ANALYTICAL
BIOCHEMISTRY
204,
181-184
(1992)
A Microassay for Proteases Using Succinylcasein as a Substrate Tomomitsu Hatakeyama, Hidetsugu Kohzaki, and Nobuyuki Yamasaki Laboratory
Received
of Biochemistry,
November
Faculty
of Agriculture,
Kyushu
University,
Hakozaki
All rights
used: TCA, trichloroacetic TNP, trinitrophenyl;
$5.00 1992
812, Japan
that they require the specialized fluorometer or the acid-precipitation step. In this report, we present a microassay system for determination of protease activity that is carried out in the microtiter plate wells. This method employs as a substrate a casein derivative whose amino groups are chemically modified by succinylation, and amino groups appearing by the hydrolysis of the substrate are quantitatively detected with trinitrobenzene sulfonate (TNBS). Since whole reactions can be carried out in the microtiter plate wells, the amounts of the samples and the reagents are small, and the following measurement of absorbance can be rapidly done by using a microtiter plate reader. We also demonstrate the proteolytic activity in short-necked clam Tapes philippinarum tissues conveniently detected by this method.
MATERIALS
Casein has been widely used in the protease assays as a substrate, since it is readily hydrolyzed by most of the proteases with various specificities. While TCA’ soluble peptides resulting from protease action can be simply detected by measurement of the absorbance at 280 nm, which is due to aromatic amino acid residues in the peptides (l), the sensitivity can be further enhanced by means of the substrates with covalently attached chromophores (2,3) or those labeled with radioactive isotopes (45). However, such assays are sometimes very time-consuming and require expensive substrates. Some assays for proteases using a microtiter plate in which fluorescence or absorption resulting from the hydrolysis of the substrates was measured by automatic instruments, thereby facilitating the procedure, have been reported (6,7). However, there are still problems in
0003.2697/92 Copyright 0
Fukuoka
18, 1991
A photometric assay for proteases has been developed. A chemically modified casein whose amino groups were succinylated was used as a substrate. After incubation with trypsin, chymotrypsin, thermolysin, and subtilisin, the extent of hydrolysis of the substrate was determined with trinitrobenzene sulfonate (TNBS). The whole procedure of the assay was performed in the microtiter plate wells and the increase in the absorbance resulting from the reaction between TNBS and newly formed amino groups in the substrate was able to be determined with a high sensitivity by a microtiter plate reader, enabling the simultaneous measurement of a number of samples. Application of this method to the measurement of proteolytic activity contained in the protein extract of Tapes philippinarum is B 1992 Academic Press, Inc. demonstrated.
’ Abbreviations benzene sulfonate; fate.
6-10-1,
SDS,
acid; TNBS, trinitrosodium dodecyl sul-
AND
METHODS
Trypsin, chymotrypsin, subtilisin Carlsberg, and azocasein were purchased from Sigma (St. Louis, MO). Thermolysin was from Wako (Osaka, Japan). Casein (Hammarsten) was obtained from Merck (Darmstadt, Germany). TNBS and succinic anhydride were products of Nacalai Tesque (Kyoto, Japan). Other reagents used were of analytical grade.
Preparation
of the Succinylcasein
Casein (500 mg) was dissolved in 100 ml of 50 mM sodium borate buffer (pH 8.0) by heating in a boiling water bath. To this solution was added 400 mg of succinic anhydride. During the reaction, the pH was kept at 8.0 by addition of 1 M NaOH. After 60 min, the modified protein was dialyzed against deionized water and lyophilized. The extent of the modification of amino groups in the protein was estimated to be over 98% by the method of Fields (8). 181
by Academic
of reproduction
Press, Inc.
in any form
reserved.
182
HATAKEYAMA,
KOHZAKI.
