用于在动物天冬氨酸蛋白酶酶原存在的情况下测定其活性的发色肽底物：一种新的测定方法。

Chromophoric peptide substrates for activity determination of animal aspartic proteinases in the presence of their zymogens: a novel assay.

作者信息

Pohl J, Baudys M, Kostka V

出版信息

Anal Biochem. 1983 Aug;133(1):104-9. doi: 10.1016/0003-2697(83)90228-2.

Abstract

Solid-phase synthesis was used for the preparation of pyroglutamyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (I) and glycyl-glycyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (II), two water-soluble and sensitive chromophoric substrates of chicken pepsin, hog pepsin A, and bovine spleen cathepsin D. The kinetic constants of hydrolysis of the p-nitrophenylalanyl-phenylalanyl bond of the substrates were measured by difference spectrophotometry at 308 nm (delta epsilon = 860 M-1 cm-1) and by ninhydrin colorimetry (substrate I, epsilon 570 = 2.31 X 10(4) M-1 cm-1). The pH optimum of cleavage is 5 for the pepsins and 3.7 for cathepsin D. Since all three proteinases still have a significant activity at pH 5.5-6 a new, simple assay was designed for submicrogram quantities of pepsins in the presence of pepsinogens without interference of the latter. The method is particularly suitable for the analyses of the zymogen activation mixtures.

摘要

采用固相合成法制备了焦谷氨酰 - 组氨酰 - 对硝基苯丙氨酰 - 苯丙氨酰 - 丙氨酰 - 亮氨酸酰胺（I）和甘氨酰 - 甘氨酰 - 组氨酰 - 对硝基苯丙氨酰 - 苯丙氨酰 - 丙氨酰 - 亮氨酸酰胺（II），这是鸡胃蛋白酶、猪胃蛋白酶A和牛脾组织蛋白酶D的两种水溶性且灵敏的发色底物。通过在308 nm处的差示分光光度法（Δε = 860 M⁻¹ cm⁻¹）以及茚三酮比色法（底物I，ε₅₇₀ = 2.31×10⁴ M⁻¹ cm⁻¹）测定了底物中对硝基苯丙氨酰 - 苯丙氨酰键的水解动力学常数。胃蛋白酶的最佳裂解pH为5，组织蛋白酶D的最佳裂解pH为3.7。由于所有这三种蛋白酶在pH 5.5 - 6时仍具有显著活性，因此设计了一种新的简单测定方法，用于在存在胃蛋白酶原的情况下对亚微克量的胃蛋白酶进行测定，且不受胃蛋白酶原的干扰。该方法特别适用于酶原激活混合物的分析。