Isolation of Human Carbonic Anhydrase B and C and Apocarbonic Anhydrase by Affinity Chromatography

Human carbonic anhydrase B and C have been purified in high yields by affinity chromatography, using two different affinity gels, incorporating the potent benzenesulfonamide inhibitor, but coupled to Sepharose through different ,spacer-arms~. Although both affinity gels bind carbonic anhydrase B and C quantitatively, they exhibit different binding capacities. Sepharose-glycyi-L-tyrosine-azobenzenesulfonamide was used to separate the mixture of isoenzymes from the bulk hemoglobin in the hemolysate of human erythrocytes. This affinity gel had a capacity of 15 mg of enzyme pr. ml packed gel. The isoenzymes were eluted with thiocyanate an inhibitor of carbonic anhydrase. The mixture of hemoglobin free isoenzymes was separated efficiently on a Sepharose-ethyl fp-carboxybenzenesulfonamide) carboxamide affinity gel by exploiting the difference in binding strengths for the Band Cenzyme, respectively. Furthermore, the affinity gels could be used to separate the apoenzyme from the zinc enzyme.