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Note on the determination of chymotrypsin and chymotrypsin inhibitor activity using casein
ANALYTE~L
BIOCHEMISTRY
Note
33, 255-258 (1970)
on the
Determination
Chymotrypsin
Inhibitor
M. L. KAKADE, Department University
of Chymotrypsin Activity
D. H. SWENSON, of Biochemistry, of Minnesota, Received
AND
Using IRVIN
and Casein
E. LIENER
College of Biological Sciences, St. Paul, ikfinnesota 55101 September
4, 1969
Proteolytic enzymes acting on protein substrates such as casein or denatured hemoglobin have been generally shown to exhibit a curvilinear response to enzyme concentration (1). The casein digestion method of Kunitz (2) for the determination of the activity of trypsin or chymotrypsin is a typical example of this. It has been our experience that, in the case of chymotrypsin, modifications of this method involving the incorporation of calcium ions as suggested by Laskowski (3)) or the use of mathematical transformations as proposed by Schlitz (4) or Miller and Johnson (5) likewise fail to produce a linear enzyme response. However, we have found that it is possible to obtain a linear relationship between the activity of chymotrypsin on casein and enzyme concentration by judicious choice of experimental conditions, the most important of which are the concentration of calcium ions, the time of digestion, and the use of buffered trichloroacetic acid solution (6) for precipitation of the undigested protein. Such a linear response was found to be an important prerequisite for obtaining reliable and reproducible measurements of the chymotrypsin inhibitory activity of crude soybean extracts. REAGENTS
Casein solution. 1 gm of casein (Hammersten quality, Nutritional Biochemical Corp., Cleveland, Ohio) was suspended in 80 ml of 0.1 M borate buffer, pH 7.6, and completely dissolved by heating on a steam bath for 15 min. The solution was cooled, the pH adjusted to 7.6, and the volume brought to 100 ml with borate buffer. Chymotrypsin stock solution (40 pug/ml). 4 mg of chymotrypsin (3 X crystallized, Worthington Biochemical Corp., Freehold, N. J.) was dissolved in 100 ml of 0.001 M HCl containing 0.08 M CaCI,*2H20. Trichloroacetic acid (TCA) reagent. This solution was prepared according to Hagihara et al. (6) and contains 18.0 gm TCA, 18.0 gm anhydrous sodium acetate, and 20 ml glacial acetic acid per liter. 255
256
KAKADE,
SWENSON, AND LIENER PROCEDURE
Various levels of the stock solution of chymotrypsin (0.2 to 1.0 ml) were pipetted into a triplicate set of tubes (one set for each level of enzyme), and the volume made up to 1.0 ml with 0.001 M HCI containing 0.08 M Cat+. Then 1 ml of 0.1 M borate buffer, pH 7.6, was added to each tube, and the tubes were set in a water bath at 37”. To one of the triplicate tubes was added 6 ml of TCA reagent, this tube serving as a blank for the other two. To each tube was added 2 ml of casein solution, prewarmed to 37”. The tubes were allowed to remain at 37” for exactly 10 min, at which time the reaction was stopped by the addition of 6 ml of TCA reagent to the experimental tubes. After standing at room temperature for at least 30 min, the suspension was filtered, and the absorbance of the filtrate was measured at 275 rnp against appropriate blanks. One chymotrypsin unit (CU) is arbitrarily defined as an increase of 0.01 absorbance unit at 275 rnp in 10 min per 10 ml of the reaction mixture under the conditions described herein. RESULTS
AND
DISCUSSION
Figure 1 compares the standard curves obtained with the Kunitz (2) and Laskowski (3) procedures, curves A and B respectively, and the present method, curve C. In contrast to the other two methods, the
8 LO54 cu 0.8% N19 0.6-
0
8
16 CHYMOTRYPSIN
24
32
40
( pg )
FIQ. 1. Standard curves for assay of chymotrypsin on casein substrate using methods of Kunitz (A), Laskowski (B), and the present procedure (C). Absorbance measured at 280 mp in the case of curves A and B and at 275 mp in the case of curve C.
