Naphtoyl-glycyl-glycyl-glycine: A new substrate for Angiotensin Converting Enzyme (ACE) assay using HPLC

Abstract

Background: Several in vitro assays are used to determine Angiotensin Converting Enzyme (ACE) activity. The purpose of the present investigation, was developing a practical and extraction-free chromatographic method to determine ACE activity. Methods: The method relies on UV-detection of Naphthoyl-glycine (NG), which is resulted from enzymatic hydrolysis of the synthetic substrate, Naphthoyl-glycyl-glycyl-glycine (NGGG), applying a reverse phase chromatographic separation. In this regard, experimental conditions for the assay such as Enzyme/Substrate (E/S) ratio and incubation time were optimized. Chromatographic separation was performed on a reverse phase C18 column (250 x 4.6 mm), using a mobile phase consisting of acetonitrile/water (20:80, v/v), pH = 5.0 with a flow rate of 2.0 mL/min and a detection wavelength of 280 nm. Results: The optimized Enzyme/Substrate (E/S) ratio and incubation time were 10 mU/nmol and 35 min respectively. The results indicated that the calibration curve was linear (r2 = 0.994) and the average recovery (n = 6) of NG was 99.5 ± 1.3% (mean ± RSD). Conclusion: In this study, we introduced a method which is an efficient approach to determine ACE activity due to its sensitive, accurate, and reliable performance with great repeatability.