of approximately 80% of the enzyme activity. In addition, regardless of the mode ~0y. Nagai and H. Hori, Biochim. Biophys..4cta 263, 564 (1972). l~Proctase is an acid proteinase obtained from the broth filtrate of cultures of Aspergillus niger var. macrosporus. Presumably, the nonhelical amino terminus of the collagen molecule could be removed just as well by using pepsin.
424
AMINO ACIDS AND PEPTIDES
[48]
of elution the enzyme preparations obtained contain multiple bands on polyacrylamide gel electrophoresis. Although this method may provide an excellent initial purification step, it must be followed by chromatography on collagen-Sepharose or another chromatographic procedure to obtain highly purified collagenase. Acknowledgment This work was supported by U.S. Public Health Service Research Grants AM 12129, AM 05611, and HD 05291.
[48] Plasminogen B y B. A. K. CHIBBER, D. G. DEUTSCH, and E. T. MERTZ
Plasminogen is the zymogen of the proteolytic enzyme plasmin (EC 3.4.4.14), the enzyme responsible for the dissolution of fibrin clots in blood. In a continuing effort to resolve problems of homogeneity, yield, stability, reproducibility, solubility at physiological pH, and isolation of the native form of the zymogen, various methods have evolved over the three decades following the original euglobulin precipitation procedure for concentrating plasminogen from p l a s m a ? These efforts culminated in combining gel filtration and ion-exchange chromatography techniques as the procedures of choice. 2,3 The development of affinity chromatography in recent years' provided an attractive alternative to conventional multistep purification procedures as a gentle and effective technique for the purification of plasminogen. The application of affinity methods to the purification of plasminogen was based upon the ability of this zymogen to bind various aliphatic ,0-amino acids. ~-9 These amino acids inhibit the activation of the zymoIH. A. Milstone, 1. Immunol. 42, 109 (1941). 2K. C. Robbins, L. Summaria, D. Elwyn, and G. H. Barlow, J. Biol. Chem. 240, 541 (1965). K. C. Robbins and L. Summaria, this series, Vol. 19, p. 184. ' P. Cuatrecasas and C. C. Anfinsen, this series, Vol. 22, p. 345. aN. Alkjaersig, A. P. Fletcher, and S. Sherry, J. Biol. Chem. 234, 832 (1959). 6 F. B. Ablondi, J. J. Hagan, M. Phillips, and E. C. DeRenzo, Arch. Biochem. Biophys. 82, 153 (1959). F. Markwardt, H. Landmann, and A. Hoffman, Hoppe-Seyler's Z. Physiol. Chem. 340, 174 (1965). AM. Maki and F. K. Ellery, Thromb. Diath. Haemorrh. 16, 668 (1966). 0y. Abiko, M. Iwamoto, and M. Tomikawa, Biochim. Biophys. Acta 185, 424 (1969).