Molecular Pharmacology, Vol 9, 61-73, Copyright © 1973 by the American Society for Pharmacology and Experimental Therapeutics

A Spin Label Study of Human Erythrocyte Carbonic Anhydrases B and C

¹ Laboratory of Chemical Pharmacology, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014

The topography of the active sites of human erythrocyte carbonic anhydrases (EC 4.2.1.1) B and C has been studied by means of a series of spin-labeled sulfonamide inhibitors. Electron spin resonance measurements indicated that the nitroxide group of 2,2,5,5-tetramethyl-3-[(p-sulfamoylphenyl)carbamoyl]-1-pyrrolidinyloxyl was highly immobilized when this inhibitor bound to the active site of human erythrocyte carbonic anhydrase C. As the chain length between the aromatic and the pyrrolidine rings was increased, the mobility of the nitroxide group of the enzyme-bound inhibitor progressively increased until, with 2,2,5,5-tetramethyl-3-[[[(p-sulfamoylphenyl)carbamoyl]methyl]carbamoyl]-1-pyrrolidinyloxyl, there was only minimal interaction between the free radical and the active site. These results suggest that the active site of human erythrocyte carbonic anhydrase C is a cleft approximately 140 nm deep. Similar experiments indicated that the active site of human erythrocyte carbonic anhydrase B had the same general shape as the C isozyme. Human erythrocyte carbonic anhydrase B, after reaction with a nitroxide analogue of bromacetamide, showed spectral evidence for the presence of fairly mobile and highly immobilized covalently bound spin labels. When spin labeling was carried out in the presence of the inhibitor acetazolamide, the highly immobilized component of the spectrum was abolished, which suggested that it arose from a nitroxide group attached to the active site of the enzyme. When a nitroxide analogue of N-ethylmaleimide reacted with human erythrocyte carbonic anhydrase C, the spin label exhibited a high degree of mobility. Since prior treatment of the enzyme with p-chloromercuribenzoate abolished all labeling by this reagent, it appeared that the spin label had reacted with a sulfhydryl group on the surface of human erythrocyte carbonic anhydrase.

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ACKNOWLEDGMENT One of us (RHE) would like to thank Hoffmann-La Roche, Inc. for a postdoctoral fellowship.