高效液相色谱测定ACE抑制率方法改进的研究

Improved Method for High-performance Liquid Chromatography Assay of Angiotensin I Converting Enzyme Inhibitory Activity

摘要:

本文是在原有的高效液相色谱测血管紧张素转化酶（ACE）抑制率方法的基础上进行改进，用于测定马氏珍珠贝蛋白酶解液的ACE抑制率。改进的方法消除了紫外228 nm时样品本身在马尿酸（HA）保留时间处的吸收峰的影响。对于本身在HA保留时间处有吸收峰（以下称干扰峰）的样品而言，明显提高了测定精确度。本文研究了酶解液干扰峰面积随酶解时间的变化趋势，发现干扰峰面积随酶解时间增加而减小。对于自制酶酶解液，在酶解24 h后，其干扰峰面积减小至零。比较了马尿酰-组氨酰-亮氨酸（HHL）和ACE加入不同顺序对结果的影响，发现采用先加HHL的方法得到的ACE抑制率全部高于采用先加ACE的方法。因此，在在测定过程中，应由始至终地采用同一加入顺序，数据才具有可比性。

Abstract:

A modified method for determining the inhibitory activity of angiotensin-converting enzyme (ACE) from Pinctada martensi hydrolysate by HPLC was proposed. This method eliminated the effect of absorption peaks of some samples at the retention time of hippuric acid (HA) at 228 nm, and improved the measurement accuracy. The interfering peak area of the hydrolyzate decreased with increasing hydrolysis time. The interfering peak area of the hydrolysate by endogenous enzymes decreased to zero after 24 hours. The effects of different adding sequences of N-Hippuryl-His-Leu hydrate (HHL) and ACE on the assay were compared. Results showed that ACE inhibitory activity when HHL was added firstly was higher than those when ACE was added firstly. Therefore, in determination of ACE inhibitory activity, the same addition sequence of ACE and HHL should be adopted.