PURIFICATION OF SWINE SERUM ANGIOTENSIN-CONVERTING ENZYME WITH HIGH RECOVERY OF ACTIVITY USING LISINOPRIL COUPLED TO EPOXY-ACTIVATED SEPHAROSE 6B

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 comm ercial 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 dev eloped, based on the hydrolysis of synthetic FAPGG (N-[3-(2-furyl) acr yloyl] 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 +/- 0.05 2 mM and 5830 s(-1), respectively, and the I-50 values for captopril a nd lisinopril, two specific ACE inhibitors, are 5.7 +/- 0.67 nM and 1. 0 +/- 0.29 nM, respectively.