ANALYTICAL

BIOCHEMISTRY

97, 11-16 (1979)

A New Method ~A~ORU SUGIURA, Department

for Protease

Activity

Measurement1

MASAO ISHIKAWA,” MASANORI SASAKI,~, KAZUYUKI HIRANO, YOSHIMASA ITO, AND SHOJI AWAZU*

of Pharmacy, Gifu College of Pharmacy, Mitahorahigashi S-6-1, Gifu 502, *Department of Pharmacy, Tokyo College of Pharmacy, Horinouch 1432-1, Hachioji 192-03, and tDeparttnent of Pharmacy, Niigata College of Pharmacy. Kamishinei-cho 5829, Niigata 950-21, Japan

Received October t 1, 1978 A new method for protease activity measurement is described. In the presence of excess leucine aminopeptidase from Aspergillus japonica, action of protease on succinyl-casein results in the production of L-amino acids and their amino acids are simultaneously determined by L-amino acid oxidase-peroxidase system. Our proposed method is less time consuming and has a much higher sensitivity than the casein-Folin method. The present method is suggested to be suitable for the assay of neutral or alkaline proteases from animals and microorganisms.

A number of assay methods for protease activities using protein substrates such as hemoglobin (I), casein (Z), albumin (3), protamine (4), fibrinogen (5), ribonuclease S (6), and luciferase (7) had been presented. The method by Anson (1) or Kuniz (2) has been commonly used as the ultimate determination of protease activity by use of protein substrate. In this, the enzyme reaction is usually terminated by trichloroacetic acid (TCA).Z After separating the precipitate by centrifugation or filtration, quantitative determination of enzyme activity is achieved by measuring the amount of digested substrate in the acid-soluble fraction. In the method by Anson (I), Folin-Ciocalteu phenol reagent (8) is used as a color indicator for TCA-soluble hydrolysates. More complicated procedures are involved in the 2,4,6-t~nit~benzene sulfonic acid (9) and fluoresamine methods (IO). Since these methods require discontinuous operation, they are complicated and time consuming. ’ This report forms a Part CLVIII of “Studies on Enzymes” by M. Sugiura. * Abbreviations used: TCA, trichloroacetic acid; DFP, diisopropyl fluorophosphate.

In this paper, we described a new, rapid, simple, and sensitive one-step assay method for protease activity using leucine aminopeptidase, L-amino acid oxidase, and peroxidase . MATERIALS

AND METHODS

Enzymes. L-Amino acid oxidase from the venom of Ag~istrodu~ caliginosus and leucine aminopeptidase from Aspergilhs japonica were purified to homogeneous state according to the method described in our previous papers (1 I, 12). Horseradish peroxidase (type II) and crystalline carboxypeptidase A from bovine pancreas were obtained from Sigma Chemical Company, swine kidney leucine aminopeptidase from Boehringer/Mannheim GmbH and yeast carboxypeptidase Y from Oriental Yeast Company. As~erg~llus ~e~~e~s crystalline semialkaline protease was presented from Amano Pharmaceutical Company, crude serratiopeptidase from Takeda Pharmaceutical Ind. Company, crude trypsin and a-chymotrypsin from Eisai Company, crude protease E from Kaken Chemical Ind. Company, crystalline thermolisin 11

0003-2697/79/l 1001 l-06$02.00/0 Copyright

0 1979 by Academic

Press. Inc.

