Arazoformyl peptide surrogates as spectrophotometric kinetic assay substrates for carboxypeptidase a
Arazoformyl peptide surrogates as spectrophotometric kinetic assay substrates for carboxypeptidase a
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DOI：
10.1006/abio.1996.0318
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发表时间：
1996-08-01
影响因子：
2.9
通讯作者：
Stanford, DJ
中科院分区：
文献类型：
--
作者：
Mock, WL;Liu, YY;Stanford, DJ
N-（4-甲氧基苯基偶氮甲酰基）-L-苯丙氨酸被牛羧肽酶A有效地裂解成苯甲醚、分子氮、碳酸盐和苯丙氨酸片段，在此过程中底物的强光谱吸收（λ（350）= 19，000 M（-1）cm（-1））完全消失。这证明了蛋白酶的灵敏分光光度检测。稳态催化速度以正常方式取决于酶和底物浓度，在pH 7.5的盐水溶液中测得的Michaelis-Menten Km值为0.11 mM，k（cat）值为44 s（-1）。这些参数具有酶的典型pH依赖性。在饱和量的底物下，含有10 nM酶的溶液在350 nm处产生-0.03 a.u/min（1 mm光程）的光谱吸收率变化，适用于测定目的。可替代地，可以在长达400 nm的波长下监测催化。(C)出版社：Academic Press，Inc.
N-(4-Methoxyphenylazoformyl)-L-phenylalanine is efficiently cleaved by the enzyme bovine carboxypeptidase A into fragments anisole, molecular nitrogen, carbonate, and phenylalanine, in the course of which an intense spectral absorption of the substrate (epsilon(350) = 19,000 M(-1) cm(-1)) disappears completely. This furnishes a sensitive spectrophotometric detection of the protease. Steady-state catalytic velocity depends upon enzyme and substrate concentrations in the normal manner, and a Michaelis-Menten K-m value of 0.11 mM and a k(cat) value of 44 s(-1) were measured at pH 7.5 in saline solution. These parameters have a pH dependence typical for the enzyme. With saturating amounts of substrate, a solution containing 10 nM enzyme produces a spectral absorptivity change at 350 nm of -0.03 a.u/min (1-mm path-length), suitable for assay purposes. Catalysis may alternatively be monitored at wavelengths as long as 400 nm. (C) 1996 Academic Press, Inc.