An improved method for the purification of carbonic anhydrase isozymes by affinity chromatography.

Methods for purifying carbonic anhydrase (EC 4.2.1.1.) isozymes, carbonic anhydrase B (or I) and C (or IIL by affinity chromatography have been described by Falkbring et al. (1) and Whitney (2), in which affinity gels are formed by coupling, respectively, p-aminobenzenesulfonamide, and p-(aminomethyl)benzenesulfonamide to Sepharose polysaccharides by means of cyanogen bromide activation. Attempts to repeat these methods in our laboratory were not satisfactory as the reproducibility and yields were variable. It is possible that the susceptibility of the linkage to esterase hydrolysis by carbonic anhydrase could account for this variability. It has been suggested (3) that the linkage given by cyanogen bromide activation is of an ester type, i.e., -C-O--CO--NH--R, where R represents a functional group, and it I was possible to hydrolyse this linkage by heating at about 80°C and pH values between 8 and 12. When purified carbonic anhydrase at pH 9.0 was passed through columns of this type of gel, cleavage of the sulfonamides occurred; and in particular, the cleavage products were readily observed when highly colored azosulfonamides were coupled. Also the total distance of the coupled inhibitor from the gel matrix, given by these methods, does not exceed the depth of the active site cleft (4). Therefore, to overcome these factors, gels were prepared by coupling sulfonamides to CM Sephadex using a water-soluble carbodiimide (5) to form a peptide bond between the carboxyl and amino groups. Two sulfonamide inhibitors were used, p[(2,4-diaminophenyl)azo]benzenesulfonamide (Prontosil), and p-(aminomethyl)benezenesuifonamide. The azosulfonamide coupled gel had the advantages of a higher capacity and a red-colored product that enabled visual estimation of coupling efficiency. For recent reviews of carbonic anhydrase isozymes see Lindskog et ah (6), and Carter (7).