12

SUGIURA

from Daiwakasei Company, Bacillus crystalline neutral, and alkaline protease from Seikagaku Kogyo Company. Ficin from the latex of fruits of the fig tree (Fiscus carica var. Horaishi) was used (13). Reagents. 4-Amioantipyrine, N,N-dimethylaniline, succinic anhydride, hammersten milk casein, and bovine serum albumin were purchased from Wako Pure Chemical Ind. Company, diisopropyl fluorophosphate (DFP) from Sigma Chemical Company and p-chloromercuribenzoate from Tokyo Chemical Ind. Company. The other reagents used were of reagent grade. Succinylation of casein. Casein was succinylated as follows. Three grams of milk casein was dissolved in 0.2 M sodium phosphate buffer (pH 7.5). The pH was m~ntained at pH 7.5 with 2 N NaOH, occasionally while 600 mg of succinic anhydride was added to the solution. The reaction was allowed to take place for 6 h at room temperature. Then the reaction mixture was dialyzed against 20 mM sodium phosphate buffer (pH 7.2) for 48 h at 4°C. Standard assay of protease activity. Substrate solution was prepared as follows. One gram of succinyl casein was dissolved in 100 ml of 20 mM sodium phosphate buffer (pH 7.2) containing 80 units of L-amino acid oxidase and 50 units of leucine aminopeptidase from A. japonica. As a color reagent solution, 300 units of peroxidase, 20 /*l of N,N-dimethylaniline, and 8 mg of 4-aminoantipyrine were dissolved in 100 ml of 50 mM sodium phosphate buffer (pH 7.2). One milliliter of the substrate solution and 2 ml of color reagent solution were preincubated at 37°C for 5 min, and then 0.5 ml of enzyme solution was added to the reaction mixture. The enzyme reaction was carried out at 37°C for 10 min and terminated by the addition of 0.5 ml of 5 mM a-phenanthroline containing 1.5 mM DFP. The absorbance was measured at 550 nm. One unit of protease activity was defined as 1 pg of L-leutine which was released from the substrate per minute by the enzyme under the above conditions.

ET AL.

Protease assay by the casein-Folk method. The protease assay was carried out according to the method of Matsubara (14) with minor modifications. A reaction mixture containing 5 ml of 1% casein (pH 7.2) and 1 ml of the enzyme was incubated for 10 min at 37°C and the reaction was stopped by the addition of 5 ml of precipitating solution composed of 0.11 M TCA, 0.22 M sodium acetate, and 0.33 M acetic acid. After 30 min at 37”C, the reaction mixture was filtered with filter paper (Toyo No. 131). To 5 ml of 0.55 M sodium carbonate, 1 ml of filtrate and threefold diluted Folin reagent were added. Standing at 37°C for 30 min, absorbance was measured at 660 nm. One unit of the enzyme activity was defined as 1 pg of tyrosine which was released from the substrate per minute by the enzyme under the above conditions. RESULTS Effect of Saccinylation Protease Activity

of Casein on

It is well known that casein denatured by ganidine hydrochloride or urea was more susceptible to protease action than native casein. Therefore, casein was subjected to denaturation by succinylation, considering that leucine aminopeptidase was involved in the assay system. The effect of succinylation of casein on protease was examined in comparison with that of native casein. The results are shown in Table 1. Protease activity toward succinyl casein was 1.2- 1.9 times higher than that toward native casein. Since succinyl casein was not hydrolyzed by coexistent leucine aminopeptidase, the blank value at 550 nm was negligible as compared with native casein. From these results, succinyl casein was decided to use as a substrate. Concentration

of Reagents

and Substrate

Figure 1 shows the correlation between concentration of reagents and absorbance at 550 nm. From the results, it was decided

NEW

PROTEASE

to use the following concentration of reagents: 1% succinyl casein as a substrate solution; 80 units of L-amino acid oxidase, 300 units of peroxidase, 20 ~1 of N,N-dimethylaniline, and 8 mg of 4-aminoantipyrine were dissolved in 100 ml of 50 mM phosphate buffer (pH 7.2) as a color reagent solution. The concentration and the kind of peptidase were also examined using the peptidases described under Materials and Methods. From this investigation, it was found that aminopeptidases from A. japonica and swine kidney were available for use. However, the unit of aminopeptidase from A. japonica was used less than that of the kidney aminopeptidase in order to obtain the maximal constant absorbance. Therefore, it was decided that aminopeptidase from A. japonica would be used for the determination of protease activity. Stopper of the Reaction

To establish the endpoint assay for protease activity, the stopper solution was investigated. The enzyme reaction was not stopped by the addition of 0.5 ml of the mixture, which contained 50 mM EDTA and 10 mM DFP, but completely stopped by the addition of the mixture which contained 10 mM o-phenanthroline and 3 mM DFP.

