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[6] Chymotrypsin C (porcine pancreas)
[6]
CHYMOTRYPSIN c (PORCINE PANCREAS)
109
[6] Chymotrypsin C (Porcine Pancreas) By J. E. FOLX R'R"CHCOR'" ---* R'R~CHC00 - -t- +HsNR or H0R where R' is (usually) an acylamido group; R" is the side chain of a variety of neutral amino acids; and R'" is --NHR or --OR.
Assay Method1 Principle. The method is based upon measurement of change in ultraviolet absorbancy that occurs as a result of enzymatic hydrolysis of N-benzoyl-L-leucine ethyl ester to N-benzoyl-L-leucine and ethanol. Reagents Tris(hydroxymethyl) aminomethane(Tris)-HC1 buffer, pH 7.8, 0.08 M in Tris N-Benzoyl-L-leucine ethyl ester, 1 0.01 M in absolute methanol Absolute methanol Enzyme. Dissolve enzyme in cold water and dilute with cold water to obtain a solution containing 1 to 10 units/ml.
Procedure. The incubation mixture contains 1.5 ml of buffer, 0.6 ml of substrate, 0.3 ml of methanol, 0.55 ml of water, and 0,05 ml of enzyme. The change in absorbancy is measured in a 0.5-em quartz cuvette at 254 nm and 25 °. The hydrolysis of 1 mieromole of substrate causes an increase in absorbancy of 0.222. Definition o] Unit and Specific Activity. One unit of enzyme activity is defined as that amount of enzyme t h a t catalyzes the hydrolysis of 1 micromole of substrate per minute in the above assay. Specific activity is the number of units per milligram of protein; protein concentration is determined by the trichloroacetic acid turbidimetric procedure ~ in early ~1% of 25.01 in the chromatogstages of purification and by the use of an ~278 raphy steps. Purification Procedure 1
Step 1. Extraction of Acetone Powder. One hundred grams of an acetone powder of autolyzed swine pancreas, prepared as described in this volume for the isolation of carboxypeptidase B [32] is extracted with 1 liter of distilled water at room temperature by gently stirring for 1j. E. Folk and E. W. Schirmer, J. Biol. Chem. 240, 181 (1965). : T. Biicher, Biochim. Biophys. Acta 1, 292 (1947).
110
THE SERINE PROTEASES
[5]
30-45 minutes. The extract is obtained as a clear supernatant fluid after centrifugation for 30 minutes at 15,000 g. Step 2. Ammonium Sul]ate Fractionation. The extract is cooled to 2o-5 ° and adjusted to pH 7 to 7.2 with 1 N N a 0 H . All further operations are performed in this temperature range. Solid (NH4)2SO4 (243 g/liter) is added gradually with stirring while the pH is maintained between 7 and 7.2. The precipitate is removed by eentrifugation for 15 minutes at 15,000 g.a Solid (NH4)2S04 (132 g/liter) is added to the supernatant fluid while the pH is maintained as before. The suspension is stirred for 30 minutes and the precipitate is collected by centrifugation for 30 minutes at 15,000 g. This precipitate is dissolved in about 50 ml of distilled water and is dialyzed against distilled water for 18 hours. Step 3. First DEAE-Cellulose Chromatography. The dialyzed protein solution is applied to a column, 3.5 X 20 cm, of DEAE-cellulose which has previously been equilibrated with 5 mM Tris-HC1, pH 8.0. The protein solution is washed into the column with about 20 ml of the same buffer and the chromatogram is developed with a linear gradient of NaC1 from 0 to 0.45 M NaC1 in 1500 ml of 5 mM Tris, pH 8.0. Fractions of 10 ml are collected at a flow rate of 6-8 ml/minute. The ehymotrypsin C emerges from the column between 0.3 and 0.4 M NaC1. The fractions containing the enzyme are combined