AND
YAMASAKI
Protease Assay Fifty microliters of the succinylcasein (l-10 mg/ml) in 50 InM sodium borate buffer (pH 8.0) was dispensed into each of the microtiter plate wells. The proteolytic reaction was started by the addition of 50 ~1 of the solution containing proteases. After incubation at 37°C for various times, 50 ~1 of 0.1 M sodium tetraborate/O.l M NaOH containing 5% SDS was added. In the case of subtilisin, addition of this solution was not sufficient to terminate the proteolytic reaction. Therefore, 10 ~1 of 0.5 M HCl was added, followed by 50 ~1 of 0.1 M sodium tetraboratei0.2 M NaOH containing 5% SDS. Ten minutes after the addition of 50 ~1 of 50 mM TNBS, 50 ~1 of freshly prepared 0.2 M NaH,PO, containing 5 mM Na,SO, was added, and the absorbance at 405 nm was directly read using a Immunoreader NJ-2000 (Intermed, Tokyo, Japan). -0
Comparison
of the Sensitivity
of the Protease Assays
The protease assay reported here was compared with the other protease assays that use casein or its derivative, azocasein, for their sensitivity. Aqueous trypsin solution (250 ~1) and the same volume of casein or azocasein (10 mg/ml) dissolved in 50 mM sodium borate buffer (pH 8.0) were mixed and incubated at 37°C for 60 min. After addition of 500 ~1 of 5% TCA and following centrifugation, the supernatant was examined for the extent of hydrolysis by measurement of the absorbance at 280 nm (1) or 450 nm [for azocasein (a)] or by the ninhydrin method (9). Preparation of Crude Protein philippinarum
Extract from T.
All steps were carried out at 4°C. Short-necked clam (T. philippinarum) was obtained from a local market. Tissues of T. philippinarum were homogenized with a Waring blender in 3 vol of 0.9% NaCl solution. After removal of debris by centrifugation, solid ammonium sulfate was added to the solution to give 60% saturation. Precipitated proteins were collected by centrifugation, dialyzed against deionized water for 4 h, and used in the assay. The concentration of protein was determined by the method of Bradford (10). RESULTS
Proteolytic activity of trypsin, chymotrypsin, thermolysin, and subtilisin toward the succinylcasein is demonstrated in Fig. 1. As a result of hydrolysis of peptide bonds in the substrates, the absorbance at 405 nm arising from the reaction between TNBS and newly appearing a-amino groups increased. While casein itself gave strong background absorption (A,, = 1.2 at 5 mg/ml) due to the original free amino groups, the succinylcasein showed little background and the increase in the absor-
10
20
30
TIME
40
50
60
(min)
FIG. 1. Hydrolysis of the succinylcasein by the proteases. Fifty microliters of trypsin (0) (2.5 pg/ml), chymotrypsin (0) (2.5 pg/ml), thermolysin (0) (1.0 pglml), and subtilisin (m) (1.0 ag/ml) were mixed with 50 ~1 of the succinylcasein (5 mg/ml) dissolved in 50 XnM sodium borate buffer (pH 8.0) and incubated at 37°C in microtiter plate wells. The extent of hydrolysis of the substrates was determined with TNBS in duplicate as described in the text. The A,, value without the protease and the substrate was taken as a blank.
bance resulting from the hydrolysis was clearly detected. A higher increase in the absorbance was observed with thermolysin and subtilisin, reflecting their relatively low substrate specificity, while chymotrypsin and trypsin gave a lower increase due to their limited hydrolyzing sites. The hydrolysis of the succinylcasein by trypsin proceeded rapidly until 10 min, but after that the rate of reaction decreased considerably. Since most of the c-amino groups of lysine residues are blocked, the sites in the succinylcasein susceptible to the action of trypsin should mostly be confined to arginine residues, which could be rapidly hydrolyzed within a short period at the initial stage of the reaction. Figure 2 shows the increase in the absorbance at 405 nm with the protease concentrations when incubated with the substrate at 37°C for 1 h. The extent of hydrolysis varied depending on the four proteases used and their concentrations. The highest increase in absorbance was observed with subtilisin, reflecting its low substrate specificity. While trypsin gave the smallest increase at higher concentrations, relatively rapid hydrolysis of the substrate was observed at low concentration of the enzyme up to 0.3 pg/ml. Although this assay seems less sensitive for proteases with high specificity than for those with low specificity as described above, sensitivity could be further enhanced by using higher concentrations of the substrate.
