716
N£TURALLY OCCURRING PROTEASE INIIIBITORS
[51]
Inhibitor II is a strong inhibitor of elastase when assayed with Congo red-elastin system. It inhibits both trypsin and elastase simultaneously and independently. Inhibitors I and IIIb also inhibit elastase, but with specific activities less than one-quarter that of inhibitor II. Reactive Site Residues. 1,~ Isoinhibitor I has lysine in the trypsinreactive site while isoinhibitor II has arginine. The location of the trypsin reactive site in isoinhibitor II is at Args~-Ser~4, and that of the elastase reactive site is at Ala2s-Ser27. Amino Acid Composition? The amino acid composition of isoinhibitors I, II, and IIIb is shown in Table II. The inhibitors contain no free --SH groups and no carbohydrates. Peptide Maps? The peptide maps of inhibitors I and IIIb are very similar; much less similarity is apparent between inhibitor II and either I or IIIb. Isoinhibitors I and II have no peptides in common. Partial Sequence. ~ The partial amino acid sequence of isoinhibitor II is 3O - (Val,Cys) Thr-Ala-Ser-Ile-Pro-Pro-Gln (Cys,Ile, Cys,Thr,Asx,Val) 40 50 Arg-Leu-Asx-Ser-Cys-His-Ser-Ala-Cys-Lys-Ser-Cys-Met-Cys-Thr-Arg-Ser6o 7o Met-Pro-Gly-Lys-Cys-Arg-Cys-Leu-Asx-Thr-Thr-Asx-Tyr-Cys-Tyr-Lys-SerCys-Lys-Ser-Asx-Ser- Gly-Glx-Asx-Asx
[51] A T r y p s i n a n d C h y m o t r y p s i n I n h i b i t o r f r o m Groundnuts (Arachis hypogaea) B y YEHUDITH BIRK Assay Methods I
Inhibition of Proteolysis Principle. Inhibition of proteolysis is determined by Kunitz's casein digestion method, 2 which has been described in a previous volume of this treatise? 1 A. Tur-Sinai, Y. Birk, A. Gertler, and M. Rigbi, Biochim. Biophys. Acta 263, 666 (1972). 2 M. Kunitz, J. Gen. Physiol. 30, 291 (1947). a M. Laskowski, Sr., this series Vol. 2 [3].
[51]
GROUNDNUTS T R Y P S I N - AND CHYMOTR Y P S IN -IN H IB ITO R
717
Definition o] Unit. Kunitz ~ defined 1 tryptic or chymotryptic unit as the enzyme activity which causes an increase of 1 unit of absorbance at 280 nm per minute of digestion of casein, under the standard conditions. The specific activity is expressed as units per microgram of enzyme protein. Enzyme-inhibiting activity is expressed as units of enzyme inhibited, and specific activity as units of enzyme inhibited per absorbance unit, at 280 nm, of inhibitor. Inhibition o] Esterolysis Principle. Inhibition of the esterolytic activity of trypsin is determined on the substrate p-toluenesulfonyl-L-arginine methyl ester (TAME) either spectrophotometrically or by a potentiometric method. Inhibition of the esterolytic activity of chymotrypsin is determined on the substrate acetyl-L-tyrosine methyl ester (ATEE) in a similar manner. Spectrophotometric Assay Reagents Buffer: Tris-chloride, 46 mM, pH 8.1, containing 11.5 mM CaCI~ Substrate: Dissolve 37.9 mg of TAME (Sigma) in 10 ml of water. Mix 3 ml of this solution with 26 ml of the buffer. Enzyme: Trypsin (Worthington), 1 mg/ml in 1 mM HC1
Procedure. The reaction is performed in a Gilford 2400 spectrophotometer, at constant temperature (25°). Preincubate for 5 min 25 ~1 of enzyme solution with the inhibitor (up to 150 ~l) in 1 ml of the buffer, at room temperature. In each of two 1-cm quartz cuvettes, pipette 3 ml of substrate and place one of them as a blank into the spectrophotometer. Set the wavelength to 247 nm. Start the reaction by adding 100 td of the enzyme-inhibitor mixture (after 5 min of preincubation) to the second cuvette. Mix thoroughly and record change in absorbance for about 4 min. Definition o] Units. One trypsin unit is defined as 1 t~mole of substrate hydrolyzed per minute of reaction. One inhibition unit is defined as unit of enzyme inhibited. Specific activity is defined as inhibition units per absorbance unit, at 280 nm, of the inhibitor. Chymotrypsin. Inhibition of the esterolytic activity of chymotrypsin is determined on ATEE in the same way as described above for trypsin except for the following changes.
