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 × 10-7MHA. ACE
activities of different tissues using this method were
comparable to the standard high-performance liquid
chromatography (HPLC) method. Kinetic studies
showed a Km of 30.8 ( 0.1 × 10-6 M for HHL and
Vmax 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.