ANALYTICAL
BIOCHEMISTRY
65,
548-551
(1975)
Substrate
Solubilization cu-Chymotrypsin
for
the
Hummel
Assay
An improvement of the Hummel ff-chymotrypsin assay (1) has been recently reported by Kang and Fuchs (2), whereby the sensitivity of the method was increased more than 2-fold by adjusting the concentration of methanol and of the substrate, N-benzoyl-L-tyrosine ethyl ester (BTEE). We have developed another modification of the Hummel procedure with which an equal or even greater increase in the sensitivity is obtained by reducing the concentration of methanol in the reaction mixture to 0.5% (v/v) and solubilization of BTEE with a 0.2% (w/v) solution of Triton X-100 in Tris buffer. MATERIALS
AND
METHODS
Activity was measured by following the increase in absorbancy at 256 nm at 25°C using a Gary spectrophotometer Model 15. The substrate BTEE was obtained from Schwarz-Mann, Triton X-100, and bovine a+ chymotrypsin Type II, 3 x crystallized from Sigma Chemical Company, and enterokinase from Miles Laboratories. All other materials were of reagent grade. Homogenates of mouse pancreas were prepared in 0.01 M Tris (hydroxymethyl) aminoethane-HCl buffer, pH 8.5, containing 0.15 M NaCl and 0.1% (w/v) Triton X-100. The pH of the homogenates so prepared was 7.8. A stock solution of 1.O mglml of chymotrypsin was prepared in 0.00 I M HCl and appropriate dilutions were made for the assay so that 0.1 ml contained the desired concentration of the enzyme. Pancreas homogenates were diluted loo-fold with 0.08 M Tris-HCl buffer, pH 7.8 containing 0.10 M CaCl,. Diluted homogenates, 0.1 ml, were incubated at 37°C for 60 min with 0.1 ml of a 1% solution of enterokinase in the same buffer used for diluting the homogenates. Under these conditions, activation of chymotrypsinogen to chymotrypsin was found to be complete. The activated homogenates (0.2 ml) were assayed for chymotrypsin. BTEE, 16.0 mg, was dissolved in 0.5 ml of methanol in a 50 ml volumetric flask and the volume brought to mark by slow addition with shaking of a 0.2% Triton X-100 solution in 0.08 M Tris buffer, pH 7.8. containing 0.10 M CaCl,. Alternatively, BTEE was prepared as recommended in the original Hummel method (1). All assays were performed in a final volume of 3 ml and were monitored in standard quartz cuvettes 548 Copyright @ 197.5 by Academic Press, inc. All rights of reproduction in any form reserved.
549
SHORT COMMUNICATIONS
of l-cm path length. The reaction was started by the addition of chymotrypsin or of substrate. Chymotrypsin specific activity was calculated by a standard formula (3). RESULTS
The a-chymotrypsin used in these experiments was reputed by the supplier to have a specific activity of 45, and assay 1 (Table 1) confirmed this value. However, when the modified BTEE solution was used (assay 2) the specific activity of the enzyme rose to 119. Assays 3-5 were performed with the modified BTEE solution in the presence of increasing concentrations of methanol added to the reaction mixture before starting the reaction by the addition of chymotrypsin. The results of these assays, but especially those of assay 2 and 6, show clearly that methanol has an inhibitory action on the enzyme. On the other hand, Triton X-100, in a final concentration of 0. l%, had no effect on the enzyme activity as shown by the results of assay 6 which was carried out according to the original Hummel procedure after addition of Triton X-100 to the reaction mixture. Rates of hydrolysis using the modified solution of BTEE were linear for at least 3 min with up to 3 Fg of purified a-chymotrypsin (results not presented). The rate of hydrolysis was also directly proportional to the concentration of the enzyme and was maximal, or near maximal, with 300 pg of BTEE (Table 2). Following Kang and Fuchs approach (2), attempts were made to see whether the sensitivity of the assay could be enhanced even further by increasing substantially the concentration of TABLE EFFECT
OF METHANOL
1
CONCENTRATION
PURIFIED
ON THE ACTIVITY
OF
(u-CHYMOTRYPSI~~
Assay no.