718
NATURALLY
OCCURRING
PROTEASE
INHIBITORS
[61]
Substrate: Dissolve 24.5 mg of ATEE (Sigma) in 10 ml of water. Mix 6 ml of this solution with 24 ml of the buffer Enzyme: Chymotrypsin (Worthington), 1 mg/ml in 1 mM HC1 The preincubation mixture contains 40 td of the enzyme with up to 150 ~l of the inhibitor in 1 ml of the buffer. The change in absorbance is recorded at 237 nm.
Potentiometric Assay
Reagents Buffer: Tris-chloride 3 raM, pH 8.0, containing 45 mM KC1 and 150 mM CaC12 Substrate: TAME (Sigma) 56.85 mg in 10 ml of water Enzyme: Trypsin (Worthington), 250 ~g/ml in 1 mM HC1
Procedure. Assays are performed in a Radiometer T T T l c pH stat at a constant temperature (25°). The syringe is filled with standardized 0.1 M NaOH, and the titrator is set to raise the pH to 8.0. In the reaction vessel preincubate for 2 min 50 ~l of the enzyme with up to 200 ~l of the inhibitor, in 1 ml of the buffer. Start the reaction by adding 1 ml of substrate. Record the rate of NaOH uptake for 2 min. The slope of the linear relationship between NaOH consumed at the time of reaction, gives a direct measure of micromoles of substrate consumed per minute. Definition o] Unit. The units are the same as for the spectrophotometric method. Chymotrypsin. Inhibition of esterolytic activity of chymotrypsin is determined on ATEE in the same way as described above for trypsin, except for the following changes: Substrate: Dissolve 48.56 mg of ATEE (Sigma) in 10 ml of water Enzyme: Chymotrypsin (Worthington) 200 t~g/ml in HC1, 1 mM Purification Procedures
Method A 1 The starting material is defatted groundnut flour prepared by two consecutive ether extractions, in a Soxhlet aparatus, of coarsely ground groundnuts, Virginia Sihit Meshubahat variety. The defatted meal is then finely ground to flour. All operations are performed at room temperature. The purification is summarized in Table I.
[61]
GROUNDNUTS
TRYPSIN-
AND
719
CHYMOTRYPSIN-INttIBITOR
¢4¢'~
c',l cq
¢",1
5"~
¢t5¢~ ['-- ,"-~
, ~ l'-,. ['-- "~
,~ ~
ce5 ¢D
© Z
~
¢ v ¢.D ¢D
g~ ~
Z
~a0
Z
r~ 5~
Z
'~
"
t[
720
NATURALLY OCCURRING PROTEASE INHIBITORS
[~1]
Step 1. Extraction and Ammonium Sul]ate Fractionation. Suspend 370 g of the starting material in 3.7 liters of 0.02 M HC1, yielding a pH of 4.8. Stir for 1 hr. Remove the undissolved residue by centrifugation for 20 min at 1000 g. Bring the supernatant to pH 7.0 with 1 M NaOH and clarify by filtration. Add solid ammonium-sulfate to 70% saturation, collect the resulting precipitate by centrifugation, and dissolve in 180 ml of distilled water. Dialyze swiftly against distilled water (12 changes, every 45 rain, against 5 liters of distilled water, each). Step 2. Chromatography on a DEAE-Cellulose Column. Prepare a DEAE-cellulose column (35 X 3.6 cm) equilibrated with 3 mM ammonium acetate buffer pH 7.0---the starting buffer. Centrifuge the solution from step 1 and treat the yellowish active supernatant (about 240 ml) with ammonium acetate, pH 7.0, to 3 mM concentration. Apply to the column and elute first with about 800 ml of the starting buffer and then with ammonium acetate buffer, 0.06 M, pH 7.0. Collect 13-ml fractions at a flow rate of 260 ml/hr. Pool the first active fraction that emerges with the hold-up volume of the starting buffer (approximately tubes 14-45), and dialyze swiftly against 1 mM phosphate buffer pH 6.8. Step 3. Chromatography on a Calcium Phosphate Column. Prepare a calcium phosphate column (16 X 3.3 cm) according to the procedure described in a previous volume of this treatise. 