A high-throughput fluorimetric assay for angiotensin I-converting enzyme

Angiotensin I-converting enzyme (ACE) assays are commonly used for measuring enzymatic activity in clinical and biological samples. the fluorimetric procedure described is sensitive, rapid and involves unsophisticated procedures and inexpensive reagents. It uses the substrate hippuryl-L-histidyl-L-leucine, and the fluorescent adduct of the enzyme-catalyzed product L-histidyl-L-leucine is quantified fluorimetrically. This assay has been adapted for a 96-well plate format that produces comparable data to previously described assays and has the advantage of greater efficiency with respect to both time and reagents. the protocol can be used for routine purposes or for more detailed kinetic analyses. the apparent K(m) and k(cat) values for purified testis ACE determined from a double reciprocal plot were 3.0 mM and 195.7 s(-1), respectively. the protocol can be completed within 4 h.Univ Cape Town, Inst Infect Dis & Mol Med, Div Med Biochem, ZA-7925 Cape Town, South AfricaUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, São Paulo, BrazilWeb of Scienc