CHYMOTRYPSIN
ACTIVITY
ON
257
CASEIN
present procedure shows a linearrelationship betweenthe velocity of thereaction, asmeasured bytheincreasein absorbanceat 275 mp, and the concentrationof the enzyme.It is not clear why the relatively minor changes incorporated into the present procedure produces this linear relationship, whereas other procedures do not. Compared to Laskowski’s procedure (3), the present method provides for a 4-fold increase in the concentration of calcium, a reduction in digestion time from 20 to 10 min, and the use of buffered TCA instead of a solution of TCA. None of these changes was individually capable of producing linearity; only the combination of these various modifications was found to be effective. In our laboratory the method described here has been adopted for the routine determination of the chymotrypsin inhibitor activity present in crude extracts of the soybean and other legumes (7). For this purpose 1 ml of the stock chymotrypsin solution is added to various levels of the inhibitor solution in 0.1 M borate buffer, pH 7.6 (0 to 1.0 ml), and the total volume adjusted to 2 ml with the borate buffer. The remainder of the procedure is essentially the same as described above. The chymotrypsin inhibitor activity is then defined as the number of chymotrypsin units inhibited (GUI). As originally pointed out by Kunitz (2) and subsequently emphasized by Vogel et al. in their monograph on natural proteinase inhibitors (8)) the advantage of using such an expression of activity is that it is independent of the purity of the chymotrypsin used in the assay. Similar to observations previously reported for the trypsin inhibitor activity of crude soybean extracts using the synthetic substrate, benzoylI
0
I
I
02
0.4 VOLUME
0.6 OF EXTRACT
0.8 (ml)
1.0
FIG. 2. Plot of chymotrypsin inhibitor activity of crude soybean extract, expressed as chvmotrypsin units inhibited (GUI) per milliliter, versus volume of extract employed in assay. “True” chymotrypsin inhibitor activity, obtained by extrapolation to zero volume, is 72 GUI/ml.
258
KAKADE, SWENSON, AND LIENER
nL-arginine-p-nitroanilide (9)) the chymotrypsin inhibitor activity, expressed on a per milliliter basis, decreases as the level of inhibitor solution used in the assay system is increased. It has already been pointed out (9) that this type of behavior is a reflection of relative differences in the binding constants of the enzyme with the several protease inhibitors known to be present in crude extracts of soybeans (7) on one hand and the enzyme and synthetic substrate on the other. In such an instance the “true” chymotrypsin inhibitor activity may be obtained by extrapolation to zero volume of the inhibitor solution as shown in Figure 2. SUMMARY
A method is described which enables one to obtain a linear relationship between the activity of chymotrypsin on casein and the concentration of the enzyme. This method has been applied to an evaluation of the chymotrypsin inhibitor activity of crude soybean extracts. ACKNOWLEDGMENT
This study was supported by Agricultural Research Service, II. S. Department of Agriculture, grant 12-14-169-9180 (71), administered by the Northern Utilization Research and Development Division, Peoria, Illinois. REFERENCES 1. NORTHROP, J. H., KUNITZ, M., AND HERRIOTT, R. M., ‘Crystalline Enzy,mes,” 2nd ed. Columbia University Press, New York, 1948. 2. KUNITZ, M., .I. Gen. Physiol. 30, 291 (1947). 3. LASKOWSKI, M., in “Methods in Enzymology” (S. B. Colowick and N. 0. Kaplan, eds.), Vol. 2, p. 20. Academic Press, New York, 1955. 4. SCHULTZ, E., 2. Physiol. Chem. 9, 577 (1885). 5. MILLER, B. S., AND JOHNSON, J. A., Arch. Biochem. Biophys. 32, 269 (1951). 6. HAGIHARA, B., MATSUBARA, H., NAKAI, M., AND OKUNUKI, K., J. Biochem. (Tokyo) 45, 185 (1953). 7. LIENER, I. E., AND KAKADE, M. L., in “Toxic Constituents of Plant Foodstuffs” (I. E. Liener, ed.), p. 7. Academic Press, New York, 1969. 8. VOGEL, R., TRAIJTSCHOLD, I., AND WERLE, E., “Natural Proteinase Inhibitors,” p. 7. Academic Press, New York, 1963. 9. KAKADE, M. L., SIMONS, N., AND LIENER, I. E., Cereal Chem. 46, 518 (1969).