4 Apply the dialyzate from step 2 to the column. Elute primarily with 300 ml of 1 mM phosphate buffer, pH 6.8, and then with 400 ml of 0.1 M of the same buffer. Collect 13-ml fractions at a flow rate of 300 ml/hr. Pool the active fraction (approximately tubes, 11-35) and dialyze swiftly against distilled water. Step 4. Chromatography on a CM-Cellulose Column. Prepare a CMcellulose column (40 X 1.8 cm) equilibrated with 30 mM ammonium acetate buffer pH 5.0. Treat the dialyzate from step 3 with ammonium acetate to 30 mM and apply to the column. Elute with ammonium acetate buffer pH 5.0, consecutively, as following: 450 ml of 0.03 M, 650 ml of 0.12 M, and 455 ml of 0.2 M. Collect 13-ml fractions at a flow rate of 150 ml/hr. Pool the active fractions (approximately tubes 95-106) and dialyze for 5 hr against distilled water. Lyophilize and store in a desiccator. Method B 5
The starting material is the dialyzed solution from step 1 (Method A). All the operations are carried out at 4% Prepare a chymotrypsin40. Levin, this series Vol. 5 [2l. 5 y . Birk, S. Khalef, and M. Rotm~n, personal communication.
[61]
GROUNDNUTS TRYPSIN- ANn CHYMOTRYPSIN-INHIBITOR
721
Sepharose column 6 (3.6 X 16 cm). Equilibrate with Tris-chloride, 50 mM, pH 8.0, containing 20 mM CaC12 and 200 mM KCl--the starting buffer. Centrifuge the solution from step 1 for 20 min at 1000 g. Bring the supernatant to pH 8.0 and to the molarity of the starting buffer and apply to the column. Elute inactive proteins first with the starting buffer until no more protein appears in the effluent and then elute with 0.2 M KC1 pH 2.0. Collect 5-ml fractions at a flow rate of 100 ml/hr maintained with a peristaltic pump. Pool the active fraction and dialyze against distilled water. Lyophilize and store at room temperature.
Properties Stability. l The inhibitor is stable when kept in acetate buffer solutions of pH 2-11 for 24 hr at 5 °, or when boiled for 15 min in Trischloride buffer, pH 8.0. It is stable when incubated with trypsin or chymotrypsin at pH 8.0 for 36 hr at 5 ° and at room temperature. P u r i t y 1,~ The inhibitor prepared by Method A is a pure homogeneous protein according to polyacrylamide gel electrophoresis at pH 4.5 and pH 8.9, and by isoelectric focusing in polyacrylamide gels in the pH range of 3-10. The inhibitor prepared by affinity chromatography (Method B) gave 4 active bands--presumably isoinhibitors--in microzone electrophoresis on cellulose acetate strips at pH 6.9. Physical Properties.~ The molecular weight is 7450-7700 by sedimentation and diffusion measurements, sedimentation equilibrium, gel ehromatography and amino acid composition. The s.,0.,~ is 1.4 X 10-13, the D20.~ is 14.67 × 10-7, and the partial specific volume, as calculated from the amino acid composition, is 0.69. The specific extinction at 280 nm is ~~1~ i cm = 2.5. The inhibitor has an isoelectric pH of 8-9, estimated by isoeleetric focusing in polyacrylamide gels. Specifity. 1,7 The inhibitor inhibits both trypsin and ehymotrypsin. The enzyme-inhibitor complex is formed by 1 mole of inhibitor with 1 mole of trypsin or ehymotrypsin. Full inhibition of proteolysis and nearly full inhibition of esterolysis is attained by this ratio. The trypsin-inhibitor complex formed at pH 7.6 is devoid of tryptic activity as well as of trypsin- and chymotrypsin-inhibiting activities. Similarly the complex formed between ehymotrypsin and the inhibitor lacks chymotryptic activity and chymotrypsin- and trypsin-inhibiting activities. Incubation of the inhibitor with trypsin at pH 3.75 splits a 6S. C. March, I. Parikh, and P. Cuatrecasas, Anal. Biochem. 60, 149 (1974). 7 y . Birk, Proteinase Inhibitors, Proc. Int. Res. Conf., 2nd (Bayer Syrup. V), Grosse Ledder, 1973, p. 355. Springer-Verlag, Berlin and New York, 1974.
