Studies on neutrophil b-type cytochrome in situ by low temperature absorption spectroscopy.
The b-type cytochrome in porcine neutrophils in situ was studied by the low temperature absorption spectroscopy at 77 K. Absolute spectra of the dithionite-reduced cell suspension revealed the existence of a b-type cytochrome with alpha, beta, and Soret absorption maxima at 558, 528, and 426 nm, respectively. The alpha band was unsymmetrical and showed a main peak at 558 nm with a shoulder at around 556 nm. When the cells were anaerobically stimulated either by phorbol myristate acetate or arachidonate followed by reduction by dithionite, the alpha band split clearly into double peaks at 555.5 and 558 nm, suggesting the presence of at least two states or species of the b-type cytochrome(s) in the cell. By monitoring absolute spectra of neutrophils at 77 K, we examined the possibility of CO binding to the b-type cytochrome. The absorption spectra of reduced b-type cytochrome in the presence and absence of CO, however, were not distinguishable under various conditions including equilibration with CO under high pressure or CO treatments in a dark room or at pH 8.5, 7.0, or 5.5. In contrast, the spectra of the reduced cytochrome disappeared immediately after exposure to O2, whether or not the cells had been treated with CO. The results indicate that the cytochrome does not form a CO complex in situ but reacts with O2, either directly or indirectly.
D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism; D003573:Cytochrome b Group / Q000097:blood*; D004227:Dithionite / Q000494:pharmacology; D005615:Freezing; D007700:Kinetics; D009504:Neutrophils / Q000378:metabolism*; D011485:Protein Binding; D013053:Spectrophotometry / Q000379:methods; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D003573:Cytochrome b Group; D004227:Dithionite; D002248:Carbon Monoxide; D013755:Tetradecanoylphorbol Acetate