Purification and Properties of an Avian Carbonic Anhydrase from the Erythrocytes of Gallus domesticus

Abstract Homogeneous carbonic anhydrase has been prepared from the erythrocytes of single comb, white Leghorn hens (Gallus domesticus). The procedure involved chloroform-ethanol precipitation of the hemoglobin, gel filtration of the resulting extract on a Sephadex G-75 column, and ion exchange chromatography of the pooled active fractions from the Sephadex column by salt gradient elution on carboxymethylcellulose. The presence of reducing agents, dithiothreitol or 2-mercaptoethanol, was essential throughout the entire preparation to maintain the stability of the carbonic anhydrase. Only one form of carbonic anhydrase was found in chicken erythrocytes. The purified enzyme behaves as a single molecular entity in sedimentation and diffusion and in polyacrylamide gel electrophoresis. It is active as an esterase, with p-nitrophenyl acetate as substrate; its amino acid composition and its catalytic properties suggest that it belongs to the "high activity" class of carbonic anhydrases. Its general properties are similar to those of other vertebrate carbonic anhydrase: s20, w0 is 2.9 S, the frictional ratio is about 1.2, the molecular weight is 28,000 ± 1,200 from sedimentation equilibrium studies, and there is 1 atom of tightly bound zinc per molecule. Attempts to remove the zinc reversibly have not yet succeeded. Acetazolamide strongly inhibits both the CO2 hydration and the esterase activities (Ki ≅ 10-7 m). The esterase activity rises with increasing pH between 6.5 and 9.5. Measurements of ultraviolet absorption, optical rotatory dispersion, and circular dichroism reveal close similarities with other vertebrate carbonic anhydrases, including the presence of Cotton effects in the region between 285 and 295 nm. Chicken carbonic anhydrase differs most strikingly from the corresponding mammalian enzymes in its high half-cystine content: 7 moles of half-cystine per mole of enzyme. The avian enzyme is reversibly inhibited by p-chloromercuribenzoate, and requires the presence of a reducing agent in vitro for maximal enzymatic activity and structural integrity.