722
NATURALLY
OCCURRING
PROTEASE
INHIBITORS
[611
single A r g - X bond, which results in loss of chymotrypsin-inhibiting activity, whereas the ability to inhibit trypsin is not. affected even upon removal of the newly formed carboxy-terminal Arg by carboxypeptidase B. Incubation of the inhibitor with chymotrypsin at pH 3.75 did not open any peptide bond in the inhibitor and did not affect its trypsin- or chymotrypsin-inhibiting activity. All four isoinhibitors prepared by method B form distinct complexes with trypsin and with chymotrypsin when submitted to elcctrophoresis on paper or on cellulose acetate strips at pH 6.9. Amino acid composition. 1 The amino acid composition of the inhibitor prepared by method A is given in Table II. No free -SH groups or carbohydrates could be detected. TABLE II AMINO ACID COMPOSITmNOF TRYPSIN-
AND CHYMOTRYPSININHIBITOR FROM GROUNDNUTS a'b
Amino acid Aspartic acid Glutamic acid Glycine Alanine Valine Leucine Isoleucine Serine Threonine Italf-cystine Methionine Proline Phenylalanine Tyrosine Histidinc Lysine Arginine Tryptophan
Number of residues per molecule 8 6 4 3 5 1 0 5 7 14 0 7 2 1 2 2 7 0 74
a A. Tur-Sinai, Y. Birk, A. Gertler, and M. Rigbi, Bwchim. Biophys. Acta 263, 666 (1972). b Values are given in molar ratios; the molecular weight of the inhibitor was taken as 7580.
N£TURALLY OCCURRING PROTEASE INIIIBITORS
[51]
Inhibitor II is a strong inhibitor of elastase when assayed with Congo red-elastin system. It inhibits both trypsin and elastase simultaneously and independently. Inhibitors I and IIIb also inhibit elastase, but with specific activities less than one-quarter that of inhibitor II. Reactive Site Residues. 1,~ Isoinhibitor I has lysine in the trypsinreactive site while isoinhibitor II has arginine. The location of the trypsin reactive site in isoinhibitor II is at Args~-Ser~4, and that of the elastase reactive site is at Ala2s-Ser27. Amino Acid Composition? The amino acid composition of isoinhibitors I, II, and IIIb is shown in Table II. The inhibitors contain no free --SH groups and no carbohydrates. Peptide Maps? The peptide maps of inhibitors I and IIIb are very similar; much less similarity is apparent between inhibitor II and either I or IIIb. Isoinhibitors I and II have no peptides in common. Partial Sequence. ~ The partial amino acid sequence of isoinhibitor II is 3O - (Val,Cys) Thr-Ala-Ser-Ile-Pro-Pro-Gln (Cys,Ile, Cys,Thr,Asx,Val) 40 50 Arg-Leu-Asx-Ser-Cys-His-Ser-Ala-Cys-Lys-Ser-Cys-Met-Cys-Thr-Arg-Ser6o 7o Met-Pro-Gly-Lys-Cys-Arg-Cys-Leu-Asx-Thr-Thr-Asx-Tyr-Cys-Tyr-Lys-SerCys-Lys-Ser-Asx-Ser- Gly-Glx-Asx-Asx
[51] A T r y p s i n a n d C h y m o t r y p s i n I n h i b i t o r f r o m Groundnuts (Arachis hypogaea) B y YEHUDITH BIRK Assay Methods I
Inhibition of Proteolysis Principle. Inhibition of proteolysis is determined by Kunitz's casein digestion method, 2 which has been described in a previous volume of this treatise? 1 A. Tur-Sinai, Y. Birk, A. Gertler, and M. Rigbi, Biochim. Biophys. Acta 263, 666 (1972). 2 M. Kunitz, J. Gen. Physiol. 30, 291 (1947). a M. Laskowski, Sr., this series Vol. 2 [3].
