A modified method for the purification of erythrocuprein.

A copper-containing protein named haemocuprein was isolated from the erythrocytes of different mammals by MANN AND KEILIN 1. The preparation from ox-blood contained 0.34 % Cu, and there was evidence that the protein was obtained in a highly purified state. The authors also tried to purify a corresponding protein from human erythrocytes and obtained a preparation with a copper content of o.21%, A copper protein of human erythrocytes was recently highly purified and characterized~, 3 and it was shown to have the same copper content and probably the same molecular weight as ox-blood haemocuprein. The authors* suggested the name erythrocuprein for the human-erythrocyte protein, and they showed that erythrocuprein seems to account for all or nearly all of the copper in the erythrocytes. The preparation method for erythrocuprein described by MARKOWITZ et al.* is to a great part identical with that used by MANN AND KEILIN 1. KIMMEL et al. ~ simplified this method by replacing some of the later purification steps by an electrophoretic fractionation. As in the procedure of KIMMEL et al. ~, the present method of purification retains steps 1-3 in the method of MARKOWlTZ et al. 2 but the further purification is achieved by chromatography on the chloride form of DEAE-cellulose 4. The erythrocuprein prepared in this manner shows a slightly higher copper content than the one obtained previously and appears homogeneous in the ultracentrifuge and in free electrophoresis. The starting material consists of human blood stored for 1-4 weeks at 4 ° in acid citrate-dextrose solution. Typical runs were performed with 6 1 blood. All buffers and salt solutions were made from reagent-grade chemicals in deionized water. After removal of the hemoglobin by treatment with the chloroform-ethanol mixture, the remaining protein solution was treated with aq. lead acetate, and the precipitate was extracted with 0.33 M phosphate buffer, pH 6.0 (for details see MARKOWITZ et al.2). The phosphate solution was dialyzed against deionized water at 4 ° until free from phosphate. To the dialyzed solution, Tris-HC1 buffer (pH 7.4) was then added to a final concentration of 0.005 M. The column used was jacketed and cooled with tap water (8°). I t was packed with DEAE-cellulose (from Distillation Products Industries, Rochester 3, N.Y., U.S.A.) and equilibrated with 0.005 M Tris-HC1 buffer (pH 7.4)The protein solution was then filtered through the column. Approx. 65 % (varying between 50 and 85 %) of the protein material ran through the column. Erythrocuprein is adsorbed to the column and forms an easily detectable light greenish zone. At the top of the column a yellowish-brown zone is usually formed. After washing the column with 20o-300 ml 0.005 M Tris-HC1 (pH 7.4) the elution is performed by means of a concentration gradient (for details, see the caption to Fig. I). In the elution pattern (Fig. I) erythrocuprein was localized through its color. The peak behind the erythrocuprein is due to a yellowish-brown protein. I t shows a very high H,O2-decomposing activity and seems, at least partly, to consist of catalase. The pooled fractions of erythrocuprein from the chromatographic experiment was extensively dialyzed against deionized water and lyophilized. Usually several different chromatographic preparations of erythrocuprein were pooled and further purified by rechromatography (column dimensions, 1.3 × 18 cm; mixing chamber,