Purification of swine serum angiotensin converting enzyme with high recovery of activity using lisinopril coupled to epoxy‐activated sepharose 6B
Purification of swine serum angiotensin converting enzyme with high recovery of activity using...
Quassinti, Luana; Miano, Antonino; Bramucci, Massimo; Maccari, Ennio; Amici, Domenico
1998-04-01 00:00:00
The Authors describe the purification of swine serum ACE to molecular homogeneity with high recovery of activity (40 %) in a few stepsl. 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 prodcts 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.
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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 stepsl. 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 prodcts 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.
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