BTEE 0-4
Methanol (9%)
Triton X-100 (%)
Sp accb (Uimg)
1 2
480 480
23.0 0.5
0 0.1
44.8 t 1.8 119.4 5 4.1
3 4 5
480 480 480
1.5 2.5 3.5
0.1 0.1 0.1
114.0 2 4.7 108.3 2 0.5 102.3 + 1.3
6
480
23.0
0.1
49.2 k 1.3
a Assays 1 and 6 were performed according to the original procedure of Hummel (1). In assay 6, Triton X-100 was added to a final concentration of 0.1% to the reaction mixture before addition of the enzyme. The reaction mixture for assays 2-5 contained 1.5 ml of the Triton X-100 containing solution of BTEE, O-90 ~1 of methanol, 0.1 ml of enzyme and H,O to 3 ml final volume. B Each value represents the mean ?SEM of three determinations.
SHORT COMMUNICATIONS
550
TABLE (Y-CHYMOTRYPSIN
ASSAY SUBSTRATE
2
AS A FUNCTION
OF ENZYME
AND
CONCENTRATIONS?
Enzyme h9
0.78
1.56
3.12
BTEE pg 300 450 600
86.5 k 5.1 93.4 f 3.7 84.0 k 8.3
177.2 k 1.2 190.8 + 3.4 190.5 + 4.2
360.9 k 5.3 364.1 * 1.6 369.2 k 4.9
’ Each value represents nmoles of BTEE hydrolysed/min, and is the mean +SEM of three determinations. Reaction conditions were as indicated in Footnote a to Table 1. assay 2, except for varying amounts of enzyme or substrate.
BTEE. However, these attempts were either negative or unsuccessful. Triton X-100, 0.2%, would keep in solution 32 mg of BTEE initially dissolved in either 0.5 ml or 1.5 ml of methanol. However, the substrate came out of solution when added to the complete assay mixture. Higher concentration of Triton X-100 could not be used because of too great an absorbance at 256 nm. Units of chymotrypsinogen per total pancreas homogenates from two mice fed laboratory chow were 13 6 and 17 1 using the original Hummel procedure and 301 and 401 with the modified assay. DISCUSSLON As previously reported by Gorrill and Thomas (4), and by Kang and Fuchs (2) methanol was found to have an inhibitory effect on a-chymotrypsin activity. However, the need for methanol as a solubilizer of BTEE, the specific substrate used in the Hummel procedure, can be markedly reduced by employing a 0.2% solution of Triton X-100. With this modified substrate solution an increase in the sensitivity of the assay is achieved which is even slightly greater than that obtained by Kang and Fuchs modification (2). A further advantage afforded by the use of Triton X-100 is that a single solution can be prepared containing Tris buffer and CaClz as well as the substrate. The modified assay works satisfactorily not only with purified cY-chymotrypsin but also with pancreas homogenates. Gorrill and Thomas (4) have described the advantageous effects of Triton X- 100 in the determination of the pancreatic enzyme in homogenates of the organ. For routine determinations of a-chymotrypsin activity in pancreas homogenates we presently use 0.1 or 0.2 ml of homogenate added to 2.8 or 2.9 ml of a substrate solution prepared as follows: 16.0 mg of BTEE initially dissolved in 0.5 ml methanol and brought to a 100 ml volume with a 0.1% solution of Triton X-100 in 0.04 M Tris-HCl buffer, pH 7.8, containing 0.05 M CaCl,. In this way,
551
SHORT COMMUNICATIONS
the need for addition of Hz0 to the final reaction mixture, and thus of an additional pipetting, is avoided, with no effect on the sensitivity of the assay. REFERENCES 1. Hummel, B. C. W. (1959) Can. 1. B~~~~z~~?z. ~~~~~~~~.37, 1393. 2. Kang. S. H., and Fuchs, M. S. t 1973) Anal. Bjoc~~e~z. 54, 262. 3. Worthington Enzyme Manual, p. 129. Worthington Biochemical Corporation, New Jersey, 1971. 4. Gorrill, A. D. L., and Thomas, J. W. (1967) Anul. Biochrm. 19, 111.
