Degradation of heme by a soluble peptide of heme oxygenase obtained from rat liver microsomes by mild trypsinization.
A tryptic peptide of heme oxygenase obtained after solubilization of rat liver microsomes by mild trypsin treatment was purified. The purified peptide gave only a single protein band with a molecular mass of 28 kDa on SDS/PAGE. The tryptic peptide, like the native heme oxygenase, readily bound with substrate heme forming a hemeprotein transiently. The absorption spectra of the ferric, ferrous, ferrous-CO and ferrous-O2 forms of the resulting complex resembled those of the corresponding forms of the complex of heme and the native enzyme. Ferric heme bound to the tryptic peptide was quantitatively decomposed to biliverdin on incubation with a mixture of ascorbic acid and desferrioxamine, indicating that the tryptic peptide still retained catalytic activity. These observations suggest that heme oxygenase has two domains, a hydrophilic and a hydrophobic domain, and that the two domains are folded almost independently of each other. An NADPH-cytochrome-P-450 reductase system composed of NADPH and detergent-solubilized NADPH-cytochrome-P-450 reductase readily reduced the ferric heme bound to the tryptic peptide, but failed to transfer the second electron required for rapid heme degradation, suggesting that the hydrophobic domain of heme oxygenase is important for receiving the second electron from the reductase.
Yoshida
T
T
Department of Molecular and Pathological Biochemistry, Yamagata University School of Medicine, Japan.
Ishikawa
K
K
Sato
M
M
D000818:Animals; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000378:metabolism*; D006419:Heme Oxygenase (Decyclizing) / Q000378:metabolism*; D006882:Hydroxyapatites; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D013053:Spectrophotometry; D014357:Trypsin / Q000378:metabolism*
D006882:Hydroxyapatites; D010446:Peptide Fragments; D006418:Heme; D017886:Durapatite; D006419:Heme Oxygenase (Decyclizing); D014357:Trypsin