Establishment of in Vitro High-throughput Activity Detection Method for Angiotensin Converting Enzyme Inhibitors Based on 96 Well Plates
Lin Luo, Qingzhi Ding, Haile Ma · CHINESE JOURNAL OF ANALYTICAL CHEMISTRY (CHINESE VERSION) · 2012
A high throughput method for the determination of angiotensin converting enzyme(ACE) inhibitory activity,using 96-well plate technology,has been developed.Hydrolysis of N-[3-(2-furyl)acryloyl[L-phenylalanyl-glycyl-glycine(FAPGG) to N-[3-(2-furyl)acryloyl]-L-phenylalanine(FAP) and glycyl-glycine(GG) by ACE was quantified by measuring the decrease in absorbance at 340 nm to evaluate the activity of ACE.The percentage inhibition of angiotensin converting enzyme inhibitory(ACEI) was determined by comparing the results of control and test samples.The effects of different buffer systems,chloride ion concentration,ACE activity(ACE enzyme concentration),pH value of buffer system on the test of the activity of angiotensin converting enzyme inhibitors in vitro model reaction system of detection were investigated.The new method can detect ACE inhibitory activity of no more than 96 ACEI samples in 10 s or so on microplate-reader for ELISA.For different batches of sample,the RSD was less than 0.001%,p=0.667(0.05),which shows no significant difference between the results measured.The method is simple,accurate,stable,and reliable in antihyperten-sive peptide in vitro inhibitory activity of ACE measured.The method was used to detect a famous angiotensin converting enzyme inhibitors product named Captopril and a IC50 value(Half inhibitory concentration) of 16.19 nmol/L was obtained,which is consistent with the results have been reported in extensive literature.