PROTEASE
ASSAY
TO
MEASURE
PROTEOLYTIC
183
ACTIVITY TABLE
Comparison
of the
1
Sensitivity
of the
Protease
Assays
Substrate Casein Trypsin (rdd 0.01 0.1 1
Succinylcasein A ma 0.038 + 0.013 0.217 + 0.016 0.582 + 0.025
A mob
A mc
N.D.d 0.007 * 0.002 0.084 * 0.003
N.D. 0.014 f 0.005 0.523 f 0.012
Azocasein A mb N.D. 0.052 + 0.028 0.113 + 0.012
Note. Indicated concentrations of trypsin were mixed with the substrate (5 mg/ml for the succinylcasein and 10 mg/ml for casein and azocasein) in 50 mM sodium borate buffer (pH 8.0) at a 1:l ratio by volume and incubated at 37°C for 60 min. The assays were performed in triplicate and values are means f SD. a This method. b Absorbance of the supernatant after TCA precipitation (1, 2). ’ Ninhydrin method (9). d Not detected. 0.2
0
0.4
0.6
PROTEASE
0.8
1.0
(pg/ml)
FIG. 2. Hydrolysis of the succinylcasein at various concentrations of trypsin (0), chymotrypsin (a), thermolysin (O), and subtilisin (w). Fifty microliters of the succinylcasein (5 mg/ml) in 50 mM sodium borate buffer (pH 8.0) and 50 ~1 of the protease solutions at indicated concentrations were incubated at 37°C for 60 min. The determinations were done in duplicate. The A,,, value without the protease and the substrate was taken as a blank.
As shown in Fig. 3, the final absorbance at 405 nm increased with increasing concentration of the substrate when the activity of trypsin was assayed. Since the succinylcasein prepared here gave little background absorbance, there was no difficulty in using a substrate solution of at least 10 mg/ml. These results suggest that this
r
assay could also be applicable to proteases with high specificity by using a high concentration of the substrate. The sensitivity of this assay was compared in Table 1 with those of the other protease assays using casein or its derivative, azocasein. While trypsin clearly gave a measurable level of absorbance even at the concentration of 0.01 pgjml(O.5 ngper 50 yl used for each determination), the other three assays could not detect activity of trypsin at this concentration. As an application of this assay, proteolytic activity in crude extract of T. philippinarum tissues was determined as shown in Fig. 4. Since the crude extract itself yielded a fairly high background (A,,, = 1.9 for 64 pg/ml protein) after reaction with TNBS, data were plotted following subtraction of the blank values. The absorbance at 405 nm increased with time, depending on the protein concentration. By measuring the increase in the absorbance, the proteolytic activity in the crude extract was clearly determined within 60 min. DISCUSSION
0 0
10
20
30
TIME
40
50
60
(min)
FIG. 3. Hydrolysis of the succinylcasein by trypsin. Activity of trypsin (2.5 pg/ml) was measured using the substrate at concentrations of 1 mg/ml (0), 2 mg/ml (O), 5 mg/ml (O), and 10 mg/ml (w). The assay was done as described in the legend to Fig. 1.
