Acid Difference Spectra of Human Carbonic Anhydrases

Abstract

Human carbonic anhydrase B loses enzymatic activity as it undergoes an irreversible conformational change near pH 4 (1). Riddiford (2) further found that seven of the eleven histidyl groups, which are unreactive in the native protein, become titratable during this change. Also, five or six of the eight tyrosyl groups do not ionize freely and reversibly in the native protein (2, 3). Human carbonic anhydrase C has a much greater specific activity (4) and a higher isoelectric point than carbonic anhydrase B (l), but its titration behavior is very similar to that of enzyme B (5). Seven of the twelve imidazole groups are normalized near pH 4.5 (at a slightly higher pH than the value of 4.2 found for enzyme B), coincident with a loss in enzymatic activity. Also, as in carbonic anhydrase B, only two of the eight tyrosyl groups titrate normally. However, enzyme C is more labile above pH 12.25 than is B.l Difference spectra between native and acidified carbonic anhydrase B were first studied by Ghazanfar in this laboratory (1). He observed three characteristic peaks in the difference spectra, at 236, 285, and 291.5 mp. The first of these is the most intense. He showed that there is a very abrupt transition when AQ is plotted against pH. The resulting curve is qualitatively similar to an ordinary titration curve, but is much steeper. At an ionic strength of 0.05 the midpoint of the curve (pH,id) lies near pH 3.5, or slightly above. Here we report studies of the pH dependence of the acid difference spectra at I’/2 = 0.05 and at P/2 = 0.15 of carbonic anhydrases B and C. The difference spectra are time dependent, as Ghazanfar had already found, and we have studied the changes with time in further detail.