Ortho phthaldialdehyde opa

ACE-1 activity was measured using the method devised by Santos et al.34 (link) The assay was based on two reactions: in the first reaction, N-hippuryl-histidine-leucine (HHL) (Sigma-Aldrich, St Louis, MO, USA) was depredated into histidine-leucine (HL) (Sigma-Aldrich) by ACE-1, and in the second reaction HL and ortho-phthaldialdehyde (OPA) (Sigma-Aldrich) formed under the alkaline conditions of a fluorescent complex, which was measured at excitation/emission =365/495 nm. The assay buffer contained 0.1 M sodium borate and 0.4 M sodium chloride (pH 8.3). HHL (10 μM) and HL (0.05–15 μM) were diluted in the assay buffer. Pure HL was used as the standard. OPA (2%) was diluted in methanol. ACE-1 activity was measured from cell lysate using tripeptide (H-4632/H-4634; Bachem, Bubendorf, Switzerland) concentrations 0.1, 1.0, and 3.3 μM. Purity of tripeptides was >98%, but they were a mixture of cis/trans configurations. The reaction was stopped using acid after a 60 minute-long incubation and centrifuged at 3,000 rpm for 10 minutes. Fluorescence was measured using a fluorescence spectrophotometer (Varian Cary Eclipse; Agilent Technologies, Santa Clara, CA, USA) from the supernatant shortly after centrifugation. Captopril (1 nM) was used as a positive control for ACE-1 inhibition.35 (link),36 (link) All concentrations reported were the final concentrations in the reaction.