Sensitive fluorogenic substrates for the detection of trypsinlike proteases and pancreatic elastase

ANALYTICAL

BIOCHEMISTRY

Sensitive

126,447-455

(1982)

Fluorogenic Substrates for the Detection Proteases and Pancreatic Elastase PAUL J. BRYNES,

Departments

PATRICIA

of Pharmacological Stony

Sciences Brook,

ANDRADE,

AND DAVID

and Chemistry State New York 1 I794

of Ttypsinlike

GORDON

University

of New

York,

Received June 24, 1982 Several new fluorogenic substrates capable of measuring the proteolytic activities of pancreatic elastase, trypsin, and trypsinlike enzymes were prepared. The fluorophore utilized in these substrates is 6-aminoquinoline (6-AQ), a leaving group that affords significant improvement over the use of other chromogenic moieties in that (i) the appearance of 6-AQ can be measured fluorimetrically at its excitation and emission maxima, while at these wavelengths the substrate is essentially nonfluorescent; (ii) the reduction of background emission confers an enhanced detection limit to the substrate; and (iii) the amino group of 6-AQ very readily undergoes acylation by peptides and enables substrates to be synthesized in good yields.

Proteolytic enzymes play an important role in a diverse range of physiological processes characterized by limited proteolysis, including the coagulation of blood (I), fibrinolysis (2), the synthesis and turnover of peptide hormones (3), ovulation (4), fertilization (5), inflammation (6), and the remodeling of tissues during development (7). Less completely understood, but of no less significance, are the many processes mediated by unscheduled or uncontrolled destruction by proteases of structural proteins. Examples of this are the breakdown of articular cartilage by elastase (8) and collagen (9,10) during rheumatoid arthritis, the destruction of pulmonary elastin by elastase during emphysema (1 l), and in some cases, the invasion by tumor cells of healthy tissues, which has been associated with the activation of plasminogen (12). Proteases are often detected in vitro by the use of synthetic, chromogenic, or fluorogenic substrates that are composed of peptidyl amides of aromatic amines. Upon hydrolysis, the spectroscopic properties of the leaving group usually shift to longer wavelengths of absorption and/or emission that are char441

acteristic for the particular fluorophore. We recently described the preparation of a new class of synthetic substrates that employs 6aminoquinoline (6-AQ)’ as the fluorogenic ’ Abbreviations used: 6-AQ, 6-aminoquinoline; TLC, thin-layer chromatography; Cbz-Arg (MBS)-6AQ, 6(N”-benzyloxycarbonyl-N”-p-methoxybenzensulfonylL-arginylamino)quinoline; Cbz-Arg(MBS), N”-benzyloxycarbonyl -NW - p - methoxybenzenesulfonyl - L - arginine; Arg(MBS)-6AQ, 6-(NW-pmethoxybenzenesulfonylL-arginylamino)quinoline; Bz-Arg(MBS)-6AQ, 6-(NObenzoyl - N * - p - methoxybenzenesulfonyl - L - arginylamino)quinoline; Bz-Arg-6AQ, 6-(N”-benzoyl-L-arginylamino)quinoline; Bz-Arg(N0&6AQ, 6-(N”-benzoylN “-nitro-DL-arginylamino)quinohne; Bz-Arg(NOz), N abenzoyl-N “-nitro-L-arginine; Bz-DL-Arg-6AQ, 6-(N L1benzoyl-DL-arginylamino)quinoline; Cbz-Ala-6AQ, 6-(Nbenzyloxycarbonyl-L-alanyiamino)quinoline; Cbz-AlaAla-Ala-6AQ, 6-(N-benzyloxycarbonyl-L-alanyl-L-alanylL-alanylamino)quinoline; Sue-Ala-Ala-Ala-6AQ, 6-(Nsuccinylamino-L-alanyl-L-alanyl-L-alanylamino~uinoline; RR], relative fluorescence unit; Bz-DL-Arg-AMC, 4methyl-7-(N”-benzoyl-DL-arginylamino)coumarin;BzDL-A~~-~NA, N*-benzoyl-DL-arginine-pnitroanilide;SucAla-Ala-Ala-pNA, N-succinylamino-L-alanyl-L-alanyl-Lalanine-p-nitroanilide; MeO-Sue-Ala-Ala-Pro-Val-AMC, 4-methyl-7-(N-methoxysuccinylamino-L-alanyl-L-alanylL-prolyl-L-valylamino)coumarin; Bz-DL-Arg+JNaph, 2(N “-benzoyl-DL-arginylamino)naphthalene; i-BuOCOCl,isobutylchloroformate. 0003-2697/82/160447-09$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

