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Monocarboxamidomethyl Carbonic Anhydrase Purified by Affinity Chromatography

From the ¹ From the Department of Biochemistry, University of Miami School of Medicine, Miami, Florida, 33152

A monocarboxamidomethyl derivative of human erythrocyte carbonic anhydrase B was purified by affinity chromatography. The modified enzyme possesses 3% of the CO₂-hydrating activity and 30% of the esterase activity of the native enzyme. The esterase activity is inhibited by the usual carbonic anhydrase inhibitors although the K_i values are, in general, higher than for native enzyme. The pH dependence of esterase activity for modified enzyme has a pK of 8.3 compared to 7.3 for native enzyme.

Zinc dissociates so slowly from modified enzyme that it would take about 3 months to remove 90% of the metal. After half of the metal was removed from the enzyme, pure apoenzyme was separated by affinity chromatography.

Carboxamidomethyl Co²⁺-enzyme was prepared from apoenzyme and its visible spectrum was determined at several pH values. The general shapes of the spectra were similar to those for native Co²⁺-enzyme. However, the major pH-dependent transition has a pK of 8.3 compared to 7.3 for native enzyme. In addition, a secondary pH-dependent transition which occurs in native enzyme below pH 7 is absent in the modified enzyme, and a transition with a pK of 10.8 occurs in modified enzyme but is absent in native enzyme.

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