Assay of carbonic anhydrase by titration at constant pH

1. A method is described for measuring accurately the initial velocity of the hydration reaction catalysed by the enzyme carbonic anhydrase (EC 4.2.1.1); the method depends on the titration of H(+) ions at constant pH. 2. Human erythrocyte carbonic anhydrase, isoenzyme C, was used to illustrate the method. Under the experimental conditions employed (0 degrees , pH7.0, in the presence of 45mm-sodium chloride and 5mm-sodium phosphate) isoenzyme C obeyed the Michaelis equation over the range of substrate concentration 1-16mm-carbon dioxide. The kinetic constants found were: K(m)=8.2mm; V/[E(0)]=5.0x10(4) sec.(-1).