00 0 0 8 0

0.25 I 0.5

TABLE

(O.D.

Semialkaline protease Pronase E Bacillus neutral protease Trypsin cu-Chymotrypsin

Activity at 550 nm)

Succinyl casein

Native casein*

1.5 5.0

0.2% 0.442

0.246 0.136

5.0 1.0 5.0

0.624 0.831

0.326 0.436 0.153

Volume” (/a

Protease

0.264

o The indicated amount of the enzymes was added to the reaction system. b One gram of milk casein was dissolved in 100 ml of 20 mM sodium phosphate buffer @H 7.2).

This mixture was adopted as a stopper. When this stopper solution was used, it was found that the color developed in the assay medium was stable within 60 min. Calibration Curve of A. melleus Semialkaline Protease and Trypsin

Figure 2 shows the calibration semialkaline protease by the

3

1.0

I 0.75 I 1.5

0.50

1

EFFECT OF MODIFICATION OF CASEIN ON PROTEASE ACTIVITY

2

1

13

ASSAY

4 1.00 2.0

Color Substrate LAOD

reagent

curve of proposed

a) (%I

(units/tube)

1. Effect of concentration of color reagent, succinyl casein, and L-amino acid oxidase (LAOD) on final absorbance. (a) The indicated numbers were represented as the function of reagent concentrations: 1, 8 mg of 4-aminoantipyrine, 20 ~1 of N,N-dimethylaniline, and 300 units of peroxidase in 50 mM phosphate buffer (pH 7.2). Color reagent, 0; substrate, A; LAOD, 0. The volume of the color reagent, substrate, and LAOD were described under Materials and Methods. FIG.

14

SUGIURA

Enzyme FIG. 2. Calibration curve for A. m&us Materials and Methods.

COnCentratiOII

2

REPRODUCIBILITY FOR DETERMINATION SEMIALKALINE PROTEASE ACTIVITY BYTHEPROPOSEDMETHOD

(pg)

semialkaline protease. The assay was described under

method. The plots of absorbance at 550 nm vs the amount of the enzyme (O-2.5 pg) were regressed to a straight Iine passing through the origin. The calibration curve of trypsin was also drawn and in the range of 0- 100 ng of trypsin, a linear straight line passing through the origin was obtained. When the reproducibility of the proposed method was examined using semialkaline protease, the coefficient of variation was very small as shown in Table 2. TABLE

ET AL.

OF

No.

Enzyme activity (O.D. at 550 nm)

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

0.351 0.357 0.356 0.350 0.353 0.348 0.361 0.356 0.358 0.356

Mean + SD Coefficient of variation (%)

0.355 + 0.004 1.07

a Semialkaline protease (2 pg) was added to the standard reaction medium.

Comparison of Protease Activities Determined by the Proposed and Casein-Folin Methods The units of various proteases by the proposed and casein-Folin methods are compared in Table 3. Each unit of protease E, serratio-peptidase, and trypsin showed five times higher than that by the caseinFolin method, and the respective unit of the others was lower than those by the caseinFolin method. This discrepancy was due to the definition of enzyme units calculated by both the methods. Table 3 shows the comparison between the sensitivities of the proposed and casein-Folin methods. The proposed method has 9-22 times the sensitivity of the conventional one. Moreover, the sensitivity of trypsin by the proposed method was about 100 times higher than that by the casein-Folin method. On the other hand, the sensitivity of ficin by the proposed method was only four times that by the casein-Folin method. Since the oxidative condensation reaction by H,O,peroxidase system is fairly affected by the reducing agents which the SH enzyme @in> contains, this low sensitivity may be caused by the substrate specificity. DISCUSSION

The method we have developed for the measurement of protease activity depends on the endo action of protease exposing