The assay presented is based on the reaction between TNBS and a-amino groups (11) formed by hydrolysis of peptide bonds in the substrate. All the steps in the assay can be performed in the microtiter plate wells. Therefore, it requires small amounts of the samples as well as the substrate and no centrifugation step. The yellow color arising from the reaction between TNBS and newly formed a-amino groups in the succinylcasein can be conveniently measured with the microtiter plate reader. An assay for proteases using TNBS and the casein derivative has also previously been reported by Lin et al. (12). They performed the reaction of TNBS for 30 min at 5O”C, while we shortened the reaction time to 10
184
HATAKEYAMA,
TIME
KOHZAKI,
(min)
FIG. 4. Proteolytic activity of 2’. ~~~~~pp~~~um extract. Fifty microliters of the solution of proteins extracted from !l! p~~~~pp~~ru~ tissues was mixed with 50 ~1 of the succinylcasein (5 mglml) in 50 mM sodium borate buffer (pH 8.0) and incubated at 37°C. The increase in A,,, was plotted as proteolytic activity after subtraction of the blank values for which hydrolysis of the substrate had been stopped by addition of 0.1 M sodium tetraborate/O.l M NaOH containing 5% SDS immediately after mixing with the samples. The determinations were done in triplicate, and values are expressed as means rt SD.
min at room temperature, essentially employing the method of Fields (8). In the latter method, a higher concentration of TNBS was used to accelerate the reaction and after addition of sulfite ion the absorbance of TNPamino groups was measured at 420 nm, where unreacted reagent did not interfere with the absorption of the reaction products. In the experiment presented in this report, however, measurements were done at 405 nm since this is one of the wavelengths available for the microtiter plate reader that we used, and absorption of the products is comparably high enough to be determined at that wavelength. Moreover, there is another difference in that Lin et al. used the NJV-dimethyl proteins as substrates, which were modified at amino groups by reductive methylation, whereas we used the succinylcasein. Besides ease of preparation, succinylcasein has the additional advantage of increased solubility, especially at acidic pH due to the shift of the isoelectric point to lower pH when compared with unmodified casein. Although casein and N,N-dimethylcasein are insoluble between pH 3.5 and 5.5, the succinylcasein is soluble above pH 4, enabling the assay of proteases having optimum pH at the acidic region. Since the reaction of TNBS proceeds in proportion to the number of hydrolyzed peptide bonds and the reaction products, TNP-amino groups, have a large molar absorption coefficient [22,000 M-’ cm-’ at 420 nm (S)],
AND
YAMASAKI
proteolytic activity can be determined quantitatively with high sensitivity. Since the succinylcasein is thought to be susceptible to the action of a wide variety of proteases, this assay may be especially useful in detecting a protease with broad specificity or a protease mixture as shown in the case of T. philippinarum extract (Fig. 4). Hydrolysis by proteases with high substrate specificity, such as trypsin or chymotrypsin, gives a relatively small increase in the absorbance at 405 nm. However, sensitivity could be much enhanced by using a higher concentration of the substrate as the succinyicasein gives a very low background value. In addition to its high sensitivity, this assay requires only small amounts of sample; therefore, it would also be very useful when proteolytic activity in many samples must be examined. Because of the reactivity of TNBS, however, there is the limitation that the protease solution should not contain a high concentration of amines; a concentration of Tris buffer higher than 10 mM gave a fairly strong background (data not shown). Therefore, when one is dealing with buffers containing a high concentration of amines, samples should be diluted prior to measurement so as to reduce the background level to such a degree that it can be subtracted from the value from the protease action. ACKNOWLEDGMENT We are grateful to Professor T. Kawarabata (Insect Pathology Division, Institute of Biological Control, Kyushu University) for allowing us to use the Intermed Immunoreader NJ-2000. REFERENCES 1. Kunitz, 2. Charney, 501-505. 3. Barrett, (Lorand, York. 4. Means,
M.
11947) J., and
J. Gen. Physiol. 30, B-310. Tomarelli,
R. M.
(1947)
J. Biot. Chem. 171,
A. J., and Kirschke, H. (1981) in Methods in Enzymology L., Ed.), Vol. 80, pp. 535-561, Academic Press, New G. E., and Rice,
R. H. (1970)
J. Biol.
Chem.
246,831-832.
5. Waxman, L. (1981) in Methods in Enzymology (Lorand, Vol. 80, pp. 664-680, Academic Press, New York. 6. Dresden, M. H., Rotmans, J. P., Deelder, A. M., Koper, Ploem, J. S. (1982) Anal. Hiochem. 126, 170-173.