448

BRYNES,

ANDRADE,

moiety (13). One of the most significant properties of this heterocyclic amine is that its excitation maximum does not overlap the excitation band of the substrate. That feature of 6-AQ enables it to overcome a problem that generally limits the sensitivity of other fluorophores such as 4-methyl-7-aminocoumarin and P-naphthylamine, namely, the high levels of interfering fluorescence arising from the unhydrolyzed substrate if it is exposed to the Xgs, of the leaving group. It. therefore, becomes necessary to select less efficient, submaximal wavelengths of light to excite the product, which in turn decreases the sensitivity of the substrate. In this report we describe the synthesis and kinetics of hydrolysis of new 6-AQ-containing substrates for trypsin and porcine pancreatic elastase and demonstrate that, because of the unique spectroscopic properties of the leaving group, they are exceedingly sensitive, convenient reagents for the detection and study of these proteases. EXPERIMENTAL

PROCEDURES

Materials. Amino acid derivatives, trypsin, and porcine pancreatic elastase were purchased from Sigma Chemical Company; 6aminoquinoline was prepared as described previously (13). Analytical and preparative thin-layer chromatography was conducted using Analtech silica-gel plates. [I]. 6 - (N* - BenzyloxycarbonylN” - p methoxybenzenesulfonylL - arginylamino) quinoline. To a solution of 800 mg (1.67 mmol) of N “-benzyloxycarbonyl-N “-pmethoxybenzenesulfonyl-L-arginine ( 14) and 184 ~1 ( 1.67 mmol) of N-methyl morpholine in 5 ml of dry dimethylformamide at -20°C was added 217 ~1 (1.67 mmol) of isobutyl chloroformate. The resulting suspension was stirred at this temperature for 45 min and then 185 mg (1.28 mmol) of 6-AQ was added in 1 portion. The cooling bath was removed and the solution was then stirred for 24 h at room temperature. The product was isolated by diluting the reaction solvent with 20 ml

AND

GORDON

of water, adjusting the pH to 9 with 5% NaHC03, and extracting several times with ethyl acetate. The combined organic extracts were then dried over MgS04 and evaporated in vacua to afford 798 mg of tan solids. Purification on preparative TLC plates (2000 pm) using chloroform:methanol (20: 1) gave 605 mg (78% yield) of compound [l], mp 154-l 55°C. Rf = 0.5 1 (chlorofoimmethanol, 7: I). [2]. 6-(NW-p-Methoxybenzenesulfonyl-Larginylamino)quinoline. To a solution of 300 mg (0.50 mmol) of [ I] in 3 ml ofglacial acetic acid was added 5 ml of 30% HBr in acetic acid. After 30 min the solution was diluted with 100 ml of anhydrous diethyl ether, which resulted in the precipitation of product as white flocculent crystals. These were washed several times by decantation from diethyl ether and dried overnight in vacua. Attempts to purify the dihydrobromide salt of [2] were not successful, owing to its deliquescence, and it was therefore used directly in the following reaction. [3]. 6-(N*-Benzoyl-N”-p-methoxybenzenesulfonyl-L-arginylamino)quinoline. Dihydrobromide salt [2] (216 mg, 0.36 mmol) was added to a solution of tetrahydrofuran:water (9:l) and cooled to -5°C. Triethylamine (205 ~1, 1.48 mmol) and benzoyl chloride (46 ~1, 0.40 mmol) were added sequentially and the solution was stirred for periods of 1 h each at -5,O, and 25°C. The product was isolated by removing the solvent in vacua, dissolving the residue in 1 ml of ethanol, and precipitating the benzoylated product by the addition of diethyl ether. Preparative TLC using chlorofor;m:methano1 (20: 1) for elution gave 176 mg (85% yield) of compound [3], mp 167’C decomposes. Rf = 0.59 (chloroforrnmethanol, 5: 1). [4]. 6 - (N” - Benzoyl- L - arginylamino) quinoline. To 33 mg (0.06 mmol) of compound [3] was added 850 ~1 of methanesulfonic acid containing 15 ~1 of anisole. After stirring at room temperature for 40 min, the product was precipitated by the addition of 50 ml of diethyl ether, washed several times

