A simple method for determination of proteinase activity

A simple method for determination of proteinase activity

Journal of Cereal Science 8 (1988) 69-82 A Simple Method for Determination of Proteinase Activity* P. R. MATHEWSONtt, B. w. SEABOURN§ and Y. POMERANZ...

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Journal of Cereal Science 8 (1988) 69-82

A Simple Method for Determination of Proteinase Activity* P. R. MATHEWSONtt, B. w. SEABOURN§ and Y. POMERANZt tUSDA -Agricultural Research Service, U.S. Grain Marketing Research Laboratory, Manhattan, KS 66502 and §Kansas State University, Department of Grain Science and Industry, Manhattan, KS 66502, U.S.A. Received 4 January 1988 A method has been developed that is applicable for the determination of both exoand endo proteinase activity in cereal extracts. The procedure is based on measurement of the fluorescent adduct formed between the ex-amino group resulting from proteolysis and o-phthaldialdehyde in the presence of ethanethiol. This adduct can be measured fluorometrically at its emission wavelength (450 nm) or spectrophotometrically at its excitation wavelength (340 nm). Both methods respond similarly though, as expected, the fluorescence measurement is more sensitive. This assay procedure is simple, does not require separation of hydrolysis product from substrate, and overcomes many of the limitations associated with previously reported proteinase assays involving precipitation with tricholoracetic acid, dialysis or viscometric analysis.

Introduction The study of proteinase enzymes in cereals has been hampered by the lack of an assay procedure with sufficient sensitivity to both exo- and endoproteolysis. The largely uncharacterized proteinase activity in cereals is usually assayed using a non-cereal substrate protein such as hemoglobin or Cl-casein. To separate unhydrolyzed protein from hydrolysis products, trichloroacetic acid (TCA) is commonly used l -4. In principle, the TCA-insoluble material is assumed to be unhydrolyzed substrate protein while hydrolysis products are assumed to be TCA-soluble. The extent of hydrolysis is determined by measuring the amount of TCA-soluble material. In practice, however, the separation is not that precise. Endoproteinase activity may result in large peptides that are still TCA-insoluble. This activity would not be detected. Exoproteinase activity produces TCA-soluble amino acids, whose detectability depends on the analysis method used. The terminal amino acid may unduly affect the results. This is true for hemoglobin,

* Contribution 85-80-J, Department of Grain Science and Industry, Kansas Agricultural Experiment Station, Manhattan, KS 66506, U.S.A. Mention of firm names or trade products does not constitute endorsement by the U.S. Department of Agriculture over others not mentioned. :I: To whom correspondence should be addressed at Nabisco Brands, Inc., Corporate Technology Center, 100 DeForest Avenue, PO Box 1943, East Hanover, NJ 07936, U.S.A. Abbreviations used: TCA = trichloroacetic acid; OPA = o-phthaldialdehyde; CBZ-PAL = N-carbobenzoxY-L-phenylalanyl-L-leucine, CBZ-PAA = CB2-phenylalanylalanine; LAP = leucine amino peptidase; LTMAPA = LEU-TRP-MET-ARG-PHE-ALA-acetate; SDS = sodium dodecyl sulphate; BME = ~-mer­ captoethanol. ET = ethanethiol; CBPASE Y = carboxypetidase Y. 0733-5210/88/040069+ 14 $03.00/0

© 1988 Academic Press Limited

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P. R. MATHEWSON ET AL.

which has a C-terminal penultimate tyrosine residue that, when released, will produce a rapid increase in absorbance at 280 nm. Thus, this TCA precipitation method for separating substrate from hydrolysis products tends to bias the results toward detection of exoproteinase activity while being relatively insensitive to endoproteolysis. Schwabe 5 reported a proteinase assay using a fluorochrome (fluorescamine), which specifically labeled a-amino groups. For this test, the substrate protein was not separated but was succinylated in order to block the terminal amino groups and thereby lower the blank fluorescence. Preston 6 applied this concept to an automated fluorometric proteinase assay, choosing to separate unhydrolyzed substrate by dialysis. This procedure results in dilution of the analytes and is, in principle, similar to TCA precipitation in that small compounds (amino acids) will dialyse faster than larger peptides. This procedure would also be biased toward exo-proteinase detection. In later reports 7 ,8 this automated fluorometric assay is, in fact, referred to as an exoproteinase assay. Several alternative fluorometric compounds have been utilized by other investigators, the most widely used being o-phthaldialdehyde (OPA)9-u. We report here a modification of the reported technique that provides a sensitive assay for determination of both exoand endoproteinase activity. The assay has been demonstrated to respond to proteinases of known specificity as well as to the uncharacterized proteinases present in a wheat extract. Experimental

