naphtoyl-glycyl-glycyl-glycine: a new substrate for angiotensin converting enzyme (ace) assay using hplc

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 × 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.