FXUOROMETRIC

PROTEASE

ASSAY

449

by decantation from this solvent, and dried bonate (0. l- 1 M). The blue fluorescent fracunder high vacuum. The crude product was tions were collected and lyophilized to afford then purified by passage through an Amber203 mg (84% yield) of substrate [7], mp lite CG-50 ion-exchange column (carboxyl117°C expands, 156°C decomposes. The ate form) using a discontinuous gradient of product gave a positive Sakaguchi test and ammonium carbonate (0.1-I M). The blue migrated as a single spot in several TLC solfluorescent fractions were combined and ly- vents. R,= 0.41 (methanokacetic acid, 10: 1). ophilized to afford 18.5 mg (7 1% yield) of Analysis. Calculated for CZ2HZ3N602. compound [4] as its bicarbonate salt, mp HzC03: C, 59.22; H, 5.62; N, 18.02. Found: 135- 140°C decomposes. The product gave C, 59.66; H, 6.16; N, 18.11. a positive Sakaguchi test and migrated as a [8]. 6 - (N - Benzyloxycarbonyl- L - alanylsingle spot in several TLC solvents. Rf = 0.4 1 amino)quinoline. Into 25 ml of anhydrous (methanol:acetic acid, 1O:l). [(u]b = +4.0” tetrahydrofuran was added 3.03 g (18.7 (c = 1 g/ml, methanol). Analysis. Calculated mmol) of 1, l’carbonyldiimidazole and 4.19 for CZ2HZ4N602. H2COj. 2 H20: C, 54.97; g ( 18.7 mmol) of N-benzyloxycarbonyl-L-alH, 5.82; N, 16.72. Found: C, 54.66; H, 5.38; anine. After stirring for 30 min at room temN, 16.98. perature, 466 mg (3.23 mmol) of 6-AQ was [6]. 6- (N”- Benzoyl- N”- nitro- DL- argi- added and the solution allowed to stir for 72 nylamino)quinoline. A solution of 734 mg h. The product was isolated by diluting the (2.26 mmol) of N*-benzoyl-N”-nitro-L-arorganic solution with 15 ml of water, adginine [5], prepared by the method of Nishi justing the pH to 10 with concentrated et al. (15), and triethylamine (3 15 gl., 2.26 NaOH, and extracting several times with mmol) in 5 ml of anhydrous dimethylformchloroform. The combined organic phases amide was cooled to -20°C whereupon iso- were washed thoroughly with water, dried butyl chlorofonnate (295 ~J2.26 mmol) was over MgS04, and evaporated under reduced added. After stirring at this temperature for pressure to afford a white solid (unreacted 45 min, 6-aminoquinoline (164 mg, 1.15 amino acid was recovered from the aqueous mmol) was added in 1 portion and the so- phase by first acidifying it to pH 2 and then lution allowed to stir at room temperature extracting it with ethyl acetate). Recrystallifor 24 h. The product was then isolated by zation of the crude product from acetonediluting the solution with water, raising the petroleum ether produced 1.11 g (98% yield) pH to 9 with 5% NaHCO,, and extracting of white needles, mp 17 1- 172°C. Analysis: with several portions of ethyl acetate. The Calculated for CZOH,9N303: C, 68.77; H, combined organic extracts were dried over 5.44; N, 12.03. Found: C, 69.05; H, 5.64; N, MgS04 and evaporated in vacua to afford 485 11.94. [9]. 6-(N-Benzyloxycarbonyl-L-alanyl-Lmg of a beige powder. Recrystallization from ethanol-ethyl acetate produced 37 1 mg (72% alanyl-L-alanylamino)quinoline. To a soluyield) of compound [6], mp 173.5-175°C. tion of 230 mg (0.66 mmol) of monopeptide [ 7j. 6 - (N” - Berzzoyl- DL - arginylamino) - [8] in 2 ml of glacial acetic acid was added quinoline. To 250 mg (0.56 mmol) of com- 5 ml of 30% HBr in acetic acid. After 30 min pound [6] and 1 ml of anisole at 0°C was at room temperature, the solution was diadded 10 ml of hydrogen fluoride that had luted with 50 ml of anhydrous diethyl ether, been previously dried over CoF3. After stir- which precipitated the intermediate amine ring at this temperature for 1 h, the acid was dihydrobromide salt. These solids were removed in vacua, and the crude product was washed several times by decantation from eluted through an Amberlite CG-50 ion-ex- diethyl ether and dried overnight in vacua change column (carboxylate form) using a and used in the following reaction without discontinuous gradient of ammonium car- further purification. To 1.00 g (3.40 mmol)

