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

Abstract

1. 1. A procedure employing mild conditions has been developed for purifying S-protein (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-60.2. 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[mu], and the dithionite-reduced spectrum shows maxima at 426, 530, and 559 m[mu]. 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 b5.3. 3. The reduced form of the protein binds CO, and the resulting complex shows absorbance maxima at 421, 540, and 568 m[mu]. 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-420, the altered form of the microsomal hydroxylase, hemoprotein P-450. The spectral properties, together with other physical properties, suggest that S-protein is P-420 from erythrocytes.