Materials Carboxypeptidase Y, trypsin, chymotrypsin and hemoglobin were from Worthington Biochemical Corp., Freehold, NJ. The trypsin had been treated with L-(tosylamido-2-phenyl) ethyl chloromethyl ketone to inhibit contaminating chymotryptic activity. Pronase -CB and papain were from Calbiochem-Behring Corp., La Jolla, CA. Amino acids, cytochrome-C (equine), ~­ lactoglobulin, insulin (bovine), albumin (BSA), OPA, leucinamide, N-carbobenzoxy-L-phenylalany1-L-1eucine (CBZ-PAL), CBZ-pheny1alanylalanine (CBZ-PAA) and' good' buffers (HEPES, EPPS and MES) were from Sigma Chemical Co., St Louis, MO. Leucine aminopeptidase (LAP) was from Boehringer Mannheim Biochemicals, Indianapolis, IN. Ribonucliease-A was from Pharmacia Fine Chemicals, Piscataway, NJ. Glucagan (porcine) and a peptide, LEU-TRP-METARG-PHE-ALA-acetate (LTMAPA), were from Research Plus, Inc., Bayonne, NJ. Reduced and pyridylethylated purothionin was a gift from Dr B. L. Jones (USDA, Madison, WI) and was prepared as described by Mak and Jones 12 • Sodium dodecyl sulfate (SDS) and ~-mercaptoethanol (BME) were electrophoresis grade from Bio-Rad Laboratories, Richmond, CA. Ethanethiol (ET) was from Pierce Chemical Co., Rockford, IL and Wheatpro was from Manildra Protein Corp., Myrtle Beach, SC. All other chemicals were reagent grade and all water used was distilled and deionized.

Instrumentation Instruments used in this study were a Pye-Unicam 1750B UV-VIS spectrophotometer and a Farrand Model A4 fluorometer. A 1'0 cm square cuvette was used for all absorbance readings. Fluorometric readings were done in 75 x 10 mm round cuvettes. The fluorometer allows for selection of one of six aperture openings; the smallest aperture (no. I) and the next largest (no. 2) were used as noted. Interference filters having a band pass of less than 10 nm were used for excitation at 340 nm and emission at 450 nm.

DETERMINATION OF PROTEINASE ACTIVITY

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Volumetric transfers of less than 500 ~l were done using microsyringes (Hamilton Co., Reno, NV). Larger transfers, up to 5'0 ml, were done using pipettors (Rainin Instrument Co., Woburn, MA).

Proteinase assay A test tube (13 x 100 mm) containing substrate solution (usually 1·0 mg/ml) in an appropriate buffer (see Table I) was equilibrated in a water bath at 40 "c. To begin the assay, proteinase was TABLE 1. Conditions used for Proteinase assaysa Enzyme Pronase Trypsin [EC 3 .4.21 .4] Chymotrypsin [EC 3 .4.21 . 1] Carboxypeptidase Y [EC 3 .4. 12.-] Papain [EC 3 . 4 . 22 . 2] Leucine aminopeptidase [EC 3 .4. 11 . 1]

Enzyme (mg/m1)

Substrate b

Buffer

pH

1·0 1'0

Aile ~- Lactoglobulin

7·0 7-8

1·0 1'0

All others

1·0 0·1

All others CBZ-PAL

5mMP04 5mMHEPES, 2mMCaCl 2 5 mMP0 4 5 mMEPPSP, 2mM CaCI 2 5 mMP0 4 5mMMES 5 mMP0 4 1 mMHEPES, 5mMBME 1 mMEDTA 25 mM Bicine 50 mM Mg acetate