450

BRYNES,

ANDRADE,

of N-benzyloxycarbonyl-L-alanyl+alanine in 15 ml of dry tetrahydrofuran at room temperature was added 551 mg (3.40 mmol) of 1, l’-carbonyldiimidazole. After 30 min, the dihydrobromide salt (250 mg, 0.66 mmol) was added to the solution, followed by 2 equivalents of triethylamine to ensure the formation in situ of the free base. The solution was stirred for 72 h at room temperature and the product was then isolated by filtration of the solid precipitate. Recrystallization from methanol afforded 111 mg (35% yield from [8]) of trialanylaminoquinoline [9], mp 263-265°C. Analysis. Calculated for CZ6H29N505: C, 63.45; H, 5.90; N, 14.25. Found: C, 63.06; H, 5.90; N, 14.25. [IO]. 6 - (N - SuccinyIamino - L - alanyl- Lalanyl-L-alanylamino)quinoline. To a solution of 111 mg (0.226 mmol) of tripeptide [9] in 1 ml of glacial acetic acid was added 3 ml of 30% HBr in acetic acid. After 30 min at room temperature, the solution was diluted with 100 ml of anhydrous diethyl ether, which precipitated the product as white flocculent crystals. The solids were filtered, washed thoroughly with ether, and dried overnight under high vacuum to afford 103 mg of a deliquescent dihydrobromide salt. This was dissolved in 5 ml of methylene chloride containing 2.2 equivalents of triethylamine; succinic anhydride (22 mg, 0.22 mmol) was then added and the solution was allowed to stand overnight at room temperature. At the end of this period, a white precipitate had formed which was then collected and recrystallized from methanol to afford 88 mg (97% yield) of substrate [lo], mp 193-194.5”C. Analysis. Calculated for C22H27N506-H20: C, 55.81; H, 5.75; N, 14.79. Found: C, 56.15; H, 5.95; N, 14.76. Enzyme assays. Assays for trypsin were conducted in 46 mM Tris-Cl buffer, pH 8. I, containing 11 mM CaC12 at 25°C in a thermostatically controlled cuvette. For the determination of kinetic parameters, Bz-Arg6AQ concentrations ranged from 5 X lop5 to 3 X lop4 M, Bz-DL-Arg-6AQ concentrations ranged from 5 X lop5 to 2 X 10m4 M,