L., Ed.), G., and
7. Plantner, J. J. (1991) Anal. Ricdzem. 195,129-131. 8. Fields, R. (1972) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N., Eds.), Vol. 25, pp. 464-468, Academic Press, New York. E. H., and Klostermeyer, H. (1976) in Methods in 9. Reimerdes, Enzymology (Lorand, L., Ed.), Vol. 45, pp. 26-28, Academic Press, New York. 10. Bradford, M. M. (1976) Anal. Bioehem. 72,248-254. 11. Okuyama, T., and Satake, K. (1960) 12. Lin, Y., Means, G. E., and Feeney, 244, 789-793.
J. Biochem. 47,454-466. J. Biol. Chem.
R. E. (1969)
BIOCHEMISTRY
204,
181-184
(1992)
A Microassay for Proteases Using Succinylcasein as a Substrate Tomomitsu Hatakeyama, Hidetsugu Kohzaki, and Nobuyuki Yamasaki Laboratory
Received
of Biochemistry,
November
Faculty
of Agriculture,
Kyushu
University,
Hakozaki
All rights
used: TCA, trichloroacetic TNP, trinitrophenyl;
$5.00 1992
812, Japan
that they require the specialized fluorometer or the acid-precipitation step. In this report, we present a microassay system for determination of protease activity that is carried out in the microtiter plate wells. This method employs as a substrate a casein derivative whose amino groups are chemically modified by succinylation, and amino groups appearing by the hydrolysis of the substrate are quantitatively detected with trinitrobenzene sulfonate (TNBS). Since whole reactions can be carried out in the microtiter plate wells, the amounts of the samples and the reagents are small, and the following measurement of absorbance can be rapidly done by using a microtiter plate reader. We also demonstrate the proteolytic activity in short-necked clam Tapes philippinarum tissues conveniently detected by this method.
MATERIALS
Casein has been widely used in the protease assays as a substrate, since it is readily hydrolyzed by most of the proteases with various specificities. While TCA’ soluble peptides resulting from protease action can be simply detected by measurement of the absorbance at 280 nm, which is due to aromatic amino acid residues in the peptides (l), the sensitivity can be further enhanced by means of the substrates with covalently attached chromophores (2,3) or those labeled with radioactive isotopes (45). However, such assays are sometimes very time-consuming and require expensive substrates. Some assays for proteases using a microtiter plate in which fluorescence or absorption resulting from the hydrolysis of the substrates was measured by automatic instruments, thereby facilitating the procedure, have been reported (6,7). However, there are still problems in
0003.2697/92 Copyright 0
Fukuoka
18, 1991
A photometric assay for proteases has been developed. A chemically modified casein whose amino groups were succinylated was used as a substrate. After incubation with trypsin, chymotrypsin, thermolysin, and subtilisin, the extent of hydrolysis of the substrate was determined with trinitrobenzene sulfonate (TNBS). The whole procedure of the assay was performed in the microtiter plate wells and the increase in the absorbance resulting from the reaction between TNBS and newly formed amino groups in the substrate was able to be determined with a high sensitivity by a microtiter plate reader, enabling the simultaneous measurement of a number of samples. Application of this method to the measurement of proteolytic activity contained in the protein extract of Tapes philippinarum is B 1992 Academic Press, Inc. demonstrated.
’ Abbreviations benzene sulfonate; fate.
6-10-1,
SDS,
acid; TNBS, trinitrosodium dodecyl sul-
AND
METHODS
Trypsin, chymotrypsin, subtilisin Carlsberg, and azocasein were purchased from Sigma (St. Louis, MO). Thermolysin was from Wako (Osaka, Japan). Casein (Hammarsten) was obtained from Merck (Darmstadt, Germany). TNBS and succinic anhydride were products of Nacalai Tesque (Kyoto, Japan). Other reagents used were of analytical grade.
