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

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.
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.
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).
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.