AND

GORDON

and trypsin was used at 1.O X 10e6 M. The specific activity of this enzyme was determined using p-nitrophenyl-p ‘-guanidinobenzoate according to the method of Chase and Shaw ( 16). Assays for pancreatic elastase were conducted in 67 ITIM T&-Cl buffer, pH 8.8, in a thermostatically controlled cuvette at 37°C. The specific activity of elastase was determined prior to the experiment using orcein-elastin (17) and was used at a final concentration of 1.4 X low6 M. Concentration of Sue-Ala-Ala-AladAQ ranged from 5 X 10m5 to 2.5 X 10e4 M. Fluorescence measurements were conducted with a PerkinElmer Model MPF-44A recording fluorescence spectrophotometer that was standardized prior to each experiment such that a 6.7 X 10e6 M solution of quinine sulfate in 0.1 N H2S04 produced fluorescence intensity equal to 1.O relative fluorescence unit (RFU). Excitation and emission wavelengths used to monitor the appearance of 6-AQ were 355 and 550 nm, respectively. Correlation coefficients for all kinetic data were 0.998 or higher. RESULTS AND DISCUSSION

Proteolytic enzymes that selectively hydrolyze substrates at arginyl amides are the subject of much current research. Well-known examples of serine proteases in this class include trypsin (18), thrombin (19), various blood factors (20,21), kallikreins (22), urokinase (23) and other plasminogen activators (24). In addition, there are certain proteolytic enzymes, such as cathepsin B (25) and cathepsin H (26) which employ the sulthydryl group of cysteine as the active site nucleophile, that also show a strong preference for this amino acid. Pancreatic elastase, an endopeptidase that digests a wide variety of protein substrates, has been shown to catalyze the hydrolysis of synthetic substrates at the C-terminal of alanine and other uncharged, nonaromatic amino acids (27). This specificity has led several laboratories to prepare synthetic tri-

FLUOROMETRIC

@

Cbz-ArgtMBS)

(I) i-BuOCOCI (2)

PROTEASE

l

451

ASSAY HBf

Cbr-Arg(MBS)-GAO

6-A0 1’1

Arg(MBS)-GAO

BzCI

CH3S03H >

Bz-Arg(MBS)-GAO

[21

@

Bz-Arg(N02)

(I) i-BuOCOCI (2) 6-A0

>

Br-DL-Arg(N02)-6A0

1. (A) Scheme for the synthesis of Bz-Arg-6AQ.

alanyl substrates capable of measuring the esterolytic (28) and amidolytic (29) activities of this enzyme. We recently introduced 6-AQ as a new fluorogenic leaving group and demonstrated its utility with a substrate capable of detecting chymotryptic activity (13). The present report describes the synthesis and rates of hydrolysis of several sensitive agents that also employ this leaving group and which can detect very low concentrations of trypsinlike enzymes and pancreatic elastase. The synthesis of Bz-Arg-6AQ is shown in Fig. IA. We found that the p-methoxybenzenesulfonyl protecting group ( 14) provides a convenient method for masking the reactive guanidino moiety of arginine, while at the same time it enhances the solubility of synthetic intermediates in organic solvents. In notable contrast to the syntheses of other chromogenic arginyl substrates, the acylation of 6AQ proceeded smoothly and in good yield due to its relatively nucleophilic amino group. The N”-benzyloxycarbonyl group was used at this stage in the synthesis of the L-isomer because amino acids having urethane protecting groups at this position are not as prone to racemize in the coupling step as are the corresponding N*-acyl derivatives (30). After removing the benzyloxycarbonyl group of [ 1] by exposure to HBr in acetic acid, the

Br-Arg-GAO

[41

[31

151

FIG.

>

[61

HF,

Bz-DL-Arg-6AQ [71

(B) Scheme for the synthesis of Bz-DL-A~~-~AQ.