Preparation
of the Succinylcasein
Casein (500 mg) was dissolved in 100 ml of 50 mM sodium borate buffer (pH 8.0) by heating in a boiling water bath. To this solution was added 400 mg of succinic anhydride. During the reaction, the pH was kept at 8.0 by addition of 1 M NaOH. After 60 min, the modified protein was dialyzed against deionized water and lyophilized. The extent of the modification of amino groups in the protein was estimated to be over 98% by the method of Fields (8). 181
by Academic
of reproduction
Press, Inc.
in any form
reserved.
182
HATAKEYAMA,
KOHZAKI.
AND
YAMASAKI
Protease Assay Fifty microliters of the succinylcasein (l-10 mg/ml) in 50 InM sodium borate buffer (pH 8.0) was dispensed into each of the microtiter plate wells. The proteolytic reaction was started by the addition of 50 ~1 of the solution containing proteases. After incubation at 37°C for various times, 50 ~1 of 0.1 M sodium tetraborate/O.l M NaOH containing 5% SDS was added. In the case of subtilisin, addition of this solution was not sufficient to terminate the proteolytic reaction. Therefore, 10 ~1 of 0.5 M HCl was added, followed by 50 ~1 of 0.1 M sodium tetraboratei0.2 M NaOH containing 5% SDS. Ten minutes after the addition of 50 ~1 of 50 mM TNBS, 50 ~1 of freshly prepared 0.2 M NaH,PO, containing 5 mM Na,SO, was added, and the absorbance at 405 nm was directly read using a Immunoreader NJ-2000 (Intermed, Tokyo, Japan). -0
Comparison
of the Sensitivity
of the Protease Assays
The protease assay reported here was compared with the other protease assays that use casein or its derivative, azocasein, for their sensitivity. Aqueous trypsin solution (250 ~1) and the same volume of casein or azocasein (10 mg/ml) dissolved in 50 mM sodium borate buffer (pH 8.0) were mixed and incubated at 37°C for 60 min. After addition of 500 ~1 of 5% TCA and following centrifugation, the supernatant was examined for the extent of hydrolysis by measurement of the absorbance at 280 nm (1) or 450 nm [for azocasein (a)] or by the ninhydrin method (9). Preparation of Crude Protein philippinarum
Extract from T.
All steps were carried out at 4°C. Short-necked clam (T. philippinarum) was obtained from a local market. Tissues of T. philippinarum were homogenized with a Waring blender in 3 vol of 0.9% NaCl solution. After removal of debris by centrifugation, solid ammonium sulfate was added to the solution to give 60% saturation. Precipitated proteins were collected by centrifugation, dialyzed against deionized water for 4 h, and used in the assay. The concentration of protein was determined by the method of Bradford (10). RESULTS
Proteolytic activity of trypsin, chymotrypsin, thermolysin, and subtilisin toward the succinylcasein is demonstrated in Fig. 1. As a result of hydrolysis of peptide bonds in the substrates, the absorbance at 405 nm arising from the reaction between TNBS and newly appearing a-amino groups increased. While casein itself gave strong background absorption (A,, = 1.2 at 5 mg/ml) due to the original free amino groups, the succinylcasein showed little background and the increase in the absor-
10
20
30
TIME
40
50
60
(min)
FIG. 1. Hydrolysis of the succinylcasein by the proteases. Fifty microliters of trypsin (0) (2.5 pg/ml), chymotrypsin (0) (2.5 pg/ml), thermolysin (0) (1.0 pglml), and subtilisin (m) (1.0 ag/ml) were mixed with 50 ~1 of the succinylcasein (5 mg/ml) dissolved in 50 XnM sodium borate buffer (pH 8.0) and incubated at 37°C in microtiter plate wells. The extent of hydrolysis of the substrates was determined with TNBS in duplicate as described in the text. The A,, value without the protease and the substrate was taken as a blank.
