Carbonic anhydrase. Purification and nature of the enzyme.

UNDER the name of carbonic anhydrase Meldrum & Roughton [1933] 2 described a new enzyme present in red blood corpuscles which catalyses both phases of the reversible reaction: H2CO3, CO2+ H20. They purified this enzyme from mammalian erythrocytes and gave a detailed account of its properties and the kinetics of the reactions which it catalyses. The greatly purified enzyme preparation obtained by them had a very high catalytic activity, was a colourless substance free from haematin and from other known enzymes, stable within pH 3-12, thermolabile, and very sensitive to KCN, H2S, NaN3 and to several heavy metals. The amouht ofthe purified enzyme obtained by these workers was, however, very small and hardly sufficient to recognize it with certainty as a protein compound. The true nature of this enzyme and especially of its active group remained unknown until very recently. While studying the metallo-protein compounds present in the red Nood corpuscles we have found that the fractions of our preparations left after complete removal ofhaemocuprein [Mann & Keilin, 1938] had a very high content of Zn and a high carbonic anhydrase activity. By adapting our methods of purification to preparations on a larger scale, taking special care to increase the yield of the Zn fraction, we have obtained from the red blood corpuscles of the ox a highly active preparation of carbonic anhydrase as a colourless protein contAining 0-31-034% Zn. These results, together with some other considerations, have led us to the conclusion, put forward in a preliminary communication [Keilin & Mann, 1939], that the carbonic anhydrase is a Zn-protein compound. In this paper we propose to give a detailed account ofthe methods of purification ofthis enzyme and to bring forward fresh evidence of its Zn-protein nature.