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[52] Bovine β-trypsin
448
AMINO ACIDS AND PEPTIDES
[52]
m a n is o r t h a t of T h o m p s o n a n d Davie. 1'~ S c h m e r 1~- has r e p o r t e d trace quantities of p l a s m i n in affinity-purified t h r o m b i n which c o u l d b e r e m o v e d b y S E - S e p h a d e x c h r o m a t o g r a p h y either b e f o r e or after the affinity c h r o m a t o g r a p h y step. Such c o m b i n e d c h r o m a t o g r a p h y p r o d u c e s t h r o m b i n with 2 0 0 0 N I H u n i t s / m g . 28D. J. Baughman, this series, Vol. 19, p. 145.
[52]
Bovine
fl-Trypsin
B y ALBERT LIGHT a n d JURIS LIEPNIEKS
Schroeder and Shaw 1 reported in 1968 that chromatography of trypsin and activated trypsinogen on columns of SE-Sephadex C-50 showed the presence of almost equal amounts of two active forms of trypsin and a small amount of inert protein. One active form is B-trypsin, an intact molecule; the second is a-trypsin, a product of autolysis with Lys 131Ser 132 cleaved. Further slow proteolysis produces ~-trypsin with both Lys 131-Ser 132 and Lys 176-Asp 177 hydrolyzed."- It is clear from these studies that commercially available trypsins are mixtures of species and that studies on the properties of trypsin should use the B-form. High yields of B-trypsin can be obtained from bovine trypsinogen if enterokinase is used as activator since the enzyme is highly specific for the Lys 6-Ile 7 peptide bond. 3 However, autolysis of B-trypsin must be prevented; the presence of soybean trypsin inhibitor effectively inhibits B-trypsin but does not interfere with the activity of enterokinase. If the inhibitor is bound to Sepharose, affinity chromatography of the activation mixture provides a rapid procedure for the production of highly purified B-trypsinY" This procedure is described here. Assay Methods
Measurement oJ Trypsin Activity Trypsin activity is measured at pH 7.9 and 25 ° using 10 mM N-tosylL-arginine methyl ester in 10 mM Tris chloride and 50 mM calcium chloride. 4 -~D. D. Schroeder and E. Shaw, J. Biol. Chem. 243, 2943 (1968). -"R. L. Smith and E. Shaw, J. Biol. Chem. 244, 4704 (1969). 3 S. Maroux, J. Baratti, and P. Desnuelle, J. Biol. Chem. 246, 5031 (1971). 3, j. j. Liepnieks and A. Light, Anal. Biochem., in press. 4K. A. Walsh and P. E. Wilcox, this series, Vol. 19 [3].
[52]
BOVINE fl-TRYPSIN
449
Measurement of Enterokinase Activity
Enterokinase activity is determined by activation of 0.1 mg of trypsinogen per milliliter of 0.1 M sodium acetate, pH 5.0, and 50 mM calcium chloride. Samples of 0.5-7.0 enterokinase units are used in a 30-minute incubation at 35 °. Tryptic activity is measured in 0.5 ml fractions with N-tosyl-L-arginine methyl ester. An e~nterokinase unit is defined as the amount of enzyme which produces 1 trypsin unit per milliliter of activation mixture. Estimation of Aminopeptidase Activity
A spot test for the detection of aminopeptidase is used to examine chromatographic fractions? Usually, 10-~l samples and 0.5 ml of 0.3 mM leucyl fl-naphthylamide in 50 mM Tris chloride at pH 8.5, containing 5 mM calcium chloride are incubated at room temperature for 30 minutes in depressions of a porcelain spot plate. One adds 0.2 ml of naphthanil diazo blue (5 mg per milliliter of water), and the immediate appearance of a stable pink color is indicative of aminopeptidase activity. The relative intensity of color is used to judge maximum activity. Preparation of fl-Trypsin A suitable amount of enterokinase for the activation of approximately 300 mg of trypsinogen is prepared as follows. A crude enteropeptidase preparation (3.25 g, Miles Laboratories) is extracted with 325 ml of l0 mM ammonium acetate at pH 6.0. After stirring for 1 hour at room temperature, the suspension is centrifuged and the supernatant fluid is dialyzed exhaustively against water at 4 °. After lyophilization, 500 mg of a light brown powder is recovered. The powder is dissolved in 100 ml of l0 mM Tris chloride at pH 6.0, containing 50 mM sodium chloride. The insoluble material is removed by centrifugation, and the supernatant liquid is chromatographed on DEAE-cellulose (Whatman microgranular DE-52). Aminopeptidase activity is detected in the first half of the peak containing enterokinase activity. Removal of almost all the aminopeptidase contaminant is achieved by combining appropriate fractions. After dialysis against water at 4 ° and then lyophilization, 44 mg of a white powder is obtained containing approximately 72 units/rag. Sepharose-bound soybean trypsin inhibitor is prepared by a slight modification of a procedure previously described. ~ Settled Sepharose 4B, ~J. E. Folk, J. A. Gladner, and T. Viswanatha, BiochmT. B~phys. Acm 36, 256 (1959). P. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, Proc. Nat. Acad. Sci. U.S. 61, 636 (1968).
