Modification of carboxyl groups in bovine carboxypeptidase A. I. Inactivation of the enzyme by N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K).
Modification of carboxyl groups in bovine carboxypeptidase A. I. Inactivation of the enzyme by N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K).
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牛羧肽酶 A.I 中羧基的修饰。N-乙基-5-苯基异恶唑鎓-3-磺酸盐（伍德沃德试剂 K）使酶失活。
DOI：
10.1021/bi00793a001
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发表时间：
1971
期刊：
影响因子：
2.9
通讯作者：
P. Pétra
中科院分区：
文献类型：
--
作者：
P. Pétra
MethodsKinetic Measurements. Enzyme stock solutions were prepared by dissolving the crystals in 5 m NaCl-0.005 m potassium phosphate (pH 7.5) at 0, and diluting with distilled water to the proper concentration. Protein concentration was estimated at 278 nm with the Zeiss PMQ-II spectrophotometer using c278= 6.49 X 104 m_1 cm-1 (Bargetzi et al., 1963) or by amino acid analysis in the presence of a norleucine standard for the calculation of extinction coefficients. The standard assays for peptidase (CGP) and esterase (HPLA) activities have been previously described (Whitaker et al., 1966; Bargetzi et al., 1963). Peptidase activities and absorption spectra were mea-sured using Cary Model 16 and Model 15 spectrophotometers, respectively. Esterase activity was measured by titration in the pH-Stat (Radiometer TTT-1). The pseudo-first-order kinetic constants of inhibition, A:» PP, were calculated from the slope of plots of log of percent of activity remaining vs. time. The first-order rate constant of inhibition, k2, as well as the steadystate constant, Ki, app, were calculated from the reciprocal plot l/&app vs. 1/[I] as described below (Kitz and Wilson, 1962; Shaw and Glover, 1970).Amino acid analyses were performed by the accelerated amino acid analyzer system described by Spackman (1967). Samples were hydrolyzed for 22 hr at 110 in evacuated tubes. Ethylamine was determined by use of a 6.0-cm column of AA-27 Beckman spherical resin at 510 with a buffer and ninhydrin flow rate of 68 and 34 ml per hr, respectively. The buffer used was 0.21 n sodium citrate (pH 5.20). The constant for ethyl-amine was calculated from the value reported by Bodlaender et al.(1969) 2 after correcting for the difference in the ninhydrin constants of the basic amino acids. The ratio of ninhydrin constants of lysine¡ histidine agreed within 3% of that re-ported by these workers. The relatively low color value of ethylamine necessitated the application of large amounts of protein hydrolysate to the short column (3-4 mg/analysis). Reaction of CPA with Woodward's Reagent K (K-CPA). An enzyme stock solution was prepared by dissolving the crystals in 5 m NaCl-0.005 m potassium phosphate (pH 7.5) at 0C and diluting with distilled water to a protein concentration of 35 mg/ml in 1 m NaCl. This solution was stable for at least 2 weeks at 4. The pseudo-first-order rate constants for the hydrolysis of CGP and the pseudo-zero-order rate constant for the hydrolysis of HPLA were, respectively, 20 and 120 sec 1 (Pétra, 1970). An aliquot of enzyme stock solution was added to a given volume of 1 m NaCl-0.005 m potassium phosphate (0.64 mg/ml, 1.85 X 10-s m) and allowed to equilibrate for 10 min at the desired pH at 25. A small aliquot of freshly prepared Woodward’s reagent K (stock solution: 120 mg/ml in 10-3 n HCJ, equivalent to 0.47 m) was quickly added with stirring, resulting in a final concentration of 1.9 X 10 “3 m of reagent in the reaction mixture. The pH was maintained constant by the addition of 0.033 n NaOH to 10 ml of the reaction mixture.(The normality of the base was increased when higher concentrations of the reagent or larger reaction volumes were used.) At prescribed times, aliquots (10-80 µ were removed to measure enzymatic activity using standard esterase (HPLA) and peptidase (CGP) assays. Other samples