and treated with solid (NH,),SO, (43 g/100 ml). The precipitate obtained upon centrifuging this suspension is dissolved in 5-10 ml of distilled water. This solution of partially purified enzyme is dialyzed against distilled water for 18 hours. Step 4. Second DEAE-CeUulose Chromatography. The enzyme solution obtained above is applied to a column, 3.5 X 10 cm, of DEAEcellulose which has previously been equilibrated with 5 mM Tris-acetate, pH 6.0. The protein solution is washed into the column with 20 ml of the same buffer and the chromatogram is developed with a linear gradient of NaC1 from 0 to 0.45 M in 750 ml of Tris-acetate, pH 6.0. Fractions of 5 ml are collected at a flow rate of 4-5 ml/minute. The enzyme emerges from the column between 0.3 and 0.4 M NaC1. The fractions containing the enzyme are combined and treated with (NH4)~SO, (43 g/100 ml). The precipitate of purified enzyme is dissolved in a minimum volume of distilled water and dialyzed against several changes of distilled water. In order to obtain the purest preparations of chymotrypsin C, the dialyzed enzyme may be rechromatographed as outlined in this step. The salt-free dialyzed enzyme solution is lyophilized and stored at 5 °. SThe precipitate obtained upon 0.4 saturation with (NI-L)~04 may be conveniently used for the purification of porine carboxypeptidase A according to J. E. Folk • and E. Schirmer, J. Biol. Chem. 238, 3884 (1963).
[5]
111
CHYMOTRYPSIN C (PORCINE PANCREAS) PURIFICATION OF CHYMOTRYPSIN C FROM ACETONE POWDER OF AUTOLYZED PORCINE PANCREASa
Step 1. Acetone powder extract 2. (NH~)2SO4fractionation 3. First DEAE-cellulose chromatography, pH 8.0 4. Second DEAE-cellulose chromatography, pH 6.0
Units
Specific activity (units/mg protein)
Yield (%)
12,000 1,600 333
4000 2360 1000
0.33 1.5 3
100 59 25
150
600
Protein (mg)
4
15
100 g of acetone powder is used.
The results of a typical purification are summarized in the table. This procedure has been repeated many times with similar results. The large losses in benzoyl-L-leucine ethyl ester hydrolyzing activity in the first DEAE-cellulose chromatography step may be explained by the fact that this ester is not a specific substrate for chymotrypsin C. Chymotrypsin A, which also hydrolyzes the ester, does not chromatograph on DEAE-cellulose under the present conditions and appears in the forepeak of the first chromatogram.
Properties Stability. The dry purified enzyme powder has been kept frozen for periods up to 4 years without detectable loss in activity. Solutions of the enzyme in water or Tris buffer at pH 6-8.5 (5-20 mg/ml), under toluene vapors to prevent bacterial contamination, may be kept stored for several weeks without loss in activity. The purified enzyme in solution loses activity rapidly at acid pH. At pH 3 and 0 ° a 12% loss in activity occurs in 20 minutes. Purity and Physicochemical Properties. The enzyme prepared by this procedure contains no protein impurities detectable by the usual physical procedures, including disc gel electrophoresis. There is, however, a small amount of contaminating carboxypeptidase A activity present in all preparations. This activity may be eliminated in digestion experiments by addition of 5-10 mM 1,10-phenanthroline, an effective inhibitor of the carboxypeptidases. 4 1,10-Phenanthroline does not inhibit chymotrypsin C.l~ 4
A preliminary estimate of molecular weight of 23,800 has been made 4j. E. Folk and P. W. Cole, J. Biol. Chem. 240~ 193 (1965).