bance resulting from the hydrolysis was clearly detected. A higher increase in the absorbance was observed with thermolysin and subtilisin, reflecting their relatively low substrate specificity, while chymotrypsin and trypsin gave a lower increase due to their limited hydrolyzing sites. The hydrolysis of the succinylcasein by trypsin proceeded rapidly until 10 min, but after that the rate of reaction decreased considerably. Since most of the c-amino groups of lysine residues are blocked, the sites in the succinylcasein susceptible to the action of trypsin should mostly be confined to arginine residues, which could be rapidly hydrolyzed within a short period at the initial stage of the reaction. Figure 2 shows the increase in the absorbance at 405 nm with the protease concentrations when incubated with the substrate at 37°C for 1 h. The extent of hydrolysis varied depending on the four proteases used and their concentrations. The highest increase in absorbance was observed with subtilisin, reflecting its low substrate specificity. While trypsin gave the smallest increase at higher concentrations, relatively rapid hydrolysis of the substrate was observed at low concentration of the enzyme up to 0.3 pg/ml. Although this assay seems less sensitive for proteases with high specificity than for those with low specificity as described above, sensitivity could be further enhanced by using higher concentrations of the substrate.
PROTEASE
ASSAY
TO
MEASURE
PROTEOLYTIC
183
ACTIVITY TABLE
Comparison
of the
1
Sensitivity
of the
Protease
Assays
Substrate Casein Trypsin (rdd 0.01 0.1 1
Succinylcasein A ma 0.038 + 0.013 0.217 + 0.016 0.582 + 0.025
A mob
A mc
N.D.d 0.007 * 0.002 0.084 * 0.003
N.D. 0.014 f 0.005 0.523 f 0.012
Azocasein A mb N.D. 0.052 + 0.028 0.113 + 0.012
Note. Indicated concentrations of trypsin were mixed with the substrate (5 mg/ml for the succinylcasein and 10 mg/ml for casein and azocasein) in 50 mM sodium borate buffer (pH 8.0) at a 1:l ratio by volume and incubated at 37°C for 60 min. The assays were performed in triplicate and values are means f SD. a This method. b Absorbance of the supernatant after TCA precipitation (1, 2). ’ Ninhydrin method (9). d Not detected. 0.2
0
0.4
0.6
PROTEASE
0.8
1.0
(pg/ml)
FIG. 2. Hydrolysis of the succinylcasein at various concentrations of trypsin (0), chymotrypsin (a), thermolysin (O), and subtilisin (w). Fifty microliters of the succinylcasein (5 mg/ml) in 50 mM sodium borate buffer (pH 8.0) and 50 ~1 of the protease solutions at indicated concentrations were incubated at 37°C for 60 min. The determinations were done in duplicate. The A,,, value without the protease and the substrate was taken as a blank.
As shown in Fig. 3, the final absorbance at 405 nm increased with increasing concentration of the substrate when the activity of trypsin was assayed. Since the succinylcasein prepared here gave little background absorbance, there was no difficulty in using a substrate solution of at least 10 mg/ml. These results suggest that this
r
assay could also be applicable to proteases with high specificity by using a high concentration of the substrate. The sensitivity of this assay was compared in Table 1 with those of the other protease assays using casein or its derivative, azocasein. While trypsin clearly gave a measurable level of absorbance even at the concentration of 0.01 pgjml(O.5 ngper 50 yl used for each determination), the other three assays could not detect activity of trypsin at this concentration. As an application of this assay, proteolytic activity in crude extract of T. philippinarum tissues was determined as shown in Fig. 4. Since the crude extract itself yielded a fairly high background (A,,, = 1.9 for 64 pg/ml protein) after reaction with TNBS, data were plotted following subtraction of the blank values. The absorbance at 405 nm increased with time, depending on the protein concentration. By measuring the increase in the absorbance, the proteolytic activity in the crude extract was clearly determined within 60 min. DISCUSSION
0 0
10
20
30
TIME
40
50
60
(min)
FIG. 3. Hydrolysis of the succinylcasein by trypsin. Activity of trypsin (2.5 pg/ml) was measured using the substrate at concentrations of 1 mg/ml (0), 2 mg/ml (O), 5 mg/ml (O), and 10 mg/ml (w). The assay was done as described in the legend to Fig. 1.
