Further characterization of human erythrocyte superoxide dismutase.
1. A simplified procedure for the preparation of highly purified human superoxide dismutase from erythrocytes was developed which avoided extremes of pH and ionic strength and the use of organic solvents; the properties of human and bovine proteins, prepared by the method, were compared. 2. Using the two dimensional electrophoretic procedure of O'Farrell, the human superoxide dismutase was found to consist of a single type of polypeptide. 3. The human protein was found to have a total of eight half-cystine residues per mole of protein, compared to six such residues for the bovine protein. The human protein has two sulfhydryl groups which are reactive toward mercurials when dissolved in 1M guanidine-hydrochloride and approximately 3 reactive sulfhydrls when the protein is dissolved in 6 M guanidine hydrochloride. The distribution of the eight sulfur atoms appears to consist of four involved in disulfide linkages, two deeply buried within the molecule and unreactive except under strongly denaturing conditions, and two which are reactive under mildly denaturing conditions. No zero-valent sulfur was found. 4. The visible optical absorption, the visible circular dichroism, and the electron paramagnetic resonance spectra are essentially identical with those of the bovine protein. No unusual absorbance was found at 330 nm. The near ultraviolet spectrum is different from that of the bovine protein, and this appears to be due to differing amino acid compositions. 5. Two fractions of superoxide dismutase activity were observed during chromatography of partially purified solutions on diethylaminoethyl-cellulose. The minor, less mobile form, was found to revert to the less mobile species on aging; the reverse process was not observed to occur. The minor component was found to contain equimolar amounts of Zn and Cu and to have a specific dismutase activity somewhat higher than that of the purified major fraction.
D000596:Amino Acids / Q000032:analysis; D002942:Circular Dichroism; D003300:Copper / Q000032:analysis; D004220:Disulfides / Q000032:analysis; D004578:Electron Spin Resonance Spectroscopy; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D008970:Molecular Weight; D011487:Protein Conformation; D013053:Spectrophotometry; D013482:Superoxide Dismutase / Q000097:blood / Q000302:isolation & purification; D015032:Zinc / Q000032:analysis
D000596:Amino Acids; D004220:Disulfides; D003300:Copper; D013482:Superoxide Dismutase; D015032:Zinc