[51]
GROUNDNUTS T R Y P S I N - AND CHYMOTR Y P S IN -IN H IB ITO R
717
Definition o] Unit. Kunitz ~ defined 1 tryptic or chymotryptic unit as the enzyme activity which causes an increase of 1 unit of absorbance at 280 nm per minute of digestion of casein, under the standard conditions. The specific activity is expressed as units per microgram of enzyme protein. Enzyme-inhibiting activity is expressed as units of enzyme inhibited, and specific activity as units of enzyme inhibited per absorbance unit, at 280 nm, of inhibitor. Inhibition o] Esterolysis Principle. Inhibition of the esterolytic activity of trypsin is determined on the substrate p-toluenesulfonyl-L-arginine methyl ester (TAME) either spectrophotometrically or by a potentiometric method. Inhibition of the esterolytic activity of chymotrypsin is determined on the substrate acetyl-L-tyrosine methyl ester (ATEE) in a similar manner. Spectrophotometric Assay Reagents Buffer: Tris-chloride, 46 mM, pH 8.1, containing 11.5 mM CaCI~ Substrate: Dissolve 37.9 mg of TAME (Sigma) in 10 ml of water. Mix 3 ml of this solution with 26 ml of the buffer. Enzyme: Trypsin (Worthington), 1 mg/ml in 1 mM HC1
Procedure. The reaction is performed in a Gilford 2400 spectrophotometer, at constant temperature (25°). Preincubate for 5 min 25 ~1 of enzyme solution with the inhibitor (up to 150 ~l) in 1 ml of the buffer, at room temperature. In each of two 1-cm quartz cuvettes, pipette 3 ml of substrate and place one of them as a blank into the spectrophotometer. Set the wavelength to 247 nm. Start the reaction by adding 100 td of the enzyme-inhibitor mixture (after 5 min of preincubation) to the second cuvette. Mix thoroughly and record change in absorbance for about 4 min. Definition o] Units. One trypsin unit is defined as 1 t~mole of substrate hydrolyzed per minute of reaction. One inhibition unit is defined as unit of enzyme inhibited. Specific activity is defined as inhibition units per absorbance unit, at 280 nm, of the inhibitor. Chymotrypsin. Inhibition of the esterolytic activity of chymotrypsin is determined on ATEE in the same way as described above for trypsin except for the following changes.
718
NATURALLY
OCCURRING
PROTEASE
INHIBITORS
[61]
Substrate: Dissolve 24.5 mg of ATEE (Sigma) in 10 ml of water. Mix 6 ml of this solution with 24 ml of the buffer Enzyme: Chymotrypsin (Worthington), 1 mg/ml in 1 mM HC1 The preincubation mixture contains 40 td of the enzyme with up to 150 ~l of the inhibitor in 1 ml of the buffer. The change in absorbance is recorded at 237 nm.
Potentiometric Assay
Reagents Buffer: Tris-chloride 3 raM, pH 8.0, containing 45 mM KC1 and 150 mM CaC12 Substrate: TAME (Sigma) 56.85 mg in 10 ml of water Enzyme: Trypsin (Worthington), 250 ~g/ml in 1 mM HC1
Procedure. Assays are performed in a Radiometer T T T l c pH stat at a constant temperature (25°). The syringe is filled with standardized 0.1 M NaOH, and the titrator is set to raise the pH to 8.0. In the reaction vessel preincubate for 2 min 50 ~l of the enzyme with up to 200 ~l of the inhibitor, in 1 ml of the buffer. Start the reaction by adding 1 ml of substrate. Record the rate of NaOH uptake for 2 min. The slope of the linear relationship between NaOH consumed at the time of reaction, gives a direct measure of micromoles of substrate consumed per minute. Definition o] Unit. The units are the same as for the spectrophotometric method. Chymotrypsin. Inhibition of esterolytic activity of chymotrypsin is determined on ATEE in the same way as described above for trypsin, except for the following changes: Substrate: Dissolve 48.56 mg of ATEE (Sigma) in 10 ml of water Enzyme: Chymotrypsin (Worthington) 200 t~g/ml in HC1, 1 mM Purification Procedures
Method A 1 The starting material is defatted groundnut flour prepared by two consecutive ether extractions, in a Soxhlet aparatus, of coarsely ground groundnuts, Virginia Sihit Meshubahat variety. The defatted meal is then finely ground to flour. All operations are performed at room temperature. The purification is summarized in Table I.
