Kinetics, equilibrium and inhibition in the Hansson histochemical procedure for carbonic anhydrase: a validation of the method.

Quantitative analysis has been made of the reactions underlying the Hansson histochemical method for carbonic anhydrase, with a view toward resolving controversies that have arisen regarding its application and specificity. The basic event is the loss of CO2 from the surface of solutions containing HCO3(-), PO42(-) and cobalt at pH 6-8. Displacement of the equilibria H2CO3 in equilibrium CO2 to the right elevates the pH, and at 6.8 a cobalt precipitate is formed. When tissue containing carbonic anhydrase is floated on the surface, the loss of CO2 and elevation of pH is accelerated at the enzyme site, leading to increased cobalt deposits. These are converted to cobalt sulphide for visualization. Study of the changes of pH and CO2 equilibria during the reaction point strongly to the fact that enzymic activity is being measured by the cobalt localization. This activity is reduced or abolished by appropriate concentrations of acetazolamide (or other sulphonamide inhibitors of carbonic anhydrase) and the powerful inorganic inhibitor, cyanate (CNO-) ion.