Bovine Erythrocyte Cupro‐Zinc Protein

Bovine erythrocyte cupro‐zinc protein was obtained by ethanol‐chloroform fractionation of red blood cells and purified by chromatography on fibrous DEAE‐cellulose, gel filtration on Sephadex G‐75, and rechromatography on microgranular DEAE‐cellulose. The protein has a molecular weight of abotu 33 000 by gel filtration, and contains 2 gram atoms of cupric copper and 2 gram atoms of zinc per mole. The amino acid composition of the protein has been determined. The protein has an isoelectric point at pH 4.95 by isoelectric focussing and is resolved into two ‘size’ isomers, one being a minor component, in polyacrylamide gel electrophoresis. The apoprotein behaves as a molecule of smaller ‘size’ than the native protein components in polyacrylamide gel electrophoresis. The ultraviolet spectra of the native and apoproteins show unusual features. These spectra together with the ultraviolet spectra of partially reconstituted apoprotein indicate a stabilizing effect of the zinc atoms on the structure of the chromophoric system. It is suggested that the copper and zinc atoms are not mutually replaceable at their respective sites in the protein. The near ultraviolet and visible spectra of the native protein have been fitted with Gaussian curves which indicate absorption bands at 340, 440 and 680 nm. The value of ɛ M /Cu 2+ for the 680 nm band has been found to be 120, indicating a cupric complex of high symmetry.