Purification and some properties of the small subunit of cytochrome b558 from human neutrophils.
We have attempted to purify the heme moiety of cytochrome b558 from human neutrophils. Cytochrome b558 was solubilized from the crude membrane fraction which was pretreated with both 1 M potassium phosphate buffer and 1% octyl glucoside at low ionic strength. The solubilization of cytochrome b558 was carried out efficiently with 1.6% octyl glucoside in the presence of 100 mM phosphate buffer. Solubilized cytochrome b558 was purified by hydroxylapatite, DEAE-Sephacel, and Mono Q fast protein liquid chromatography. The specific content of purified cytochrome b558 was 37 nmol/mg of protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified cytochrome b558 revealed a single band of 20,000 Da. The large subunit of cytochrome b558, which has been reported by others, could not be found in purified cytochrome b558 even with silver staining. The amino acid composition of the heme-containing moiety of cytochrome b558 was abundant in hydrophobic amino acids. The circular dichroism spectra of both oxidized and reduced b558-type cytochromes exhibited bilobed bands with wavelengths of crossover points closely corresponding to those of the maxima in the optical absorbance spectra at the Soret region. Furthermore, there were some differences in the shoulders and peak widths of CD spectra between oxidized and reduced b558-type cytochromes. These results indicate that this method provides the purification of the small subunit of human cytochrome b558 which is the heme-carrying subunit of cytochrome b558, and suggest that cytochrome b558 has heme-heme interaction and some conformational changes in the alternation of the redox state.
Yamaguchi
T
T
National Institute of Hygienic Sciences, Tokyo, Japan.
Hayakawa
T
T
Kaneda
M
M
Kakinuma
K
K
Yoshikawa
A
A
D000596:Amino Acids / Q000032:analysis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003573:Cytochrome b Group / Q000097:blood* / Q000302:isolation & purification; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D019255:NADPH Oxidases; D009504:Neutrophils / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation
D000596:Amino Acids; D003573:Cytochrome b Group; D046911:Macromolecular Substances; C036888:cytochrome b558; D019255:NADPH Oxidases