450
AMINO ACIDS AND PEPTIDES
[52]
20 ml, is treated with 3 g of cyanogen bromide at pH 11. The suspension is gently agitated for about 8 minutes with a magnetic stirrer. After washing with water and cold 0.1 M sodium bicarbonate, pH 9.5, 500 mg of soybean trypsin inhibitor are added and the suspension (40% by volume in 0.1 M sodium bicarbonate) is agitated by rotating slowly in a bottle for 18 hours at 4 °. The preparation is washed with 100 ml of 0.2 M sodium formate at pH 2.2, and 0.2 M Tris chloride at pH 8.0, repeating the washing cycle four times. The second Tris buffer wash contains 15 g of glycine per liter to react with residual cyanogen bromide activated sites. The preparation of Sepharose-bound soybean trypsin inhibitor binds approximately 5 mg of trypsin per milliliter of settled gel as determined operationally by applying excess trypsin at pH 8.0 and eluting bound trypsin at pH 2.2. Activation of bovine trypsinogen is accomplished by mixing 30 mg of zymogen with 4 mg of partially purified enterokinase (300 units) in 0.1 M Tris, pH 8.0, containing 50 mM calcium chloride. The activation mixture includes Sepharose-bound soybean trypsin inhibitor in approximately a 2-fold excess over the amount of trypsin that could be produced from the zymogen. The total volume of the mixture is 25 ml and it is incubated at 35° for 60 minutes with slow agitation obtained by bubbling nitrogen gas through the suspension. The activation mixture is transferred to a chromatography column (1.4 X 12 cm), and inert components are removed on washing the gel with 40 ml of 0.1 M sodium formate at pH 4.5, containing 50 mM calcium chloride, fl-Trypsin is released from the complex with 0.1 M sodium formate at pH 2.6 containing 50 mM calcium chloride or with a pH gradient using equal volumes of the formate buffers at the two pH values. The fl-trypsin fraction emerges at pH 3.5, well separated from traces of ,~-trypsin; a-trypsin is eluted at pH 3.8. Dialysis of the column fractions against 1 mM HC1 and lyophilization results in an approximately 75% overall yield of /?-trypsin. Comments The preparation of fl-trypsin by the procedure of Schroeder and Shaw employing ion-exchange chromatography in the presence of the competitive trypsin inhibitor benzamidine required almost 7 days and could not completely separate the contaminating a-form. 1 A similar resolution was found by Robinson et al. 7 on affinity chromatography of trypsin samples on Sepharose-bound chicken ovomucoid, although the operations required less than a day. In contrast, the formation of fl-trypsin from trypsinogen, 7N. C. Robinson, R. W. Tye, H. Neurath, and K. A. Walsh, Biochemistry 10, 2743 (1971).
[ 53 ]
UROKINASE
451
employing affinity chromatography on immobilized soybean trypsin inhibitor, requires 4 - 6 hours, and the fl-trypsin fraction is completely free of a-trypsin. The yield of fl-trypsin approaches the maximum possible based on the amount of trypsinogen. Furthermore, similar results were found with preparations of enterokinase that were not chromatographed on DE-52 columns if the same number of enzyme units were used. 3a Presumably, the same procedures could be applied to trypsinogens of other species.
