Purification and Properties of Human Erythrocyte Carbonic Anhydrases

Abstract Three different methods are described for separating hemoglobins from carbonic anhydrases in hemolysates from human erythrocytes. The preferred method involves adsorption on diethylaminoethyl Sephadex at pH 8.7 followed by selective elution of the carbonic anhydrases. Carbonic anhydrases A, B, and C are subsequently separated from each other on DEAE-Sephadex by elution with 0.05 m Trischloride buffer at pH 8.7. Complete amino acid analyses are reported for carbonic anhydrases B and C, with results in good accord with those of preparations obtained in other laboratories by different procedures. The partial specific volume of carbonic anhydrase B is found to be 0.731 ml per g and its intrinsic viscosity is 2.76 ml per g. Molecular weights from sedimentation, diffusion, and sedimentation equilibrium are close to 28,000 for both enzymes; estimates from the amino acid analyses are 28,730 for Enzyme B, and 30,000 for Enzyme C. The s020, w values are close to 2.75 S for both enzymes. The values for both s and D are lower than others previously reported, by a factor close to 1.2, but the resulting molecular weights are in good agreement with others. Studies of optical rotatory dispersion between 330 and 600 mµ are reported for the native and the acid-denatured carbonic anhydrases B and C, and also for solutions containing the substrate bicarbonate ion and, in other experiments, the inhibitor acetazolamide. The data in all these solvents give a good fit to the simple Drude equation, with λc values that lie below 210 mµ for native Enzyme B and average close to 210 mµ for Enzyme C. Analysis of the data in terms of the Moffitt-Yang and Shechter-Blout equations is also reported, but a computer analysis indicates that the basis for applying the former equation is dubious. There appears to be a small amount of helix (10 to 20%) in the acid-denatured proteins; the complexity of the optical rotatory dispersion spectra of the native proteins makes inferences concerning helix content uncertain (see following paper). Studies on the esterase activity of Enzyme B, with p-nitrophenyl acetate as substrate, are reported. Esterase activity rises with increasing pH between 7 and 9.5. Acetazolamide is a powerful inhibitor of the esterase activity, with Ki = 0.3 µm at pH 7, 0.9 µm at pH 8, and approximately 2.8 µm at pH 9. The inhibition appears to be noncompetitive.