Dissociation constants for carbonic anhydrase--sulfonamide binding by high-performance liquid chromatography

Carbonic anhydrase-azosulfonamide dissociation constants (Kd) were determined by gel filtration with high-performance liquid chromatography. By measuring the area of the elution peak at two wavelengths, Kd values were derived without having to measure a shallow trough. The procedure proved to be fast and reliable and has a general application. The dissociation constants were measured for 7-acetamido-2-(4'-sulfamylphenylazo)-1-hydroxynaphthalene-3, 6-disulfonate (Neoprontosil) complexes with carbonic anhydrase isozymes CA I, CA II, and CA III from bovine and human sources, and chicken CA III from skeletal muscle.