[53] Urokinase B y THOMAS MACIAG, MICHAEL K. WEIBEL, and E. KENDALL PYE
Urokinase, a plasm!nogen-activating proteolytic enzyme, was first described in 19511 as occurring in trace quantities in mammalian urine. Plasminogen-activating enzymes have also been shown to occur in a number of mammalian tissues -~,:~ and recently have been obtained from cultures of cell lines derived from various tissues. 4,~ A renewed interest in urokinase has now arisen because of its therapeutic potential as an effective thrombolytic agent. ~ However, large-scale clinical trials have been delayed in the United States because the recovery and purification of urokinase from human urine by classical techniques ~ has provided inadequate amounts of the highly purified enzyme. It has recently been shown that relatively high concentrations of plasminogen activators appear in the growth medium of various human tissue culture preparations, 5 and, because of the versatility and high recoveries possible with affinity chromatography, we have applied this technique to the recovery and purification of the plasminogen-activating enzyme from these sources? Since there is a lack of conclusive proof that this enzyme is not urokinase and since the technique can also be used for the recovery of
lj. R. B. Williams, Brit. J. Exp. Pathol. 32, 530 (1951). ~O. K. Albrechtsen, Acta Physiol. Scand. 39, 284 (1957). 30. K. Albrechtsen, Brit. I. Haematol. 3, 284 (1957). ' P. Kelly, personal communication, 1972. "C. S. Kucinski, A. P. Fletcher, and S. Sherry, J. Clin. Invest. 47, 1238 (1968). " Urokinase Pulmonary Embolism Trial Study Group, J. Amer. Med. Ass. 214, 2163 (1970). ' W. F. White and G. H. Barlow, this series, Vol. 19, p. 665. ST. Maciag, M. K. Weibel, and E. K. Pye, hz "Enzyme Engineering" (E. K. Pye and L. Wingard, Jr., eds.). Plenum, New York, 1974.
AMINO ACIDS AND PEPTIDES
[52]
m a n is o r t h a t of T h o m p s o n a n d Davie. 1'~ S c h m e r 1~- has r e p o r t e d trace quantities of p l a s m i n in affinity-purified t h r o m b i n which c o u l d b e r e m o v e d b y S E - S e p h a d e x c h r o m a t o g r a p h y either b e f o r e or after the affinity c h r o m a t o g r a p h y step. Such c o m b i n e d c h r o m a t o g r a p h y p r o d u c e s t h r o m b i n with 2 0 0 0 N I H u n i t s / m g . 28D. J. Baughman, this series, Vol. 19, p. 145.
[52]
Bovine
fl-Trypsin
B y ALBERT LIGHT a n d JURIS LIEPNIEKS
Schroeder and Shaw 1 reported in 1968 that chromatography of trypsin and activated trypsinogen on columns of SE-Sephadex C-50 showed the presence of almost equal amounts of two active forms of trypsin and a small amount of inert protein. One active form is B-trypsin, an intact molecule; the second is a-trypsin, a product of autolysis with Lys 131Ser 132 cleaved. Further slow proteolysis produces ~-trypsin with both Lys 131-Ser 132 and Lys 176-Asp 177 hydrolyzed."- It is clear from these studies that commercially available trypsins are mixtures of species and that studies on the properties of trypsin should use the B-form. High yields of B-trypsin can be obtained from bovine trypsinogen if enterokinase is used as activator since the enzyme is highly specific for the Lys 6-Ile 7 peptide bond. 3 However, autolysis of B-trypsin must be prevented; the presence of soybean trypsin inhibitor effectively inhibits B-trypsin but does not interfere with the activity of enterokinase. If the inhibitor is bound to Sepharose, affinity chromatography of the activation mixture provides a rapid procedure for the production of highly purified B-trypsinY" This procedure is described here. Assay Methods
Measurement oJ Trypsin Activity Trypsin activity is measured at pH 7.9 and 25 ° using 10 mM N-tosylL-arginine methyl ester in 10 mM Tris chloride and 50 mM calcium chloride. 4 -~D. D. Schroeder and E. Shaw, J. Biol. Chem. 243, 2943 (1968). -"R. L. Smith and E. Shaw, J. Biol. Chem. 244, 4704 (1969). 3 S. Maroux, J. Baratti, and P. Desnuelle, J. Biol. Chem. 246, 5031 (1971). 3, j. j. Liepnieks and A. Light, Anal. Biochem., in press. 4K. A. Walsh and P. E. Wilcox, this series, Vol. 19 [3].