benzoylated derivative [ 31 was formed under Schotten-Baumann conditions. The optically active substrate Bz-Arg-6AQ was finally generated by hydrolysis of the p-methoxybenzenesulfonyl group by brief treatment with methanesulfonic acid. Racemic substrate [7] was prepared by the more direct route shown in Fig. lB, starting from Bz-Arg(NOz) [5]. Acylation of 6-AQ produced the doubly protected, racemic quinolylamide [6], which was then deblocked using anhydrous liquid hydrogen fluoride. The overall yields from 6-AQ of L and DL substrates were 47 and 60%, respectively. Examination of the excitation spectra of Bz-DL-Arg-6AQ and 6-AQ presented in Fig. 2 reveals that the Xg& of 6-AQ (355 nm) lies outside the excitation band of the substrate and, consequently, light of this wavelength does not cause the substrate to fluoresce. Figure 3 shows the emission spectra of these two compounds and indicates the extensive red shift of nearly 200 nm that is produced upon hydrolysis of the amide bond. Even though the emission spectrum of Bz-DL-Arg-6AQ is broad, it has only 2% of its maximal emission at 550 nm, the XFz of 6-AQ. Taken together, the spectroscopic properties of this fluorophore enable its appearance to be monitored during the assay at wavelengths where it is most fluorescent, even in the presence of ex-

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with different chromophores, only P-benzoylarginine substrates are shown. The best substrate in terms of the specificity constant, k,,JK,,,, is Bz-Arg-6AQ, which undergoes hydrolysis several times faster than its racemic isomer. Somorin and co-workers (3 1) investigated the hydrolysis by trypsin of Land DL-Bz-Arg-pNA and obtained quantitatively similar results: the optically active substrate was cleaved 3.4 times more rapidly than the racemic compound. These results suggest that the enhanced rate of breakdown of the L-isomer is the result of comparatively more nonproductive binding by the racemic isomer at or near the active site of trypsin. Among the racemic derivatives listed in Table 1, Bz-DL-Arg-6AQ is the best substrate for trypsin. Only one other substrate (BZ-DLArg-AMC) has a slightly lower Michaelis constant, but the kc,, for that compound is lower as well. One of the other substrates (BzDL-Arg-pNA) has a higher catalytic constant; however, its K,,, concentration is 10 times higher than that of Bz-DL-Arg-6AQ. A new substrate for pancreatic elastase, Sue-Ala-Ala-AladAQ, was prepared using the following sequence. Acylation of 6-AQ by IV-benzyloxycarbonylalanine using l,l’carbonyldiimidazole (32,33) as the coupling

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800

350

400

WAVELENGTH

(nm)

FIG. 2. Excitation spectra of (- - -) Bz-DL-Arg-6AQ and (-) 6-AQ. Spectra were recorded in 46 mM TrisCl buffer, pH 8.1, containing 11 mM CaC12. Concentrations of Bz-DL-A~~-~AQ and 6-AQ were 5 and 20 PM, respectively.

cess substrate, while avoiding the high levels of background emission that limit the sensitivity of other fluorogenic agents. The rates of proteolysis by trypsin of BzArgdAQ and Bz-DL-Arg-6AQ are presented in Table 1 together with kinetic constants reported previously for similar synthetic substrates having other leaving groups. To provide a basis for comparison of compounds

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GORDON

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1 400

1 500

450

WAVELENGTH FIG. 3. Emission spectra of (- - -) Bz-DL-Arg-6AQ Tris-Cl buffer, pH 8.1, containing I 1 mM CaCIZ. and 20 j.tM, respectively.

---l.550

1 600

I 650

(nm)

and (-) Concentrations

6-AQ. Spectra were recorded in 46 mM of Bz-DL-Arg-6AQ and 6-AQ were 5

FLUOROMETRIC

agent formed Cbz-Ala-6AQ in 98% yield. The protecting group was cleaved by exposure to HBr in acetic acid, and the deblocked product was converted via acylation with Nbenzyloxycarbonylalanylalanine to Cbz-AlaAla-Ala-6AQ. Unfortunately, the hydrophobicity of this tripeptide rendered it insufficiently soluble in aqueous solution for kinetic studies unless organic cosolvents were employed. To avoid their use, the substrate was converted to its N-terminal succinylated derivative, and this compound was found to undergo hydrolysis by elastase readily. The K,,, and kc,, are 5.0 X 10e4 M and 1.7 s-‘, respectively. By comparison, the chromogenic substrate Sue-Ala-Ala-Ala-pNA has K,,, = 5.9 X 1O-3 M and kc,, = 37 s-l (34). Synthetic substrates have an important clinical application for the detection of proteases in biological fluids, and we therefore established the lowest concentrations of trypsin and elastase that could be measured with the quinoline-containing substrates. Figure 4 shows that as little as 5 rig/ml of trypsin could be detected with Bz-DL-Arg-6AQ in a 5-min assay period and that the initial rate of cleavage of substrate was linear over a 1OOfold range of enzyme concentration. The lowest level of elastase that could be measured under the same conditions with SucAla-Ala-Ala-6AQ was 2.5 rig/ml. The detection limit for the spectrofluorimeter in our laboratory was 0.033 RFU/min, but this TABLE

