Mohammad Kazem Papan
1,2, Niusha Sharifi
1, Mohsen Zeeb
3, Shahla Mozaffari
2, Seyed Reza Hosseini Sedeh
4, Massoud Amanlou
1,5*1 Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Chemistry, Faculty of Science, Payame Noor University, Tehran, Iran
3 Department of Applied Chemistry, Faculty of Science, Islamic Azad University, South Tehran Branch, Tehran, Iran
4 Tehran University of Medical Sciences, International Campus (TUMS- IC), Tehran, Iran
5 Department of Medicinal Chemistry, Faculty of Pharmacy, Pharmaceutical Science Research Center, Tehran University of Medical Sciences, Tehran, Ira
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 × 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.