Hydrogen Ion Equilibria of Human Carbonic Anhydrases B and C

Abstract

Human erythrocytes contain three distinct forms of carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) which have been denoted as A, B, and C (l-3). In a previous study (4) one of us has investigated the hydrogen ion equilibria of carbonic anhydrase B at ionic strength 0.15. Here we report similar studies on enzyme C at two diierent ionic strengths, and further studies of enzyme B at low ionic strength (0.05). The two enzymes differ markedly in catalytic activity, the specific activity of enzyme C being much higher than that of enzyme B (5). In some respects they also differ markedly in amino acid composition. They are indeed closely alike in their content of histidine, arginine, and aromatic amino acids; each contains only one cysteine residue, and there are no disullide bonds in either (1, 6, 7).’ However, enzyme C contains distinctly more lysine than B, and also its content of ionizable carboxyl groups is somewhat higher. Correspondingly, the isoelectric point of enzyme C (pH 7.3) is higher than that of B, which is 5.7 (1). The minor component, enzyme A, which will not concern us further here, is almost indistinguishable from enzyme B in amino acid composition, although its isoelectric point appears to be slightly lower (1, 6). In optical rotatory dispersion studies both enzymes B and C show characteristic Cotton effects between 260 and 300 mp, which disappear on denaturation. The dispersion patterns for the two native enzymes show a certain broad general similarity but differ in detail (8). The previous titration study of enzyme B (1) showed that most of the histidyl and tyrosyl residues are masked in the native protein and become reactive only after denaturation in acid or alkali (4). Studies of difference spectra between the native and the acid-denatured enzymes B and C suggest that most or all of the tryptophanyl residues in the native protein are in the int.erior of the native molecules, and come into contact with the