通过高效液相色谱法直接测定磷酸转乙酰酶和乙酰辅酶A羧化酶

Direct assay for phosphotransacetylase and acetyl-coenzyme A carboxylase by high-performance liquid chromatography.

作者信息

De Spiegeleer B, Mannens G, Slegers G, Van den Bossche W, Claeys A

出版信息

Anal Biochem. 1986 Oct;158(1):195-200. doi: 10.1016/0003-2697(86)90609-3.

Abstract

A simple and specific assay to measure the activity of two coenzyme A derivative-processing enzymes, i.e., phosphotransacetylase (EC 2.3.1.8) and acetyl-coenzyme A carboxylase (EC 6.4.1.2), is described. The assay is based on the HPLC analysis of the short-chain coenzyme A derivatives formed by the enzymatic reaction, viz., acetyl-CoA and malonyl-CoA. For this purpose, ion-pair reversed-phase HPLC conditions are optimized. Furthermore, the influence of several variables on the enzyme reaction is studied in order to get maximum activity. Due to its short analysis time, good selectivity, and chromatogram information, HPLC proves to be an excellent method for the assay of these enzymes.

摘要

本文描述了一种简单且特异的测定方法，用于测量两种辅酶A衍生物加工酶，即磷酸转乙酰酶（EC 2.3.1.8）和乙酰辅酶A羧化酶（EC 6.4.1.2）的活性。该测定基于对酶促反应形成的短链辅酶A衍生物，即乙酰辅酶A和丙二酰辅酶A的高效液相色谱（HPLC）分析。为此，优化了离子对反相HPLC条件。此外，研究了几个变量对酶反应的影响，以获得最大活性。由于其分析时间短、选择性好且具有色谱图信息，HPLC被证明是测定这些酶的一种出色方法。