NEW PROTEASE

15

ASSAY

free amino-terminal ends in a succinyl casein, complicated and time consuming because which is not hydrolyzed by the action of the system required a two-step operation. leucine aminopeptidase from A. juponica. Succinyl casein as a substrate was seThe principle of this method is that in the lected because of high susceptibility to presence of excess leucine ~inopeptidase, protease (Table I) and insensibility to leuthe amount of released amino acids such tine aminopeptidase. The proposed method as leucine, tyrosine, tryptophan, phenylhas the advantage of simplicity, rapidity, alanine, and methionine (11) is proportional high sensibility, and good reproducibility. to the activity of protease. The H,O, pro- In the case of trypsin assay, 10 ng of the duced by the action of L-amino acid oxi- enzyme could be determined within 30 min. dase on the above amino acid was coupled This sensitivity is comparable to that of the to peroxidase-4-aminoantipyrine-N,Nluciferase (7) and protamine methods (4). dimethylaniline system to produce chromoSince the optimum pH and stability for phore possessing absorbance maximum at leucine aminopeptidase and r-amino acid 550 nm. To detect Hz02 released by the oxidase used in this assay are from neutral action of L-amino acid oxidase on L-amino to semialkaline range, the acid protease acids, a number of conventional methods activity should be measured by a two-step were available to the proposed method with reaction system. The proposed method minor modification and in the presence of a requires the three different enzymes as reducing substance or catalase, the cata- enzyme reagents and their inhibitors aplase-methanol reaction system (15) is very parently reduce protease activity. Howuseful because this system is in~uenced ever, when 100.. to lo-fold diluted envery little by the above substance. How- zyme extracted from animal tissue or microever, the catalase-methanol system was organisms was used, no detectable disturbed reaction was observed. From these results, the proposed method TABLE 3 described in this paper is considered to be COMPARISON OF PROTEASE ACTIVITY DETERMINED BY useful for the one-step assay of neutral or THE PROPOSED(A) AND CASEIN alkaline protease from animals and microFOLIN METHODS (B) organisms. Activity (units/mg) Protease Semialkaline protease Pronase E Bacillus

(A)

(B)

Sensitivity 0.D. at 550 nm (A) O.D. at 660 nm (B)

1120

2260

952

786

9.07 22.0

I. Anson, M. L. (1938)J. Gen. Physiot. 22, 79-89. 2. Kunitz, M. (1947) J. Gen. Physiol. 30, 291-310. 3. Furihata, C., Senma, T., Saito, D., Matsushima, T., and Sugiura, T. (1978) Anal. Biochem.

1370

2260

11.0

4. Ong, E. B., and Johnson, A. J. (1976) Anal.

7.57 2080

1120" 2800

12.3 13.6

sp.

neutral protease Alkaline protease Thermolysin Serratiopeptidase Trypsin a-Chymotrypsin Ficin

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Njus, D., Baldwin, T. O., and Hasting, J. W. (1974) Anal.

’ The enzyme activity was measured at pH 10. b The mixture (0.5 ml) composed of equal volumes of 10 mM o-pheanthroline and 0.1 mM PCMB was used as a stopper solution.

Bio-

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Biochem.

61, 280-287.

8. Fohn, O., and Ciocaheu, V. (1927)J. Biof. Chem. 73,627-650.

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SUGIURA

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10. Schwabe, C. (1975)Anal. Biochem. 53,484-490. 11. Sugiura, M., Sasaki, M., Ito, Y., Akatsuka, M., Oikawa, T., and Makino, M. (1975) The SNAKE 7,83-90.

12. Sugiura, M., Ishikawa, M., Suzuki, M., and Sasaki, M. (1976) Chem. Charm. Buff. ~T~kyo) 24.2026-203 1.

ET AL. 13. Sugiura, M., and Sasaki, M. (1971) Yakugaku Zasshi 91, 451-466. 14. Matsubara, H., Hagiwara, B., Nakai, M., Komaki, T., Yonetani, T., and Okunuki, K. (1958) J. Biochem. (Tokyo) 45,251-2X 15. Watana~, T., Motomura, Y., and Suga, T. (1978) Anaf. Biochem. 86,310-315.