The assay presented is based on the reaction between TNBS and a-amino groups (11) formed by hydrolysis of peptide bonds in the substrate. All the steps in the assay can be performed in the microtiter plate wells. Therefore, it requires small amounts of the samples as well as the substrate and no centrifugation step. The yellow color arising from the reaction between TNBS and newly formed a-amino groups in the succinylcasein can be conveniently measured with the microtiter plate reader. An assay for proteases using TNBS and the casein derivative has also previously been reported by Lin et al. (12). They performed the reaction of TNBS for 30 min at 5O”C, while we shortened the reaction time to 10
184
HATAKEYAMA,
TIME
KOHZAKI,
(min)
FIG. 4. Proteolytic activity of 2’. ~~~~~pp~~~um extract. Fifty microliters of the solution of proteins extracted from !l! p~~~~pp~~ru~ tissues was mixed with 50 ~1 of the succinylcasein (5 mglml) in 50 mM sodium borate buffer (pH 8.0) and incubated at 37°C. The increase in A,,, was plotted as proteolytic activity after subtraction of the blank values for which hydrolysis of the substrate had been stopped by addition of 0.1 M sodium tetraborate/O.l M NaOH containing 5% SDS immediately after mixing with the samples. The determinations were done in triplicate, and values are expressed as means rt SD.
min at room temperature, essentially employing the method of Fields (8). In the latter method, a higher concentration of TNBS was used to accelerate the reaction and after addition of sulfite ion the absorbance of TNPamino groups was measured at 420 nm, where unreacted reagent did not interfere with the absorption of the reaction products. In the experiment presented in this report, however, measurements were done at 405 nm since this is one of the wavelengths available for the microtiter plate reader that we used, and absorption of the products is comparably high enough to be determined at that wavelength. Moreover, there is another difference in that Lin et al. used the NJV-dimethyl proteins as substrates, which were modified at amino groups by reductive methylation, whereas we used the succinylcasein. Besides ease of preparation, succinylcasein has the additional advantage of increased solubility, especially at acidic pH due to the shift of the isoelectric point to lower pH when compared with unmodified casein. Although casein and N,N-dimethylcasein are insoluble between pH 3.5 and 5.5, the succinylcasein is soluble above pH 4, enabling the assay of proteases having optimum pH at the acidic region. Since the reaction of TNBS proceeds in proportion to the number of hydrolyzed peptide bonds and the reaction products, TNP-amino groups, have a large molar absorption coefficient [22,000 M-’ cm-’ at 420 nm (S)],
AND
YAMASAKI
proteolytic activity can be determined quantitatively with high sensitivity. Since the succinylcasein is thought to be susceptible to the action of a wide variety of proteases, this assay may be especially useful in detecting a protease with broad specificity or a protease mixture as shown in the case of T. philippinarum extract (Fig. 4). Hydrolysis by proteases with high substrate specificity, such as trypsin or chymotrypsin, gives a relatively small increase in the absorbance at 405 nm. However, sensitivity could be much enhanced by using a higher concentration of the substrate as the succinyicasein gives a very low background value. In addition to its high sensitivity, this assay requires only small amounts of sample; therefore, it would also be very useful when proteolytic activity in many samples must be examined. Because of the reactivity of TNBS, however, there is the limitation that the protease solution should not contain a high concentration of amines; a concentration of Tris buffer higher than 10 mM gave a fairly strong background (data not shown). Therefore, when one is dealing with buffers containing a high concentration of amines, samples should be diluted prior to measurement so as to reduce the background level to such a degree that it can be subtracted from the value from the protease action. ACKNOWLEDGMENT We are grateful to Professor T. Kawarabata (Insect Pathology Division, Institute of Biological Control, Kyushu University) for allowing us to use the Intermed Immunoreader NJ-2000. REFERENCES 1. Kunitz, 2. Charney, 501-505. 3. Barrett, (Lorand, York. 4. Means,
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