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.