ANALYTICAL BIOCHEMISTRY 99, 316--320 (1979)

A Sensitive New Substrate For Chymotrypsin ~ E. G. D E L M A R , C . LARGMAN, J. W. BRODRICK, AND M. C. GEOKAS Enzymology Research, Martinez Veteran's Administration Medical Center, Martinez, California 94553 Received February 23, 1979 A new substrate for chymotrypsin, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide has been synthesized. Kinetic analysis of the hydrolysis of this peptide by bovine chymotrypsin gave a Km of 0.043 mM and akcat of 45 s -1. This substrate is readily soluble and stable to hydrolysis in Tris buffer at pH 8. Chymotrypsin levels down to 1.0 ng can be rapidly determined spectrophotometrically.

Chymotrypsin is an important and widely studied endopeptidase, yet there exists no convenient and sensitive substrate for its determination. The commonly used substrates are either insoluble in water (1,2), unstable (1,3,4), insensitive (2,5), or require the use of specialized equipment such as a pH stat (3) or a recording spectrofluorometer (6). In the course of our study of the substrate specificity of human pancreatic elastase 2, we synthesized succinyl-AlaAla- P r o - Phe-p-nitroanilide (Suc-AAPFpNA). 2 Its synthesis and use as a chymotrypsin substrate are the subject of this report. EXPERIMENTAL Materials Bovine a-chymotrypsin and trypsin were purchased from Worthington Biochemical Corporation, Freehold, New Jersey. Amino acid derivatives were obtained from Bachem Laboratories, Torrance, California. 1 This research was supported by the Medical Research Service of the Veterans Administration and by Grant 1088 from the Council for Tobacco ResearchUSA, Inc., New York, N. Y. Abbreviations used: Suc, succinyl; pNA, p-nitroaniline; tic, thin-layer chromatography; Cbz, benzyloxycarbonyl; DMF, dimethyl formamide; ONP, p-nitrophenyl ester; OEt, ethyl ester; AMC, 7-amino-4methylcoumarin; DMSO, dimethyl sulfoxide. 0003 - 2697/79/160316-05 $02.00/0 Copyright© 1979by AcademicPress, Inc. All rightsof reproductionin any formreserved.

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Synthesis of Suc-AAPF-pNA Cbz-Ala-Pro-t-butyl ester. Pro-t-butyl ester (25 g, 0.146 mmol) was acylated with Cbz-Ala-p-nitrophenyl ester (52.5 g, 0.153 mmol) in 250 ml of methylene chloride with 1.1 eq of triethylamine. After stirring overnight at room temperature, the reaction mixture was diluted with ether and washed with 10% acetic acid and several times with NaOH. After drying over Na2SO4, evaporation of the solvents in vacuo, left 55 g of a yellow oil. This material was used in the following reaction without further purification. A l a - P r o - t - b u t y l ester. To a nitrogen purged solution of the above oil (55 g) in 500 ml of methanol was added 2.5 g of 10% Pd/C. Hydrogen gas was passed through the solution by means of a glass fritted bubbler for 4 h. The solution was again purged with nitrogen and filtered. The solvent was removed in vacuo, leaving 34.8 g of a light yellow oil. C b z - A l a - A l a - P r o - t - b u t y l ester. The Ala-Pro-t-butyl ester was acylated with Cbz-Ala-p-nitrophenyl ester as above. The resulting oil was crystallized from methylene chloride-ether to give 59.6 g (91% overall yield) of a white solid. Cbz-Ala-Ala-Pro. C b z - A l a - A l a - P r o t - b u t y l ester (54 g) was dissolved in 500 ml of 50% ~rifluoroacetic acid-methylene chlo-

SENSITIVE NEW CHYMOTRYPSIN SUBSTRATE ride and stirred for 2 h, followed by removal of the solvents in vacuo. The residue was dissolved in NaHCO3, and the resulting solution was extracted with ether-methylene chloride to remove nonacidic material. The aqueous solution was acidified and extracted with ethyl acetate-ether. The organic phase was washed several times with 1% HC1, and then with saturated NaC1. After drying over Na2SO4, the solvent was removed to give 34.2 g (60% overall yield) of a white glassy (noncrystalline) solid. This material showed a single spot on tic (methylene chloride:methanol:acetic acid, 20:1:1), R s = 0.33. The melting point was diffuse at 70-85°C. Phe-pNA. Cbz-Phenylalanine was coupled to p-nitroaniline using the phospho-azo procedure of Kasafirek et al. (7), except that the reaction was carried out at room temperature for 24 h instead of 115°C for 3 h. The Cbz group was removed by treatment with 16% HBr in acetic acid for 1 h. Ala-Ala-Pro-Phe-pNA. To a solution of Cbz-Ala-Ala-Pro (2 g, 5.11 mmol) in 50 ml of methylene chloride was added 5.62 mmol each of Phe-pNA, 1-(3-dimethylaminopropyl)- 3 - ethylcarbodiimide hydro chloride, 1-hydroxybenzotriazole, and triethylamine. After stirring overnight, the solution was washed with acid, base, and saturated NaCI. Evaporation of the solvents left 3 g (90%) of Cbz-Ala-Ala-Pro-PhepNA as a white solid. The Cbz group was removed as above with HBr in acetic acid. Suc-AAPF-pNA. To a solution of AlaAla-Pro-Phe-pNA (2.23 g, 4.4 mmol) in 50 ml of methylene chloride and 10 ml of DMF was added 5.32 mmol of succinic anhydride and triethylamine. The reaction was stirred overnight followed by evaporation of the solvents. The residual oil was triturated with 2% HC1, and the resulting solid was collected by filtration and recrystallized from tetrahydrofuran-ether. The product was obtained in 79% yield (2.1 g), and showed a single spot on tlc: R s = 0.37 (4:1 methylene chloride: methanol), mp 184-187°C. Amino

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acid analysis was consistent with the assigned structure. Anal. Calcd for C27HzsN60~: C, 57.68; H, 5.81; N, 13.45. Found: C, 57.86; H, 6.03; N, 13.38.

