用于测定血管紧张素I转换酶抑制活性的呋喃丙烯酰-L-苯丙氨酰甘氨酰甘氨酸测定法的改进

Modification of the furanacryloyl-L-phenylalanylglycylglycine assay for determination of angiotensin-I-converting enzyme inhibitory activity.

作者信息

Murray B A, Walsh D J, FitzGerald R J

机构信息

Department of Life Sciences, University of Limerick, Limerick, Ireland.

出版信息

J Biochem Biophys Methods. 2004 May 31;59(2):127-37. doi: 10.1016/j.jbbm.2003.12.009.

DOI:10.1016/j.jbbm.2003.12.009

PMID:15163524

Abstract

Angiotensin-I-converting enzyme (ACE, EC 3.4.15.1) plays a central role in the regulation of blood pressure in man. The objective of this study was to evaluate and modify the furanacryloyl-L-phenylalanylglycylglycine (FAPGG) assay method for quantification of ACE activity. The fixed time conditions developed for assay of ACE activity were as follows: 0.8 mM FAPGG, 175 + or - 10 units l(-1) ACE, incubation at 37 degrees C for 30 min and enzyme inactivation with 100 mM ethylenediaminetetra-acetic acid (EDTA). Hydrolysis of FAPGG to FAP and GG was quantified by measuring the decrease in absorbance at 340 nm. It was shown that increasing the level ACE activity in the assay from 155 to 221 + or - 15 units l(-1) resulted in a corresponding increase in the apparent IC(50) value for Captopril from 9.10 to 39.40 nM. Similar trends in the apparent IC50 values for a whey protein hydrolysate were obtained. The results demonstrate the requirement for carefully controlling ACE activity levels in the assay in order to obtained comparable and reproducible values for the inhibitory potency of ACE inhibitors.

摘要

血管紧张素转换酶（ACE，EC 3.4.15.1）在人体血压调节中起核心作用。本研究的目的是评估并改进用于定量ACE活性的呋喃丙烯酰-L-苯丙氨酰甘氨酰甘氨酸（FAPGG）检测方法。为检测ACE活性所设定的固定时间条件如下：0.8 mM FAPGG、175±10单位l⁻¹ ACE、在37℃孵育30分钟并用100 mM乙二胺四乙酸（EDTA）使酶失活。通过测量340 nm处吸光度的降低来定量FAPGG水解为FAP和GG的过程。结果表明，将检测中ACE活性水平从155单位l⁻¹提高到221±15单位l⁻¹，会导致卡托普利的表观IC₅₀值相应地从9.10 nM增加到39.40 nM。对于乳清蛋白水解物，也获得了类似的表观IC₅₀值变化趋势。结果表明，为了获得ACE抑制剂抑制效力的可比且可重复的值，在检测中需要仔细控制ACE活性水平。