112
THE SERINE PROTEASES
[5]
on the basis of sedimentation and diffusion (s~°o.w 2.67 and D2o,~ 10.1 X 10-7 cm 2 see-l). The sedimentation and diffusion behavior of the enzyme is indicative of a concentration-dependent, association-dissociation equilibrium. Chymotrypsin C shows the following amino acid composition: Asp22 Thr14 Ser2o Glu21 PrOl2 Gly25 Ala~2 ½ Cyst Vale9 Met~ Ile~2 LeUl9 Tyre Phe4 Lys7 His~ Arg7 Trps Amide N~4.1 The sequence of amino acids around the essential serine has been determined as Gly-Asp-Ser-GlyS; that around an essential histidine as Ala-Ala-His-Cys-Ile-Asp-Ser-GlyThr-Ser-Arg-Thr2 Specificity. The purified enzyme catalyzes the hydrolysis of peptide bonds that contain the carboxyl group of a variety of a-amino acids including tyrosine, phenylalanine, tryptophan, methionine, leucine, glutamine, and asparagine. ~ It has been suggested that the broad specificity of chymotrypsin C toward peptide substrates is directed by a-amino acids, the side chains of which contain at the ~-carbon atom an uncharged carbon-containing monosubstitution.~ The enzyme also catalyzes the esterolysis of a variety of N-substituted amino acid esters. 1 Activators and Inhibitors. The enzyme is active over a broad range of pH from about 5 to 9. It displays optimum activity in Tris or NH, HCO3 buffers at pH 7.8-8. It is readily inactivated by diisopropyl phosphorofluoridate,1 by diphenylcarbamyl chloride,1 and by tosyl-L-leucine chloromethyl ketone. 6 Distribution. The enzyme has been isolated only from swine pancreas glands. It exists in fresh pancreas tissue as an inactive zymogen. The zymogen has been purified and partially characterized. 1 The enzyme is similar to, if not identical with, a crystalline "esteroproteolytic" enzyme of porcine pancreas described by Gjessing and Hartnett. 7 A similar enzymatic activity of bovine pancreatic gland has been described,s
5T. Tobita and J. E. Folk, unpublished data. ST. Tobita and J. E. Folk, Biochim. Biophys. Acta 147, 15 (1967). 7E. C. Gjessing and J. C. Hartnett, J. Biol. Chem. 237, 2201 (1962). sj. R. Brown, M. Yamasaki, and H. Neurath, Biochemistry 2, 877 (1963).
CHYMOTRYPSIN c (PORCINE PANCREAS)
109
[6] Chymotrypsin C (Porcine Pancreas) By J. E. FOLX R'R"CHCOR'" ---* R'R~CHC00 - -t- +HsNR or H0R where R' is (usually) an acylamido group; R" is the side chain of a variety of neutral amino acids; and R'" is --NHR or --OR.
Assay Method1 Principle. The method is based upon measurement of change in ultraviolet absorbancy that occurs as a result of enzymatic hydrolysis of N-benzoyl-L-leucine ethyl ester to N-benzoyl-L-leucine and ethanol. Reagents Tris(hydroxymethyl) aminomethane(Tris)-HC1 buffer, pH 7.8, 0.08 M in Tris N-Benzoyl-L-leucine ethyl ester, 1 0.01 M in absolute methanol Absolute methanol Enzyme. Dissolve enzyme in cold water and dilute with cold water to obtain a solution containing 1 to 10 units/ml.
Procedure. The incubation mixture contains 1.5 ml of buffer, 0.6 ml of substrate, 0.3 ml of methanol, 0.55 ml of water, and 0,05 ml of enzyme. The change in absorbancy is measured in a 0.5-em quartz cuvette at 254 nm and 25 °. The hydrolysis of 1 mieromole of substrate causes an increase in absorbancy of 0.222. Definition o] Unit and Specific Activity. One unit of enzyme activity is defined as that amount of enzyme t h a t catalyzes the hydrolysis of 1 micromole of substrate per minute in the above assay. Specific activity is the number of units per milligram of protein; protein concentration is determined by the trichloroacetic acid turbidimetric procedure ~ in early ~1% of 25.01 in the chromatogstages of purification and by the use of an ~278 raphy steps. Purification Procedure 1
Step 1. Extraction of Acetone Powder. One hundred grams of an acetone powder of autolyzed swine pancreas, prepared as described in this volume for the isolation of carboxypeptidase B [32] is extracted with 1 liter of distilled water at room temperature by gently stirring for 1j. E. Folk and E. W. Schirmer, J. Biol. Chem. 240, 181 (1965). : T. Biicher, Biochim. Biophys. Acta 1, 292 (1947).