BIOCHEMISTRY
Note
33, 255-258 (1970)
on the
Determination
Chymotrypsin
Inhibitor
M. L. KAKADE, Department University
of Chymotrypsin Activity
D. H. SWENSON, of Biochemistry, of Minnesota, Received
AND
Using IRVIN
and Casein
E. LIENER
College of Biological Sciences, St. Paul, ikfinnesota 55101 September
4, 1969
Proteolytic enzymes acting on protein substrates such as casein or denatured hemoglobin have been generally shown to exhibit a curvilinear response to enzyme concentration (1). The casein digestion method of Kunitz (2) for the determination of the activity of trypsin or chymotrypsin is a typical example of this. It has been our experience that, in the case of chymotrypsin, modifications of this method involving the incorporation of calcium ions as suggested by Laskowski (3)) or the use of mathematical transformations as proposed by Schlitz (4) or Miller and Johnson (5) likewise fail to produce a linear enzyme response. However, we have found that it is possible to obtain a linear relationship between the activity of chymotrypsin on casein and enzyme concentration by judicious choice of experimental conditions, the most important of which are the concentration of calcium ions, the time of digestion, and the use of buffered trichloroacetic acid solution (6) for precipitation of the undigested protein. Such a linear response was found to be an important prerequisite for obtaining reliable and reproducible measurements of the chymotrypsin inhibitory activity of crude soybean extracts. REAGENTS
Casein solution. 1 gm of casein (Hammersten quality, Nutritional Biochemical Corp., Cleveland, Ohio) was suspended in 80 ml of 0.1 M borate buffer, pH 7.6, and completely dissolved by heating on a steam bath for 15 min. The solution was cooled, the pH adjusted to 7.6, and the volume brought to 100 ml with borate buffer. Chymotrypsin stock solution (40 pug/ml). 4 mg of chymotrypsin (3 X crystallized, Worthington Biochemical Corp., Freehold, N. J.) was dissolved in 100 ml of 0.001 M HCl containing 0.08 M CaCI,*2H20. Trichloroacetic acid (TCA) reagent. This solution was prepared according to Hagihara et al. (6) and contains 18.0 gm TCA, 18.0 gm anhydrous sodium acetate, and 20 ml glacial acetic acid per liter. 255
256
KAKADE,
SWENSON, AND LIENER PROCEDURE
Various levels of the stock solution of chymotrypsin (0.2 to 1.0 ml) were pipetted into a triplicate set of tubes (one set for each level of enzyme), and the volume made up to 1.0 ml with 0.001 M HCI containing 0.08 M Cat+. Then 1 ml of 0.1 M borate buffer, pH 7.6, was added to each tube, and the tubes were set in a water bath at 37”. To one of the triplicate tubes was added 6 ml of TCA reagent, this tube serving as a blank for the other two. To each tube was added 2 ml of casein solution, prewarmed to 37”. The tubes were allowed to remain at 37” for exactly 10 min, at which time the reaction was stopped by the addition of 6 ml of TCA reagent to the experimental tubes. After standing at room temperature for at least 30 min, the suspension was filtered, and the absorbance of the filtrate was measured at 275 rnp against appropriate blanks. One chymotrypsin unit (CU) is arbitrarily defined as an increase of 0.01 absorbance unit at 275 rnp in 10 min per 10 ml of the reaction mixture under the conditions described herein. RESULTS
AND
DISCUSSION
Figure 1 compares the standard curves obtained with the Kunitz (2) and Laskowski (3) procedures, curves A and B respectively, and the present method, curve C. In contrast to the other two methods, the
8 LO54 cu 0.8% N19 0.6-
0
8
16 CHYMOTRYPSIN
24
32
40
( pg )
FIQ. 1. Standard curves for assay of chymotrypsin on casein substrate using methods of Kunitz (A), Laskowski (B), and the present procedure (C). Absorbance measured at 280 mp in the case of curves A and B and at 275 mp in the case of curve C.