[52]
BOVINE fl-TRYPSIN
449
Measurement of Enterokinase Activity
Enterokinase activity is determined by activation of 0.1 mg of trypsinogen per milliliter of 0.1 M sodium acetate, pH 5.0, and 50 mM calcium chloride. Samples of 0.5-7.0 enterokinase units are used in a 30-minute incubation at 35 °. Tryptic activity is measured in 0.5 ml fractions with N-tosyl-L-arginine methyl ester. An e~nterokinase unit is defined as the amount of enzyme which produces 1 trypsin unit per milliliter of activation mixture. Estimation of Aminopeptidase Activity
A spot test for the detection of aminopeptidase is used to examine chromatographic fractions? Usually, 10-~l samples and 0.5 ml of 0.3 mM leucyl fl-naphthylamide in 50 mM Tris chloride at pH 8.5, containing 5 mM calcium chloride are incubated at room temperature for 30 minutes in depressions of a porcelain spot plate. One adds 0.2 ml of naphthanil diazo blue (5 mg per milliliter of water), and the immediate appearance of a stable pink color is indicative of aminopeptidase activity. The relative intensity of color is used to judge maximum activity. Preparation of fl-Trypsin A suitable amount of enterokinase for the activation of approximately 300 mg of trypsinogen is prepared as follows. A crude enteropeptidase preparation (3.25 g, Miles Laboratories) is extracted with 325 ml of l0 mM ammonium acetate at pH 6.0. After stirring for 1 hour at room temperature, the suspension is centrifuged and the supernatant fluid is dialyzed exhaustively against water at 4 °. After lyophilization, 500 mg of a light brown powder is recovered. The powder is dissolved in 100 ml of l0 mM Tris chloride at pH 6.0, containing 50 mM sodium chloride. The insoluble material is removed by centrifugation, and the supernatant liquid is chromatographed on DEAE-cellulose (Whatman microgranular DE-52). Aminopeptidase activity is detected in the first half of the peak containing enterokinase activity. Removal of almost all the aminopeptidase contaminant is achieved by combining appropriate fractions. After dialysis against water at 4 ° and then lyophilization, 44 mg of a white powder is obtained containing approximately 72 units/rag. Sepharose-bound soybean trypsin inhibitor is prepared by a slight modification of a procedure previously described. ~ Settled Sepharose 4B, ~J. E. Folk, J. A. Gladner, and T. Viswanatha, BiochmT. B~phys. Acm 36, 256 (1959). P. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, Proc. Nat. Acad. Sci. U.S. 61, 636 (1968).
