Carbonic anhydrase. Purification and nature of the enzyme

Abstract

I. INTROD.ICTION 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 mam- malian 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 of the 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 un- known 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 of haemocuprein [Mann & Keilin, 1938] had a very high content of Zn and a high carbonic anhydrase activity.By adapting our methods of puri- fication 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 of the methods of purification ofthis enzyme and to bring forward fresh evidence of its Zn-protein nature. II. METHOD,S OF IDENTIFICATION AND ESTIMATION OF ZnA. Qualitative tests.In purified preparations of carbonic anhydrase Zn was identified by the following three tests:(1) As Zn qUinaldinate, (CloH6N02)2Zn, H20. 25 mg.pure carbonic anhydrase in 5 ml.water are treated in a small centrifuge tube with 2 ml.20 % trichloroacetic acid; after 15 min.standing the precipitate is centrifuged off and washed with dilute trichloroacetic acid.The combined clear, protein-free extracts are adjusted with ammonia to pH 6 and treated with one drop of concentrated acetic acid and 1 Beit Memorial Research Fellow.