Freehold,
K. N. RAO BENITO LOMBARDI Department of Pathoiogy University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania 15261 Received July 26, 1974; accepted November 12, I974
BIOCHEMISTRY
65,
548-551
(1975)
Substrate
Solubilization cu-Chymotrypsin
for
the
Hummel
Assay
An improvement of the Hummel ff-chymotrypsin assay (1) has been recently reported by Kang and Fuchs (2), whereby the sensitivity of the method was increased more than 2-fold by adjusting the concentration of methanol and of the substrate, N-benzoyl-L-tyrosine ethyl ester (BTEE). We have developed another modification of the Hummel procedure with which an equal or even greater increase in the sensitivity is obtained by reducing the concentration of methanol in the reaction mixture to 0.5% (v/v) and solubilization of BTEE with a 0.2% (w/v) solution of Triton X-100 in Tris buffer. MATERIALS
AND
METHODS
Activity was measured by following the increase in absorbancy at 256 nm at 25°C using a Gary spectrophotometer Model 15. The substrate BTEE was obtained from Schwarz-Mann, Triton X-100, and bovine a+ chymotrypsin Type II, 3 x crystallized from Sigma Chemical Company, and enterokinase from Miles Laboratories. All other materials were of reagent grade. Homogenates of mouse pancreas were prepared in 0.01 M Tris (hydroxymethyl) aminoethane-HCl buffer, pH 8.5, containing 0.15 M NaCl and 0.1% (w/v) Triton X-100. The pH of the homogenates so prepared was 7.8. A stock solution of 1.O mglml of chymotrypsin was prepared in 0.00 I M HCl and appropriate dilutions were made for the assay so that 0.1 ml contained the desired concentration of the enzyme. Pancreas homogenates were diluted loo-fold with 0.08 M Tris-HCl buffer, pH 7.8 containing 0.10 M CaCl,. Diluted homogenates, 0.1 ml, were incubated at 37°C for 60 min with 0.1 ml of a 1% solution of enterokinase in the same buffer used for diluting the homogenates. Under these conditions, activation of chymotrypsinogen to chymotrypsin was found to be complete. The activated homogenates (0.2 ml) were assayed for chymotrypsin. BTEE, 16.0 mg, was dissolved in 0.5 ml of methanol in a 50 ml volumetric flask and the volume brought to mark by slow addition with shaking of a 0.2% Triton X-100 solution in 0.08 M Tris buffer, pH 7.8. containing 0.10 M CaCl,. Alternatively, BTEE was prepared as recommended in the original Hummel method (1). All assays were performed in a final volume of 3 ml and were monitored in standard quartz cuvettes 548 Copyright @ 197.5 by Academic Press, inc. All rights of reproduction in any form reserved.
549
SHORT COMMUNICATIONS
of l-cm path length. The reaction was started by the addition of chymotrypsin or of substrate. Chymotrypsin specific activity was calculated by a standard formula (3). RESULTS
The a-chymotrypsin used in these experiments was reputed by the supplier to have a specific activity of 45, and assay 1 (Table 1) confirmed this value. However, when the modified BTEE solution was used (assay 2) the specific activity of the enzyme rose to 119. Assays 3-5 were performed with the modified BTEE solution in the presence of increasing concentrations of methanol added to the reaction mixture before starting the reaction by the addition of chymotrypsin. The results of these assays, but especially those of assay 2 and 6, show clearly that methanol has an inhibitory action on the enzyme. On the other hand, Triton X-100, in a final concentration of 0. l%, had no effect on the enzyme activity as shown by the results of assay 6 which was carried out according to the original Hummel procedure after addition of Triton X-100 to the reaction mixture. Rates of hydrolysis using the modified solution of BTEE were linear for at least 3 min with up to 3 Fg of purified a-chymotrypsin (results not presented). The rate of hydrolysis was also directly proportional to the concentration of the enzyme and was maximal, or near maximal, with 300 pg of BTEE (Table 2). Following Kang and Fuchs approach (2), attempts were made to see whether the sensitivity of the assay could be enhanced even further by increasing substantially the concentration of TABLE EFFECT
OF METHANOL
1
CONCENTRATION
PURIFIED
ON THE ACTIVITY
OF
(u-CHYMOTRYPSI~~
Assay no.
