Naphtoyl-Glycyl-Glycyl-Glycine: A New Substrate for Angiotensin Converting Enzyme (ACE) Assay Using HPLC

Background:Several in vitro assays are used to determineAngiotensin Converting Enzyme (ACE) activity. The purpose of the present investigation,was developing a practical and extraction-free chromatographic method todetermine ACE activity.Methods:The method relies on UV-detection of Naphthoyl-glycine (NG), which is resultedfrom 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 andincubation time were optimized. Chromatographic separation was performed on areverse phase C18 column (250 × 4.6 mm), using a mobile phase consisting ofacetonitrile/water (20:80, v/v), pH = 5.0 with a flow rate of 2.0 mL/min and adetection wavelength of 280 nm.Results:The optimized Enzyme/Substrate (E/S) ratio and incubation time were 10 mU/nmoland 35 min respectively. The results indicated that the calibration curve waslinear (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 todetermine ACE activity due to its sensitive, accurate, and reliable performancewith great repeatability.