Preparation and Physicochemical Properties of Human Erythrocuprein

A relatively simplified method is described for preparing 220-mg quantities of highly purified human erythrocuprein, the copper-containing protein of erythrocytes. The isolated protein has been characterized and found to be similar to previously studied material except that it contains no tyrosine and no hexose and has 7 half-cystine residues per molecule as compared to 5 and 11 residues for two other preparations. The amino acid composition, the copper content (0.38%), the velocity sedimentation and diffusion data, and the results of sedimentation equilibrium experiments all indicate a molecular weight near 33,600. Purified erythrocupreins made by slightly different procedures show variations in extinction coefficients at the 265 mµ and 675 mµ absorption maxima. Erythrocuprein generally exhibits some degree of electrophoretic heterogeneity when examined in low ionic strength buffers. The more electronegative of these two variants could be generated from the other during gel filtration on Sephadex G-75. The nature of this transition has not been elucidated.