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A high throughput colorimetric assay for angiotensin I‐converting enzyme and its inhibitors
Author(s) -
Karakkat Jimsheena V,
Gowda Lalitha R
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.677.4
Subject(s) - chemistry , hippuric acid , chromatography , angiotensin converting enzyme , lisinopril , high performance liquid chromatography , captopril , enzyme , renin–angiotensin system , urine , angiotensin ii , ic50 , biochemistry , in vitro , blood pressure , medicine , receptor
Angiotensin I converting enzyme (ACE) converts angiotensin I to the potent vasoconstrictor angiotensin II which regulates blood pressure. ACE is conventionally assayed by measuring the release of hippuric acid (HA) from hippuryl‐histidyl‐leucine (HHL), either spectrophotometrically or by HPLC. These methods are time intensive and not ideally suited for high throughput screening of natural ACE inhibitors. A diagnostic colorimetric assay to measure HA in urine was transformed into an ACE assay which was sensitive and rapid. The released HA is mixed with pyridine, layered with benzene sulfonyl chloride and measured at 420 nm. The yellow color formed is directly proportional to HA released. The limits of detection and quantitation were 1.46 ?10‐10 and 4.43 ?10‐10 moles of HA respectively. The measured ACE activities of different tissues were comparable to the standard HPLC method. The Km and Vmax values of porcine ACE were 30.8±0.1 ?10‐9 M and 1.3±0.01 ?10‐9 moles/min respectively. The IC50 values for captopril and lisinopril were 1.1±0.05 ?10‐9 M and 2.5±0.03×10‐9 M respectively. A 96 well micro‐plate assay was used to screen the ACE inhibitor activity of peptides generated by the enzymatic hydrolyses of arachin, the major storage protein of peanut (Arachis hypogaea). Our results demonstrate that the application of this colorimetric method not only improves the sensitivity but also increases the sample throughput.