Enzymatic Hydrolysis of Suc-AAPF-pNA A 0.1 mM solution of Suc-AAPF-pNA in 0.1 M Tris-HC1, 0.01 M CaC12, pH 7.8, was hydrolyzed by the addition of 20 /zg of bovine chymotrypsin. The release of pnitroaniline was complete in approximately 5 min, and the initial and final absorbances at 410 nm were 0.006 and 0.843, respectively. A 0.1 mM solution of recrystallized p-nitroaniline in the same buffer had an absorbance at 410 nm of 0.848. Thus, the pnitroaniline released by total enzymatic hydrolysis was 99% of the theoretically expected amount.

Assays Chymotrypsin assays were conducted at 25°C in 0.1 M Tris-HC1, 0.01 M CaC12, pH 7.8. The absorbance of the p-nitroaniline produced was measured continuously on a Gilford 252 spectrophotometer at 410 nm. Trypsin assays were performed in 0.04 M Tris-HC1, 0.01 M CaC12, pH 8.2. The volume of all assays was 0.7 ml.

Kinetic Constants For chymotrypsin, K,, determinations were made with duplicate measurements at eight substrate levels between 0.25 and 0.01 mM. The chymotrypsin used was approximately 90% active as determined by titration with standardized bovine pancreatic trypsin inhibitor. The Km for trypsin was determined at four substrate levels from 0.25 to 0.0375 mM, and a trypsin concentration of 1.02/xM. The trypsin was previously treated with s u c c i n y l - A l a - A l a - P r o - Leu-chloromethyl ketone to remove contaminating chymotrypsin activity,3 and its concentra3 DelMar, E. G., Largman,C., Brodrick,J. W., and Geokas, M. C., unpublishedresults.

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D E L M A R E T AL.

tion was measured by p-nitrophenyl guanidobenzoate titration (8). The Km and keat were determined from the data following a least-squares analysis (9).

The low K m for Suc-AAPF-pNA is due in part to the extended peptide chain which it contains. In this regard it has been demonstrated that bovine chymotrypsin contains an extended binding site for peptide subRESULTS AND DISCUSSION strates (10,11). The rationale for designing a substrate with a proline residue in the Pz Suc-AAPF-pNA is a white solid with a uv absorption maximum at 315 nm (e3~5 position was based on studies of both por= 14,000). It is soluble to at least 10 mM cine elastase (12) and chymotrypsin (10,13) in 0.2 M Tris-HCl buffer, pH 8.0. A 1 mM which indicated that a proline in this posisolution in this same buffer hydrolyzes tion increased the binding constant, or kcai, spontaneously at a rate of approximately or both. The succinyl group was originally employed in peptide substrates (14) to in0.1% per day at 4°C. A comparison of the kinetic constants, crease their water solubility. However, Km and keat, for Suc-AAPF-pNA and several studies with peptide chloromethyl ketone other chymotrypsin substrates is presented (15) or p-nitroanilide derivatives (7) have in Table 1. As shown in the table, Suc-AAPF- indicated that peptides terminated with NpNA is a significantly better substrate for succinyl instead of N-acetyl interact more bovine chymotrypsin than most of the pre- favorably with elastases or chymotrypsin. viously described substrates in terms of This effect may be manifest in a lower Km or both Km and keat. The one substrate with a a higher kcat. The specificity of this substrate is illushigher kcat/K,~ than Suc-AAPF-pNA, acetyl-Trp-ONP, has not been widely used, trated by the fact that kcat/Km for the hydrolprobably due to the appreciable rate of hy- ysis of Suc-AAPF-pNA by bovine trypsin is drolysis ofp-nitrophenyl ester bonds at neu- only 166 M-a S-1, or 0.03% of that for chymotral pH, which would limit the sensitivity trypsin. Linearity with enzyme concentration and the lower limit of detection are preof the assay. TABLE1 KINETIC CONSTANTS FOR THE HYDROLYSIS OF SUBSTRATES BY CHYMOTRYPSIN

Km

keat/Km

Substrate

(raM)

keat (S-I)

(S-' M-1)

A s s a y conditions

Suc-Ala-Ala- Pro- Phe-pNA Acetyl-Trp-ONP ~ Acetyl-Tyr-OEt b Suc-Ala-Ala-Tyr-pNA c Benzoyl-Tyr-OEt d Benzoyl-Tyr-pNA e Ala-Ala- Phe-AMC s Acetyl-Tyr-pNA e Glutaryl-Phe-pNA ~ Sue-Phe-pNA ~

0.043 0.002 0.7 0.16 0.5 0.34 0.5 1.13 0.28 0.72

45.0 30.5 193 14.1 19.6 0.88 0.83 0.3 0.013 0.011

1,000,000 15,000,000 300,000 88,000 40,000 2,600 2,000 300 46 15

Aqueous 3.2% Acetonitrile 1.8% Acetonitrile 6.7% D M S O 30% Methanol 9.7% A c e t o n e 1% D M S O Aqueous Aqueous Aqueous

a Data Data e Data a Data e Data s Data Data

taken taken taken taken taken taken taken

from (4). from (3). from (7). from (1). from (2). from (6). from (5).

SENSITIVE NEW CHYMOTRYPSIN SUBSTRATE

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ANALYTICAL BIOCHEMISTRY 99, 316--320 (1979) A Sensitive New Substrate For Chymotrypsin ~ E. G. D E L M A R , C . LARGMAN, J. W. BRODRICK, AND M. C. G...
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