[61]
GROUNDNUTS
TRYPSIN-
AND
719
CHYMOTRYPSIN-INttIBITOR
¢4¢'~
c',l cq
¢",1
5"~
¢t5¢~ ['-- ,"-~
, ~ l'-,. ['-- "~
,~ ~
ce5 ¢D
© Z
~
¢ v ¢.D ¢D
g~ ~
Z
~a0
Z
r~ 5~
Z
'~
"
t[
720
NATURALLY OCCURRING PROTEASE INHIBITORS
[~1]
Step 1. Extraction and Ammonium Sul]ate Fractionation. Suspend 370 g of the starting material in 3.7 liters of 0.02 M HC1, yielding a pH of 4.8. Stir for 1 hr. Remove the undissolved residue by centrifugation for 20 min at 1000 g. Bring the supernatant to pH 7.0 with 1 M NaOH and clarify by filtration. Add solid ammonium-sulfate to 70% saturation, collect the resulting precipitate by centrifugation, and dissolve in 180 ml of distilled water. Dialyze swiftly against distilled water (12 changes, every 45 rain, against 5 liters of distilled water, each). Step 2. Chromatography on a DEAE-Cellulose Column. Prepare a DEAE-cellulose column (35 X 3.6 cm) equilibrated with 3 mM ammonium acetate buffer pH 7.0---the starting buffer. Centrifuge the solution from step 1 and treat the yellowish active supernatant (about 240 ml) with ammonium acetate, pH 7.0, to 3 mM concentration. Apply to the column and elute first with about 800 ml of the starting buffer and then with ammonium acetate buffer, 0.06 M, pH 7.0. Collect 13-ml fractions at a flow rate of 260 ml/hr. Pool the first active fraction that emerges with the hold-up volume of the starting buffer (approximately tubes 14-45), and dialyze swiftly against 1 mM phosphate buffer pH 6.8. Step 3. Chromatography on a Calcium Phosphate Column. Prepare a calcium phosphate column (16 X 3.3 cm) according to the procedure described in a previous volume of this treatise. 4 Apply the dialyzate from step 2 to the column. Elute primarily with 300 ml of 1 mM phosphate buffer, pH 6.8, and then with 400 ml of 0.1 M of the same buffer. Collect 13-ml fractions at a flow rate of 300 ml/hr. Pool the active fraction (approximately tubes, 11-35) and dialyze swiftly against distilled water. Step 4. Chromatography on a CM-Cellulose Column. Prepare a CMcellulose column (40 X 1.8 cm) equilibrated with 30 mM ammonium acetate buffer pH 5.0. Treat the dialyzate from step 3 with ammonium acetate to 30 mM and apply to the column. Elute with ammonium acetate buffer pH 5.0, consecutively, as following: 450 ml of 0.03 M, 650 ml of 0.12 M, and 455 ml of 0.2 M. Collect 13-ml fractions at a flow rate of 150 ml/hr. Pool the active fractions (approximately tubes 95-106) and dialyze for 5 hr against distilled water. Lyophilize and store in a desiccator. Method B 5
The starting material is the dialyzed solution from step 1 (Method A). All the operations are carried out at 4% Prepare a chymotrypsin40. Levin, this series Vol. 5 [2l. 5 y . Birk, S. Khalef, and M. Rotm~n, personal communication.
[61]
GROUNDNUTS TRYPSIN- ANn CHYMOTRYPSIN-INHIBITOR
721
Sepharose column 6 (3.6 X 16 cm). Equilibrate with Tris-chloride, 50 mM, pH 8.0, containing 20 mM CaC12 and 200 mM KCl--the starting buffer. Centrifuge the solution from step 1 for 20 min at 1000 g. Bring the supernatant to pH 8.0 and to the molarity of the starting buffer and apply to the column. Elute inactive proteins first with the starting buffer until no more protein appears in the effluent and then elute with 0.2 M KC1 pH 2.0. Collect 5-ml fractions at a flow rate of 100 ml/hr maintained with a peristaltic pump. Pool the active fraction and dialyze against distilled water. Lyophilize and store at room temperature.