1

RATESOFHYDROLYSIS OFN~-BENZOYLARGINYLAMIDES BY TRYPSIN Substrate Bz-Ars-6Aq Bz-DL-A~s-~A~ Bz-DL-A~~-AM@ Bz-DL-Arg7BNaphc Bz-DL-Arg-pNAC

0.21 0.39 0.25 2.6 3.9

0.80 0.60 0.20 0.50 2.1

3800 1500 800 180 690

a For this study;kinetic constantswere determined from initial ratesof hydrolysisby the Lineweaver-Burk method. ’ Work of Zimmerman et al. (38). ‘Work of Grant et al. (39).

453

PROTEASE ASSAY

J Trypsln.

rig/ml

FIG. 4. Initial velocity of the hydrolysis of Bz-DL-AI~6AQ as a function of trypsin concentration. Initial concentration of substrate in 46 mM Tris-Cl buffer, pH 8. I, containing I1 mM CaC12: 3.9 X 10e4 M. Time and temperature of incubation: 5 min at 25’C. The detection limit was an initial velocity of 0.033 RFU/min.

value will depend on the sensitivity of the particular instrument used. In a study of kinetic and spectroscopic advantages of different chromophoric and fluorogenic leaving groups in synthetic elastase substrates, Castillo et al. (35) reported that the amide substrate MeO-Suc-Ala-Ala-ProVal-AMC is capable of detecting the pancreatic enzyme at concentrations as low as 0.47 rig/ml, whereas the detection limit of the corresponding substrate containing 4methoxy-2naphthylamine was 12 rig/ml. Studies are currently in progress in our laboratory to compare the properties of these substrates with an analogous derivative containing 6-aminoquinoline as the leaving group.

If a substrate is to be an effective agent for the detection of low concentrations of an enzyme, it is necessary to establish its stability toward proteases of different specificity. Exposure of Bz-Arg-6AQ or Bz-DL-Arg-6AQ

454

BRYNES, ANDRADE,

for 24 h at 25 “C to a 1.4 X 1Ob6 M solution of pancreatic elastase or for 5 h at 25°C to a 1.4 X 1Oe6M solution of chymotrypsin produced no detectable increase in fluorescence due to the appearance of 6-AQ. Similarly, Sue-Ala-Ala-Ala-6AQ was not cleaved by exposure to 1.O X 1Oe6 M trypsin at 25°C for 12 h. During the course of this investigation, we observed that arginylaminoquinolines [4] and [7] are considerably more soluble in aqueous solution than are the analogous substrates containing pnitroaniline, 4-methyl-7aminocoumarin, or P-naphthylamine. Higher solubility in water confers the important advantage that solvents such as dimethylformamide or dimethylsulfoxide are not required to dissolve the substrates in the assay buffer and, consequently, the unpredictable inhibitory or stimulatory effects of these solvents (36,37) on enzymes or cells do not complicate the interpretation of kinetic data. Stock solutions of Bz-Arg-6AQ were routinely prepared at 2 mM concentration in buffer and were stored at 4°C without precipitation. ACKNOWLEDGMENTS We thank Paula Bevilacqua and Andrew Maliszewski for assisting in the preparation of synthetic intermediates, Dr. Elliott Shaw for the use of his hydrogen fluoride reactor, and the National Institutes of Health for supporting this research (Grant GM 29220).

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