6'5 8.0

1·0 10'0 1·0

~-Lactoglo

bulin

All others Cytochrome-C Leucinamide LTMAPA peptide

8·2 7-8 8'2 6'5

8·6

• All assays at 40°C except papain at 60 °e. b All substrates at I mg/ml except CBZ-PAL at I,D mM. e Substrates used were cytochrome-C, ~-Iactoglobulin, insulin, bovine serum albumin, native and acetylated hemoglobin, chymotrypsinogen-A, ribonuclease-A, glucagon and succinylated Wheatpro.

added to the substrate, mixed and a sample (usually 10-20 ~l) immediately removed and added to 200 ~l of saturated borate buffer (pH 9,4) containing 1 % SDS in a fluorometer cuvette. The combination of dilution, high pH and SDS stops the reaction. Twenty-five ~l of OPA reagent solution (consisting of 2·0 ml methanol, 220 ~I saturated borate buffer, 12·5 mg OPA and 22 III ET) was added to the tube containing sample-8D8-borate, mixed and allowed to react at room temperature for 15 min. At that time, 1·0 ml methanol was added to the cuvette and thoroughly mixed. This tube served as a control. This procedure was repeated for each sample taken from the protease-substrate reaction mixture at appropriate time intervals. The sampling intervals can be any time period as long as the time from addition of OPA reagent to addition methanol is maintained. For the assay of amino acids, peptides and proteins at various concentrations, solutions of each analyte were prepared in an appropriate buffer and diluted with the same buffer to the desired concentrations. A specific amount was added to each cuvette containing 200 ~l of SDS-borate followed by OPA reagent. After 15 min, 1·0 ml of methanol was added and thoroughly mixed. The blank consisted of an aliquot of buffer in place of analyte. The fluorometer was set at 0·00 rnA with a glass rod in the sample position of the cuvette holder. The fluorescence of the control sample was read relative to the glass rod and this value reduced

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P. R. MATHEWSON ET AL.

to zero using the blank adjust control. The fluorescence of all other samples in the series was read relative to the control, in sequence. To determine absorbance, the spectrophotometer was adjusted to read 0'000 at 340 nm with water in the reference cell and the methanol in the sample cell. All samples were then read vs. water and the value of the control subtracted from the values for subsequent readings. The reaction of OPA-reagent with ex-amino groups was monitored by adding methanol at 1 min intervals after adding OPA-reagent to the sample-SDS-borate. The fluorescence decreased for about 10 min after which it was stable at least I h. We chose to allow the reaction to proceed for 15 min before adding methanol.

Acylation oj substrate protein The succinylation of wheat gluten was based on the procedure of Wu et al,13 and Means and Feeney14. Wheatpro (I g) was suspended in 0·02 M HCI (100 ml). This mixture was homogenized using a Brinkmann Polytron for 10 min. The suspension was autoclaved at 121°C for 20 min and allowed to cool to room temperature. About 20 ml of saturated borate buffer was added while stirring with the Polytron, raising the pH of the suspension from 2·1 to 9·0. Succinic anhydride (2 g) was added slowly while homogenation continued. The pH was maintained at 9'0 by dropwise addition of 2N NaOH. After addition of succinic anhydride, the mixture was homogenized for I h at room temperature. Further succinic anhydride (l g) as added as before and allowed to stir for an additional hour. The succinylated Wheatpro was dialysed vs. water and lyophilized. Yield was about 970 mg. The number of free amino groups decreased by ~ 90 % as determined by the method of Fields l ;.

Preparation oj wheat extract All steps were carried out at 4°C. Sound, hard red winter wheat (cv. Scout 66) (10 g) was homogenized for 30 min in 0·025 M MES buffer containing 0·001 M EDTA (50 ml), and 0'002 M BME at pH 6'0, using a Brinkmann Polytron. The homogenized slurry was centrifuged at 20000 g for 30 min and filtered through a 0·2 11m filter.

Results OPA has been shown previously to produce fluorescent adducts with amino acids, peptides and proteins l 6-lB. Tests in our laboratory also showed a strong fluorescent response to these compounds. Amino acids and peptides (glucagon) showed little or no quenching effect, but larger proteins did show some quenching dependent on protein concentration. The absorbance at 340 nm, remained linear with analyte concentration while fluorescence became non-linear due to quenching. Several commercially available proteinase preparations of known specificity were used to demonstrate that this procedure provides sensitivity to both exo- and endo-proteinase activity. Pronase is a broad spectrum proteinase preparation containing endopeptidases and exopeptidases produced by a strain of Streptomyces griseus K-I, which is capable of hydrolyzing many proteins to amino acids 19 • The hydrolysis of cytochrome-C by Pronase is shown in Fig. 1. At low enzyme concentration the response was linear with time. As Pronase concentration increased, the response became non-linear due to substrate depletion or change in hydrolysis rate due to depletion of the most rapidly cleaved peptide bonds. Figure 2 shows that the response was also linear with Pronase concentration for hydrolysis times up to about 12 min.

