Purification of angiotensin-converting enzyme from rabbit lung and human plasma by affinity chromatography.
Lisinopril (N alpha-[(S)-1-carboxy-3-phenylpropyl]L-lysyl-L-proline), a potent angiotensin-converting enzyme inhibitor, is an exceptionally selective affinity chromatography ligand for this enzyme. Affinity chromatography furnishes electrophoretically homogeneous enzyme directly from crude homogenates of rabbit lung tissue, a 1,000-fold purification; also, it affords a 100,000-fold enrichment of the more rare human plasma enzyme in a single step. The affinity of angiotensin-converting enzyme for the Sepharose-spacer-lisinopril matrix (Ki matrix = 1 X 10(-5) M) is weak compared to its affinity for free lisinopril (Ki = 1 X 10(-10) M). The capacity of the affinity column is described quantitatively as a function of Ki matrix, lisinopril, and enzyme concentrations. The recovery of bound enzyme is low in chromatography of crude tissue samples (10-40%), although it approaches a reversible process (70-100%) with pure enzyme. The holoenzyme is converted to Zn2+-free apoenzyme to effect removal of lisinopril. In this process, the rate constant for spontaneous dissociation of Zn2+ from free enzyme is 1 X 10(-2) s-1 (t 1/2 = 1 min), which places a lower limit of 3 X 10(-10) M on the dissociation constant of Zn2+ at neutral pH from angiotensin-converting enzyme. The exceptional selectivity of lisinopril as an affinity chromatography ligand for angiotensin-converting enzyme suggests it is among the most specific inhibitors designed for any enzyme.
Bull
H G
HG
Thornberry
N A
NA
Cordes
E H
EH
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel; D004151:Dipeptides / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D017706:Lisinopril; D008168:Lung / Q000201:enzymology*; D008433:Mathematics; D017830:Octoxynol; D007703:Peptidyl-Dipeptidase A / Q000097:blood / Q000302:isolation & purification*; D011092:Polyethylene Glycols; D011817:Rabbits; D012995:Solubility; D015032:Zinc / Q000378:metabolism
D004151:Dipeptides; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D017706:Lisinopril; D007703:Peptidyl-Dipeptidase A; D015032:Zinc