Carbonic anhydrase. Purification and nature of the enzyme

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Abstract
Methods were descr. for purification and isolation of carbonic anhydrase from red blood corpuscles and gastric mucosa of mammals. The purest enzyme prep. obtained from erythrocytes of ox and sheep by 2 distinct methods was a colorless protein containing 14.95% N and about 0.33% Zn. It was devoid of hematin, Fe, Cu, Mn, Mg and Pb. Evidence was brought forward that this prep. was either pure or almost pure enzyme. Its catalytic activity was about 150 times higher than that of the red blood corpuscles. The maximum yield of this purified product was about 200 mg./1 of ox blood. That carbonic anhydrase was a Zn protein compound where Zn formed the active part of the enzyme molecule was strongly supported by the following considerations: (a) An immediate, strong and completely reversible inhibition of this enzyme by small concs. of KCN, H2S and NaN3 which were known to react in this way only with metals; the presence of 0.33% Zn in the purest preps. obtained from erythrocytes of ox or sheep by 2 very different methods; the presence of Zn in carbonic anhydrase obtained from gastric mucosa; the absence of other metals from purified enzyme prep.; the proportionality between the enzyme activity and Zn content of different fractions obtained from erythrocytes and gastric mucosa. The conc. of carbonic anhydrase in red blood corpuscles was very high. 100 ml. of erythrocytes contained about 0.21 g. of the purified product, which was 3-4 times higher than the conc. of hemocuprein and about 133 times lower than that of Hb. The presence of Zn in carbonic anhydrase established for the first time the physiological function of this metal in organisms.

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