DETERMINATION OF PROTEINASE ACTIVITY 0·6

./

0·5

~ (; OJ

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OJ

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0

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0·3

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0·2 0·1

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73

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24

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30

0'"

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36

Time (min)

FIGURE I. Hydrolysis of cytochrome-C with pronase using 5 j.!g, A--A, 10 j.!g, 0--0 and 20 j.!g, . - - . of Pronase. For conditions see Table 1. 0·24 0·21

.,u .,uc en

0·18

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0-12

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0·06 0·03

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8

12

16

20

Enzyme (fLl )

FIGURE 2. Fluorescence response of hydrolysis of cytochrome-C VB. Pronase concentration at 3 min, .6,--.6.; 6 min, A--.A ; 9 min, 0--0 and 12 min, . - -•.

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P. R. MATHEWSON ET AL.

0·48 0·42

0·36 <11

g

~

0·30

<11

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6

12

18

24

30

36

Time (min)

FIGURE 3. Hydrolysis of cytochrome-C with trypsin using 1 Ilg, ....- -... ; 2 Ilg, 0--0 and 5 Ilg, . - - . of trypsin. For conditions see Table 1.

Similar results were obtained for tryptic hydrolysis of cytochrome-C (Fig. 3). Trypsin is a specific endoproteinase that cleaves peptide bonds preferentially at lysyl and arginyl residues 19 • The response was linear with time at low trypsin concentration, and as for Pronase, became non-linear at higher enzyme concentration. The fluorescent response of this rather specific protease was similar to that of the non-specific Pronase though less trypsin was used. This is probably due to the high lysine content of cytochrome-C ( "'" 20 %), which provides an abundance of susceptible peptide bonds. The fluorescent response was also linear with trypsin concentration (Fig. 4).
DETERMINATION OF PROTEINASE ACTIVITY 0-48

75

=-----------------,

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0·18 0·12 0·06

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5

6

FIGURE 4. Fluorescence response of hydrolysis of cytochrome-C YS. trypsin concentration at 6 min, f).--f).; 12 min, A--A; 18 min, 0--0 and 30 min,

.--e,

0'35 0·30

..

.

0·25

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FIGURE 5. Hydrolysis of cytochrome-C with chymotrypsin using 2 ~g, A--A; 5 ~g, 0--0 and 10 ~g, . - - . of chymotrypsin. For conditions see Table 1.

P. R. MATHEWSON ET AL.

76 \·4

./. ./

1·2

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30

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-_..... --_ ..... 50

60

Time (min)

FIGURE 6. Hydrolysis ofcytochrome-C with CBPASE Y using 10 J.lg, .----. and 25 J.lg, . - - . of carboxypeptidase. For conditions see Table I.

0·7 0·6

'"

~

o

0-3

4i

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12 16 Time (min)

20

24

FIGURE 7. Hydrolysis ofCBZ-PAL with CBPASEY, using 50 ng, ...- -... ; 100 ng, 0--0 and 200 ng, . - - . of CBPASE Y. For conditions see Table I.

DETERMINATION OF PROTEINASE ACTIVITY

77

0·5

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Carboxypeptidase y (j..L9 1

FIGURE 8. Fluorescence response of hydrolysis of CBZ-PAL Ys. CBPASE Yeancentration at 4 min, ....- -.... ; 8 min, 0--0 and 16 min,

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FIGURE 9. Hydrolysis of LTMAPA using 10 J.lg, 1:::..... 6.; 15 J.lg, . - - . and 30 J.lg, 0---- 0 of leucine-amino peptidase. For conditions see Table I.

