A CRITICAL EVALUATION OF THE HISTOCHEMICAL METHODS FOR CARBONIC ANHYDRASE

The histochemical methods for carbonic anhydrase are not based on the postulated dehydration of HCO 3 – . The staining is caused by the formation of an unknown reactive Co compound in the surface layer secondary to enzyme-independent alkalinization of the medium. Kinetic analysis of the reaction shows that loss of CO 2 from the medium is rate-limiting. Carbonic anhydrase inhibitors delay the staining by interacting with Co and not by inhibiting the enzyme; they are effective when used after the reaction is complete. The reaction can also be inhibited by agents which are not carbonic anhydrase inhibitors, such as sodium lauryl sulfate and 5-aminothiadiazole, but not by in vivo administered acetazolamide. A comparison of the effect of various fixatives on the biochemical and histochemical enzyme activity shows no correlation. While carbonic anhydrase itself is stained by the reaction, the methods lack the claimed specificity for it.