110
THE SERINE PROTEASES
[5]
30-45 minutes. The extract is obtained as a clear supernatant fluid after centrifugation for 30 minutes at 15,000 g. Step 2. Ammonium Sul]ate Fractionation. The extract is cooled to 2o-5 ° and adjusted to pH 7 to 7.2 with 1 N N a 0 H . All further operations are performed in this temperature range. Solid (NH4)2SO4 (243 g/liter) is added gradually with stirring while the pH is maintained between 7 and 7.2. The precipitate is removed by eentrifugation for 15 minutes at 15,000 g.a Solid (NH4)2S04 (132 g/liter) is added to the supernatant fluid while the pH is maintained as before. The suspension is stirred for 30 minutes and the precipitate is collected by centrifugation for 30 minutes at 15,000 g. This precipitate is dissolved in about 50 ml of distilled water and is dialyzed against distilled water for 18 hours. Step 3. First DEAE-Cellulose Chromatography. The dialyzed protein solution is applied to a column, 3.5 X 20 cm, of DEAE-cellulose which has previously been equilibrated with 5 mM Tris-HC1, pH 8.0. The protein solution is washed into the column with about 20 ml of the same buffer and the chromatogram is developed with a linear gradient of NaC1 from 0 to 0.45 M NaC1 in 1500 ml of 5 mM Tris, pH 8.0. Fractions of 10 ml are collected at a flow rate of 6-8 ml/minute. The ehymotrypsin C emerges from the column between 0.3 and 0.4 M NaC1. The fractions containing the enzyme are combined and treated with solid (NH,),SO, (43 g/100 ml). The precipitate obtained upon centrifuging this suspension is dissolved in 5-10 ml of distilled water. This solution of partially purified enzyme is dialyzed against distilled water for 18 hours. Step 4. Second DEAE-CeUulose Chromatography. The enzyme solution obtained above is applied to a column, 3.5 X 10 cm, of DEAEcellulose which has previously been equilibrated with 5 mM Tris-acetate, pH 6.0. The protein solution is washed into the column with 20 ml of the same buffer and the chromatogram is developed with a linear gradient of NaC1 from 0 to 0.45 M in 750 ml of Tris-acetate, pH 6.0. Fractions of 5 ml are collected at a flow rate of 4-5 ml/minute. The enzyme emerges from the column between 0.3 and 0.4 M NaC1. The fractions containing the enzyme are combined and treated with (NH4)~SO, (43 g/100 ml). The precipitate of purified enzyme is dissolved in a minimum volume of distilled water and dialyzed against several changes of distilled water. In order to obtain the purest preparations of chymotrypsin C, the dialyzed enzyme may be rechromatographed as outlined in this step. The salt-free dialyzed enzyme solution is lyophilized and stored at 5 °. SThe precipitate obtained upon 0.4 saturation with (NI-L)~04 may be conveniently used for the purification of porine carboxypeptidase A according to J. E. Folk • and E. Schirmer, J. Biol. Chem. 238, 3884 (1963).
[5]
111
CHYMOTRYPSIN C (PORCINE PANCREAS) PURIFICATION OF CHYMOTRYPSIN C FROM ACETONE POWDER OF AUTOLYZED PORCINE PANCREASa
Step 1. Acetone powder extract 2. (NH~)2SO4fractionation 3. First DEAE-cellulose chromatography, pH 8.0 4. Second DEAE-cellulose chromatography, pH 6.0
Units
Specific activity (units/mg protein)
Yield (%)
12,000 1,600 333
4000 2360 1000
0.33 1.5 3
100 59 25
150
600
Protein (mg)
4
15
100 g of acetone powder is used.