450
AMINO ACIDS AND PEPTIDES
[52]
20 ml, is treated with 3 g of cyanogen bromide at pH 11. The suspension is gently agitated for about 8 minutes with a magnetic stirrer. After washing with water and cold 0.1 M sodium bicarbonate, pH 9.5, 500 mg of soybean trypsin inhibitor are added and the suspension (40% by volume in 0.1 M sodium bicarbonate) is agitated by rotating slowly in a bottle for 18 hours at 4 °. The preparation is washed with 100 ml of 0.2 M sodium formate at pH 2.2, and 0.2 M Tris chloride at pH 8.0, repeating the washing cycle four times. The second Tris buffer wash contains 15 g of glycine per liter to react with residual cyanogen bromide activated sites. The preparation of Sepharose-bound soybean trypsin inhibitor binds approximately 5 mg of trypsin per milliliter of settled gel as determined operationally by applying excess trypsin at pH 8.0 and eluting bound trypsin at pH 2.2. Activation of bovine trypsinogen is accomplished by mixing 30 mg of zymogen with 4 mg of partially purified enterokinase (300 units) in 0.1 M Tris, pH 8.0, containing 50 mM calcium chloride. The activation mixture includes Sepharose-bound soybean trypsin inhibitor in approximately a 2-fold excess over the amount of trypsin that could be produced from the zymogen. The total volume of the mixture is 25 ml and it is incubated at 35° for 60 minutes with slow agitation obtained by bubbling nitrogen gas through the suspension. The activation mixture is transferred to a chromatography column (1.4 X 12 cm), and inert components are removed on washing the gel with 40 ml of 0.1 M sodium formate at pH 4.5, containing 50 mM calcium chloride, fl-Trypsin is released from the complex with 0.1 M sodium formate at pH 2.6 containing 50 mM calcium chloride or with a pH gradient using equal volumes of the formate buffers at the two pH values. The fl-trypsin fraction emerges at pH 3.5, well separated from traces of ,~-trypsin; a-trypsin is eluted at pH 3.8. Dialysis of the column fractions against 1 mM HC1 and lyophilization results in an approximately 75% overall yield of /?-trypsin. Comments The preparation of fl-trypsin by the procedure of Schroeder and Shaw employing ion-exchange chromatography in the presence of the competitive trypsin inhibitor benzamidine required almost 7 days and could not completely separate the contaminating a-form. 1 A similar resolution was found by Robinson et al. 7 on affinity chromatography of trypsin samples on Sepharose-bound chicken ovomucoid, although the operations required less than a day. In contrast, the formation of fl-trypsin from trypsinogen, 7N. C. Robinson, R. W. Tye, H. Neurath, and K. A. Walsh, Biochemistry 10, 2743 (1971).
[ 53 ]
UROKINASE
451
employing affinity chromatography on immobilized soybean trypsin inhibitor, requires 4 - 6 hours, and the fl-trypsin fraction is completely free of a-trypsin. The yield of fl-trypsin approaches the maximum possible based on the amount of trypsinogen. Furthermore, similar results were found with preparations of enterokinase that were not chromatographed on DE-52 columns if the same number of enzyme units were used. 3a Presumably, the same procedures could be applied to trypsinogens of other species.
[53] Urokinase B y THOMAS MACIAG, MICHAEL K. WEIBEL, and E. KENDALL PYE
Urokinase, a plasm!nogen-activating proteolytic enzyme, was first described in 19511 as occurring in trace quantities in mammalian urine. Plasminogen-activating enzymes have also been shown to occur in a number of mammalian tissues -~,:~ and recently have been obtained from cultures of cell lines derived from various tissues. 4,~ A renewed interest in urokinase has now arisen because of its therapeutic potential as an effective thrombolytic agent. ~ However, large-scale clinical trials have been delayed in the United States because the recovery and purification of urokinase from human urine by classical techniques ~ has provided inadequate amounts of the highly purified enzyme. It has recently been shown that relatively high concentrations of plasminogen activators appear in the growth medium of various human tissue culture preparations, 5 and, because of the versatility and high recoveries possible with affinity chromatography, we have applied this technique to the recovery and purification of the plasminogen-activating enzyme from these sources? Since there is a lack of conclusive proof that this enzyme is not urokinase and since the technique can also be used for the recovery of
lj. R. B. Williams, Brit. J. Exp. Pathol. 32, 530 (1951). ~O. K. Albrechtsen, Acta Physiol. Scand. 39, 284 (1957). 30. K. Albrechtsen, Brit. I. Haematol. 3, 284 (1957). ' P. Kelly, personal communication, 1972. "C. S. Kucinski, A. P. Fletcher, and S. Sherry, J. Clin. Invest. 47, 1238 (1968). " Urokinase Pulmonary Embolism Trial Study Group, J. Amer. Med. Ass. 214, 2163 (1970). ' W. F. White and G. H. Barlow, this series, Vol. 19, p. 665. ST. Maciag, M. K. Weibel, and E. K. Pye, hz "Enzyme Engineering" (E. K. Pye and L. Wingard, Jr., eds.). Plenum, New York, 1974.