Properties Stability. l The inhibitor is stable when kept in acetate buffer solutions of pH 2-11 for 24 hr at 5 °, or when boiled for 15 min in Trischloride buffer, pH 8.0. It is stable when incubated with trypsin or chymotrypsin at pH 8.0 for 36 hr at 5 ° and at room temperature. P u r i t y 1,~ The inhibitor prepared by Method A is a pure homogeneous protein according to polyacrylamide gel electrophoresis at pH 4.5 and pH 8.9, and by isoelectric focusing in polyacrylamide gels in the pH range of 3-10. The inhibitor prepared by affinity chromatography (Method B) gave 4 active bands--presumably isoinhibitors--in microzone electrophoresis on cellulose acetate strips at pH 6.9. Physical Properties.~ The molecular weight is 7450-7700 by sedimentation and diffusion measurements, sedimentation equilibrium, gel ehromatography and amino acid composition. The s.,0.,~ is 1.4 X 10-13, the D20.~ is 14.67 × 10-7, and the partial specific volume, as calculated from the amino acid composition, is 0.69. The specific extinction at 280 nm is ~~1~ i cm = 2.5. The inhibitor has an isoelectric pH of 8-9, estimated by isoeleetric focusing in polyacrylamide gels. Specifity. 1,7 The inhibitor inhibits both trypsin and ehymotrypsin. The enzyme-inhibitor complex is formed by 1 mole of inhibitor with 1 mole of trypsin or ehymotrypsin. Full inhibition of proteolysis and nearly full inhibition of esterolysis is attained by this ratio. The trypsin-inhibitor complex formed at pH 7.6 is devoid of tryptic activity as well as of trypsin- and chymotrypsin-inhibiting activities. Similarly the complex formed between ehymotrypsin and the inhibitor lacks chymotryptic activity and chymotrypsin- and trypsin-inhibiting activities. Incubation of the inhibitor with trypsin at pH 3.75 splits a 6S. C. March, I. Parikh, and P. Cuatrecasas, Anal. Biochem. 60, 149 (1974). 7 y . Birk, Proteinase Inhibitors, Proc. Int. Res. Conf., 2nd (Bayer Syrup. V), Grosse Ledder, 1973, p. 355. Springer-Verlag, Berlin and New York, 1974.
722
NATURALLY
OCCURRING
PROTEASE
INHIBITORS
[611
single A r g - X bond, which results in loss of chymotrypsin-inhibiting activity, whereas the ability to inhibit trypsin is not. affected even upon removal of the newly formed carboxy-terminal Arg by carboxypeptidase B. Incubation of the inhibitor with chymotrypsin at pH 3.75 did not open any peptide bond in the inhibitor and did not affect its trypsin- or chymotrypsin-inhibiting activity. All four isoinhibitors prepared by method B form distinct complexes with trypsin and with chymotrypsin when submitted to elcctrophoresis on paper or on cellulose acetate strips at pH 6.9. Amino acid composition. 1 The amino acid composition of the inhibitor prepared by method A is given in Table II. No free -SH groups or carbohydrates could be detected. TABLE II AMINO ACID COMPOSITmNOF TRYPSIN-
AND CHYMOTRYPSININHIBITOR FROM GROUNDNUTS a'b
Amino acid Aspartic acid Glutamic acid Glycine Alanine Valine Leucine Isoleucine Serine Threonine Italf-cystine Methionine Proline Phenylalanine Tyrosine Histidinc Lysine Arginine Tryptophan
Number of residues per molecule 8 6 4 3 5 1 0 5 7 14 0 7 2 1 2 2 7 0 74
a A. Tur-Sinai, Y. Birk, A. Gertler, and M. Rigbi, Bwchim. Biophys. Acta 263, 666 (1972). b Values are given in molar ratios; the molecular weight of the inhibitor was taken as 7580.