78

P. R. MATHEWSON ET ALo

1-0

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FIGURE 10. Hydrolysis of leucinamide at pH 8·6, /::)..0 • of::", CBZ-PAL at pH 5,25, .--e; reduced, pyridyl-ethylated purothionin at pH 5'5, 0 ---- 0 and deamidated, succinylated wheat protein at pH 5'5, A-'-A) using an extract of sound hard red winter wheat.

much more consistent. These data (Figs 7 and 8) showed a linear response with time and CPBASE Y concentration. On the lowest sensitivity setting, about 10 ng of CPBASE Y could be detected using CBZ-PAL. Hydrolysis ofLTMAPA with LAP is shown in Fig. 9. These data are similar to those for other proteinase. Similar data were observed for hydrolysis of an LAP model substrate, leucinamide. Since Pronase contains both exo- and enda-proteinase activity, the fluorescent response suggests that both types of activity can be measured in combination as well as singly. However, the precise proteinase content of Pronase is unknown, so to verify this implication trypsin and CPBASE Y were used, alone and in combination, to hydrolyze a single substrate protein. The data showed that the fluorometric response from both enzymes in combination was significantly higher than the sum of the responses for each enzyme alone, thus clearly indicating that both types of activity were being measured. The response from the combination of enzymes was more than additive probably because each trypsin cleavage produced a new C-terminus for hydrolysis by CPBASE Y. Further examination of this assay procedure with other enzyme-substrate com-

DETERMINATION OF PROTEINASE ACTIVITY

79

0'9

0·8

j

0·7

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0·6

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t

4i

0·3

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0·2

0·1

o

4

8

12

16

20

24

Time. (min)

FIGURE 11. Relationship between relative fluorescence, - - and absorbance at 340 nrn, --- for hydrolysis of cytochrome-C using 5 IJ.g, A, 7'5 IJ.g, 0 and 10 IJ.g, • of pronase.

binations has shown its applicability to a variety of conditions. Using a plant proteinase, papain, a linear fluorescent response was observed for the hydrolysis of cytochrome-C at pH 8'0, 60°C. Succinylated wheat protein was readily hydrolyzed by both Pronase and CBPASE Y but was resistant to papain, trypsin and chymotrypsin. To determine whether proteins without blocked N-terrnini could also be used effectively, ~-lactoglobulin, insulin, hemoglobin, BSA and ribonuc1ease-A were studied and found to be resistant to the action of trypsin and chymotrypsin. These proteins did produce a higher blank value than cytochrome-C, but this value was readily reduced to zero using the blank adjust control on the fluorometer. Even with a relatively high blank value ("" 0'500 rnA), activity was easily observed using Pronase to digest these protein substrates. To demonstrate the usefulness of this assay for determining proteinase activity in cereals, an extract of sound, hard red winter wheat was used as the source of enzymes. Using an LAP substrate, leuci:Qamide, a very strong response was observed at pH 8'6, thus indicating the presence of LAP-type activity in the extract (Fig. 10). When CBZPAA was used as substrate at pH 5'25, a very strong response was observed showing that carboxypeptidase activity is high in the wheat extract (Fig. 10). Two protein substrates, RP-purothionin and deamidated-succinylated Wheatpro, a wheat protein concentrate ( '" 92 % protein), were also tested. A response was observed for both proteins at pH

P. R. MATHEWSON ET AL.

80

5·50. This response may be due either to endo- or exo-proteinases or to both types of activity which may be present in the extract. If a fluorometer is not available, this method can still be utilized, though with significantly lower sensitivity, by measuring the absorbance at 340 nm rather than fluorescence at 450 nrn. The relationship between the absorbance and fluorescence is shown in Fig. 11. This figure shows both absorbance and fluorescence for the same samples taken during the course of Pronase hydrolysis of cytochrome-C. The relative fluorescence was read on aperture no. 2. This relationship between the two methods of analysis is not constant and will be discussed in greater detail in a subsequent report. Discussion

The utility of this proteinase assay lies in its ability to respond to both exo- and endoproteinase activity. It responds to amino acids, as well as peptides and proteins of various lengths and amino acid composition. The response results from the adduct formed by the reaction of o-phthaldialdehyde (I) with an a-amino group in the presence of a thiol producing a l-thioalkyl-2-alkylisoindole (II). This compound absorbs energy at 340 nm and fluoresces at about 475 nm 21 •

