Colorimetric, High-Throughput Assay for Screening Angiotensin I-Converting Enzyme Inhibitors

Angiotensin I-converting enzyme (ACE) plays a pivotal role in blood pressure regulation. A colorimetric assay to measure hippuric acid (HA) was transformed into a rapid ACE assay wherein the released HA from the substrate hippuryl-histidyl-leucine (HHL) is mixed with pyridine and benzene sulfonyl chloride. The resulting yellow color with a λmax at 410 nm is directly proportional to the released HA. The limit of detection and limit of quantitation were 1.46 × 10−7 and 4.43 × 10−7 M HA. ACE activities of different tissues using this method were comparable to the standard high-performance liquid chromatography (HPLC) method. Kinetic studies showed a K m of 30.8 ± 0.1 × 10−6 M for HHL and V max of 1.3 ± 0.01 × 10−6 mol/min for porcine lung ACE. This assay coupled with captopril and lisinopril showed IC50 values of 1.1 ± 0.05 × 10−9 and 2.5 ± 0.03 × 10−9 M, respectively. A 96-well microplate format of this method was used to screen the ACE inhibitory potential of peptides fractionated from an enzymatic hydrolysate of arachin. The precision, accuracy, reproducibility, and excellent correlation demonstrated between the colorimetric and the often-used HPLC method renders this extraction-free method a powerful tool for high-throughput screening of ACE inhibitors.