BTEE 0-4
Methanol (9%)
Triton X-100 (%)
Sp accb (Uimg)
1 2
480 480
23.0 0.5
0 0.1
44.8 t 1.8 119.4 5 4.1
3 4 5
480 480 480
1.5 2.5 3.5
0.1 0.1 0.1
114.0 2 4.7 108.3 2 0.5 102.3 + 1.3
6
480
23.0
0.1
49.2 k 1.3
a Assays 1 and 6 were performed according to the original procedure of Hummel (1). In assay 6, Triton X-100 was added to a final concentration of 0.1% to the reaction mixture before addition of the enzyme. The reaction mixture for assays 2-5 contained 1.5 ml of the Triton X-100 containing solution of BTEE, O-90 ~1 of methanol, 0.1 ml of enzyme and H,O to 3 ml final volume. B Each value represents the mean ?SEM of three determinations.
SHORT COMMUNICATIONS
550
TABLE (Y-CHYMOTRYPSIN
ASSAY SUBSTRATE
2
AS A FUNCTION
OF ENZYME
AND
CONCENTRATIONS?
Enzyme h9
0.78
1.56
3.12
BTEE pg 300 450 600
86.5 k 5.1 93.4 f 3.7 84.0 k 8.3
177.2 k 1.2 190.8 + 3.4 190.5 + 4.2
360.9 k 5.3 364.1 * 1.6 369.2 k 4.9
’ Each value represents nmoles of BTEE hydrolysed/min, and is the mean +SEM of three determinations. Reaction conditions were as indicated in Footnote a to Table 1. assay 2, except for varying amounts of enzyme or substrate.
BTEE. However, these attempts were either negative or unsuccessful. Triton X-100, 0.2%, would keep in solution 32 mg of BTEE initially dissolved in either 0.5 ml or 1.5 ml of methanol. However, the substrate came out of solution when added to the complete assay mixture. Higher concentration of Triton X-100 could not be used because of too great an absorbance at 256 nm. Units of chymotrypsinogen per total pancreas homogenates from two mice fed laboratory chow were 13 6 and 17 1 using the original Hummel procedure and 301 and 401 with the modified assay. DISCUSSLON As previously reported by Gorrill and Thomas (4), and by Kang and Fuchs (2) methanol was found to have an inhibitory effect on a-chymotrypsin activity. However, the need for methanol as a solubilizer of BTEE, the specific substrate used in the Hummel procedure, can be markedly reduced by employing a 0.2% solution of Triton X-100. With this modified substrate solution an increase in the sensitivity of the assay is achieved which is even slightly greater than that obtained by Kang and Fuchs modification (2). A further advantage afforded by the use of Triton X-100 is that a single solution can be prepared containing Tris buffer and CaClz as well as the substrate. The modified assay works satisfactorily not only with purified cY-chymotrypsin but also with pancreas homogenates. Gorrill and Thomas (4) have described the advantageous effects of Triton X- 100 in the determination of the pancreatic enzyme in homogenates of the organ. For routine determinations of a-chymotrypsin activity in pancreas homogenates we presently use 0.1 or 0.2 ml of homogenate added to 2.8 or 2.9 ml of a substrate solution prepared as follows: 16.0 mg of BTEE initially dissolved in 0.5 ml methanol and brought to a 100 ml volume with a 0.1% solution of Triton X-100 in 0.04 M Tris-HCl buffer, pH 7.8, containing 0.05 M CaCl,. In this way,
551
SHORT COMMUNICATIONS
the need for addition of Hz0 to the final reaction mixture, and thus of an additional pipetting, is avoided, with no effect on the sensitivity of the assay. REFERENCES 1. Hummel, B. C. W. (1959) Can. 1. B~~~~z~~?z. ~~~~~~~~.37, 1393. 2. Kang. S. H., and Fuchs, M. S. t 1973) Anal. Bjoc~~e~z. 54, 262. 3. Worthington Enzyme Manual, p. 129. Worthington Biochemical Corporation, New Jersey, 1971. 4. Gorrill, A. D. L., and Thomas, J. W. (1967) Anul. Biochrm. 19, 111.
Freehold,
K. N. RAO BENITO LOMBARDI Department of Pathoiogy University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania 15261 Received July 26, 1974; accepted November 12, I974