O ~

I

I

oII

C-H

C-H

+

II

o

n

Formation of the OPA fluorescent adduct requires the presence of a thiol compound. Most workers have used BME as the thiol, but Simons and Johnson 21 reported that the hydroxyl function of BME imparts instability to the adduct and that use of ET not only increased stability, but also resulted in higher fluorescent yields for amino acid adducts. In a later report 22 these authors discussed different aspects of this reaction and the effect of pH, solvent polarity and other considerations. Based on this information, ethanethiol was chosen as the thiol and methanol was used as the diluent. It should be noted that buffers containing free amino groups (Le. Tris) cannot be utilized in this procedure, but the large number of available buffers provide many alternatives. In addition, the noxious odor of ET can be a drawback, but this can be controlled effectively by preparing the OPA-reagent solution in a septum-sealed vial and using a microliter syringe to remove and deliver the reagent solution to the sample test tube. The choice of substrate is an important one for several reasons. As has been demonstrated, using a protein substrate may be a necessary condition for proteolysis, but it is not necessarily a sufficient condition. Many proteins, in their native state, are resistant to proteolytic hydrolysis 23 • Native hemoglobin is resistant to many proteinases and becomes susceptible only after denaturation by heat, extreme pH, urea or chemical cleavage. Schwabe 5 used denatured hemoglobin, as have many others, in his fluorescent

DETERMINATION OF PROTEINASE ACTIVITY

81

proteinase assay and noted high blank values caused by the free N-tenninal amino groups. This, along with possible quenching, can lower the sensitivity of the assay. For most of this work, cytochrome-C from horse heart was used as the substrate because it is a commercially available protein that has a naturally occurring acetylated Nterminus,24 which blocks the OPA reaction at that site. This results in very low blank values compared with substrates with free N-termini. It is desirable to have blank values as low as possible in order to optimize sensitivity, but a blocked N-terminus is not required for this assay. It has been demonstrated that proteins with free N-termini can be successfully utilized. Hemoglobin can quench fluorescence, as do other proteins used as substrates for proteolysis. Both the problem of high blank value and quenching can be minimized by using the appropriate substrate at relatively low concentration. The results demonstrate potential problems in the interpretation of proteinase assays. That is, while a positive response may indicate the presence of a protease, the lack of a response does not necessarily mean that no protease is present. It may be that the conditions of the assay are not suitable for a particular proteinase or that the substrate utilized is not susceptible to proteolytic cleavage. Thus, the conditions for proteolytic hydrolysis and the substrate must be carefully chosen to avoid misinterpretation of results. It is of interest to note that of all proteins investigated as possible substrates, cytochrome-C was the only one that proved to be susceptible to all enzymes studied.

Conclusions It has been shown that the OPA procedure described here for assay of proteinase activity can be successfully utilized to determine both exo- and endoproteinase activity, using a variety of protein substrates, as well as synthetic substrates. The different modes of activity can be measured singly or in combination. The method is simple to set up and carry out and results can be analyzed either fluorometrically with high sensitivity or spectrophotometrically if a fluorometer is unavailable. This procedure is a significant improvement over existing proteinase assays in that no separation of reactants and products is required. The reaction is not stopped by adding a reagent (such as TeA) to the protease reaction mixture. Instead, a small quantity, typically 10-20 Ill, of the proteinase reaction mixture is added to SDS-borate in a separate tube. This means that high background fluorescence, due to excessive amounts of substrate protein in the solution to be analyzed, is minimized and easily blanked out. This procedure also minimizes any quenching from substrate protein. In addition, it allows the protease reaction to continue, in a separate tube, under any conditions conducive to hydrolysis, so that the reaction may be sampled at convenient intervals to follow the course of hydrolysis. The use of ET rather than BME produces fluorescent adducts, which are highly fluorescent and stable for long periods. This method should prove useful, especially in determining proteinase activity in mixtures of cereal extracts containing unknown proteinase types. It allows for convenient determination of proteinase activity in fractions resulting from various purification procedures, regardless of the mode of proteolytic cleavage.

4

CER 8

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P. R. MATHEWSON ET AL.

References 1. 2. 3. 4. 5. 6. 7. 8.

9, 10. 11. 12, 13. 14.

IS. 16. 17. 18. 19. 20. 21. 22. 23.

24,

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