The results of a typical purification are summarized in the table. This procedure has been repeated many times with similar results. The large losses in benzoyl-L-leucine ethyl ester hydrolyzing activity in the first DEAE-cellulose chromatography step may be explained by the fact that this ester is not a specific substrate for chymotrypsin C. Chymotrypsin A, which also hydrolyzes the ester, does not chromatograph on DEAE-cellulose under the present conditions and appears in the forepeak of the first chromatogram.
Properties Stability. The dry purified enzyme powder has been kept frozen for periods up to 4 years without detectable loss in activity. Solutions of the enzyme in water or Tris buffer at pH 6-8.5 (5-20 mg/ml), under toluene vapors to prevent bacterial contamination, may be kept stored for several weeks without loss in activity. The purified enzyme in solution loses activity rapidly at acid pH. At pH 3 and 0 ° a 12% loss in activity occurs in 20 minutes. Purity and Physicochemical Properties. The enzyme prepared by this procedure contains no protein impurities detectable by the usual physical procedures, including disc gel electrophoresis. There is, however, a small amount of contaminating carboxypeptidase A activity present in all preparations. This activity may be eliminated in digestion experiments by addition of 5-10 mM 1,10-phenanthroline, an effective inhibitor of the carboxypeptidases. 4 1,10-Phenanthroline does not inhibit chymotrypsin C.l~ 4
A preliminary estimate of molecular weight of 23,800 has been made 4j. E. Folk and P. W. Cole, J. Biol. Chem. 240~ 193 (1965).
112
THE SERINE PROTEASES
[5]
on the basis of sedimentation and diffusion (s~°o.w 2.67 and D2o,~ 10.1 X 10-7 cm 2 see-l). The sedimentation and diffusion behavior of the enzyme is indicative of a concentration-dependent, association-dissociation equilibrium. Chymotrypsin C shows the following amino acid composition: Asp22 Thr14 Ser2o Glu21 PrOl2 Gly25 Ala~2 ½ Cyst Vale9 Met~ Ile~2 LeUl9 Tyre Phe4 Lys7 His~ Arg7 Trps Amide N~4.1 The sequence of amino acids around the essential serine has been determined as Gly-Asp-Ser-GlyS; that around an essential histidine as Ala-Ala-His-Cys-Ile-Asp-Ser-GlyThr-Ser-Arg-Thr2 Specificity. The purified enzyme catalyzes the hydrolysis of peptide bonds that contain the carboxyl group of a variety of a-amino acids including tyrosine, phenylalanine, tryptophan, methionine, leucine, glutamine, and asparagine. ~ It has been suggested that the broad specificity of chymotrypsin C toward peptide substrates is directed by a-amino acids, the side chains of which contain at the ~-carbon atom an uncharged carbon-containing monosubstitution.~ The enzyme also catalyzes the esterolysis of a variety of N-substituted amino acid esters. 1 Activators and Inhibitors. The enzyme is active over a broad range of pH from about 5 to 9. It displays optimum activity in Tris or NH, HCO3 buffers at pH 7.8-8. It is readily inactivated by diisopropyl phosphorofluoridate,1 by diphenylcarbamyl chloride,1 and by tosyl-L-leucine chloromethyl ketone. 6 Distribution. The enzyme has been isolated only from swine pancreas glands. It exists in fresh pancreas tissue as an inactive zymogen. The zymogen has been purified and partially characterized. 1 The enzyme is similar to, if not identical with, a crystalline "esteroproteolytic" enzyme of porcine pancreas described by Gjessing and Hartnett. 7 A similar enzymatic activity of bovine pancreatic gland has been described,s
5T. Tobita and J. E. Folk, unpublished data. ST. Tobita and J. E. Folk, Biochim. Biophys. Acta 147, 15 (1967). 7E. C. Gjessing and J. C. Hartnett, J. Biol. Chem. 237, 2201 (1962). sj. R. Brown, M. Yamasaki, and H. Neurath, Biochemistry 2, 877 (1963).