CHYMOTRYPSIN
ACTIVITY
ON
257
CASEIN
present procedure shows a linearrelationship betweenthe velocity of thereaction, asmeasured bytheincreasein absorbanceat 275 mp, and the concentrationof the enzyme.It is not clear why the relatively minor changes incorporated into the present procedure produces this linear relationship, whereas other procedures do not. Compared to Laskowski’s procedure (3), the present method provides for a 4-fold increase in the concentration of calcium, a reduction in digestion time from 20 to 10 min, and the use of buffered TCA instead of a solution of TCA. None of these changes was individually capable of producing linearity; only the combination of these various modifications was found to be effective. In our laboratory the method described here has been adopted for the routine determination of the chymotrypsin inhibitor activity present in crude extracts of the soybean and other legumes (7). For this purpose 1 ml of the stock chymotrypsin solution is added to various levels of the inhibitor solution in 0.1 M borate buffer, pH 7.6 (0 to 1.0 ml), and the total volume adjusted to 2 ml with the borate buffer. The remainder of the procedure is essentially the same as described above. The chymotrypsin inhibitor activity is then defined as the number of chymotrypsin units inhibited (GUI). As originally pointed out by Kunitz (2) and subsequently emphasized by Vogel et al. in their monograph on natural proteinase inhibitors (8)) the advantage of using such an expression of activity is that it is independent of the purity of the chymotrypsin used in the assay. Similar to observations previously reported for the trypsin inhibitor activity of crude soybean extracts using the synthetic substrate, benzoylI
0
I
I
02
0.4 VOLUME
0.6 OF EXTRACT
0.8 (ml)
1.0
FIG. 2. Plot of chymotrypsin inhibitor activity of crude soybean extract, expressed as chvmotrypsin units inhibited (GUI) per milliliter, versus volume of extract employed in assay. “True” chymotrypsin inhibitor activity, obtained by extrapolation to zero volume, is 72 GUI/ml.
258
KAKADE, SWENSON, AND LIENER
nL-arginine-p-nitroanilide (9)) the chymotrypsin inhibitor activity, expressed on a per milliliter basis, decreases as the level of inhibitor solution used in the assay system is increased. It has already been pointed out (9) that this type of behavior is a reflection of relative differences in the binding constants of the enzyme with the several protease inhibitors known to be present in crude extracts of soybeans (7) on one hand and the enzyme and synthetic substrate on the other. In such an instance the “true” chymotrypsin inhibitor activity may be obtained by extrapolation to zero volume of the inhibitor solution as shown in Figure 2. SUMMARY
A method is described which enables one to obtain a linear relationship between the activity of chymotrypsin on casein and the concentration of the enzyme. This method has been applied to an evaluation of the chymotrypsin inhibitor activity of crude soybean extracts. ACKNOWLEDGMENT
This study was supported by Agricultural Research Service, II. S. Department of Agriculture, grant 12-14-169-9180 (71), administered by the Northern Utilization Research and Development Division, Peoria, Illinois. REFERENCES 1. NORTHROP, J. H., KUNITZ, M., AND HERRIOTT, R. M., ‘Crystalline Enzy,mes,” 2nd ed. Columbia University Press, New York, 1948. 2. KUNITZ, M., .I. Gen. Physiol. 30, 291 (1947). 3. LASKOWSKI, M., in “Methods in Enzymology” (S. B. Colowick and N. 0. Kaplan, eds.), Vol. 2, p. 20. Academic Press, New York, 1955. 4. SCHULTZ, E., 2. Physiol. Chem. 9, 577 (1885). 5. MILLER, B. S., AND JOHNSON, J. A., Arch. Biochem. Biophys. 32, 269 (1951). 6. HAGIHARA, B., MATSUBARA, H., NAKAI, M., AND OKUNUKI, K., J. Biochem. (Tokyo) 45, 185 (1953). 7. LIENER, I. E., AND KAKADE, M. L., in “Toxic Constituents of Plant Foodstuffs” (I. E. Liener, ed.), p. 7. Academic Press, New York, 1969. 8. VOGEL, R., TRAIJTSCHOLD, I., AND WERLE, E., “Natural Proteinase Inhibitors,” p. 7. Academic Press, New York, 1963. 9. KAKADE, M. L., SIMONS, N., AND LIENER, I. E., Cereal Chem. 46, 518 (1969).