Purification of swine serum angiotensin converting enzyme with high recovery of activity using lisinopril coupled to epoxy-activated sepharose 6B

Abstract

The Authors describe the purification of swine serum ACE to molecular homogeneity with high recovery of activity (40%) in a few steps. The purification procedure consists of affinity chromatography, using commercial activated resin (epoxy-activated sepharose 6B) and two steps of anion exchange chromatography (Resource Q) performed at different pH (pH 9.0 and pH 6.0). Furthermore, a specific and sensitive method for the accurate quantitation of ACE activity in biological fluids was developed, based on the hydrolysis of synthetic FAPGG (N-[3-(2-furyl)acryloyl] L-phenylalanyl glycyl glycine), as substrate and following the separation of products by reversed-phase HPLC. Some kinetic parameters were determined. The Km and Kcat values for FAPGG were 0.793 ± 0.052 mM and 5830 s-1, respectively, and the I50 values for captopril and lisinopril, two specific ACE inhibitors, are 5.7 ± 0.67 nM and 1.0 ± 0.29 nM, respectively.