Determination of the dissociation constant of carbonic anhydrase inhibitors: a computerized kinetic method based on esterase activity assay.

We evaluate a reliable procedure for in vitro determination of the dissociation constant, Ki, of carbonic anhydrase inhibitors. This pharmacologically important parameter is estimated, using a mathematical model derived from the Ackermann-Potter equation, by a computer-assisted nonlinear regression analysis of kinetic data from the enzyme-catalyzed hydrolysis of p-nitrophenyl acetate. The proposed method has the additional advantage, over the ones that require the use of only paper and pencil, of giving standard errors for the estimated parameters. Based on our findings, a comparison of the reported values for the inhibition of carbonic anhydrase by twelve sulfamoyl drugs is presented, and the difficulties associated with the determination of dissociation constants for carbonic anhydrase inhibitors are discussed.