Purification and properties of S-protein (hemoprotein 559) from human erythrocytes.

I. A procedure employing mild conditions has been developed for purifying Sprotein (hemoprotein 559, erythrocyte), a protein present in the stromal fraction of human red blood cells. S-protein, solubilized by extracting the stroma of red blood cells with pH 8.2 buffer, was purified by chromatography on DEAE-cellulose and Bio-Gel P-6o. 2. The oxidized and reduced forms of this protein show visible absorption spectra typical of b-type cytochromes. The oxidized form of the protein shows absorbance maxima at 412, 533, and 565 m#, and the dithionite-reduced spectrum shows maxima at 426, 530, and 559 m/z. The prosthetic group has been isolated and identified as protoheme IX by spectral and chromatographic techniques. The spectral properties of this protein differ from those of cytochrome b~. 3. The reduced form of the protein binds CO, and the resulting complex shows absorbance maxima at 421, 54 o, and 568 m#. This spectrum, the CO-reduced minus reduced difference spectrum, and the spectra of the oxidized and reduced forms of the free protein are all indistinguishable from the corresponding spectra of hemoprotein P-42o, the altered form of the microsomal hydroxylase, hemoprotein P-45 o. The spectral properties, together with other physical properties, suggest that S-protein is P-42o from erythrocytes.