Production of Carbon Monoxide by Sonication of Medium Containing Glycerol. Effect on Cytochrome P-450 Reconstituted Systems
Cytochrome P-450 is well known to be inactivated by carbon monoxide by forming a complex that absorbs at 450 nm in the reduced state. In the preparation of the cytochrome P-450 reconstituted systems, sonication has been used routinely to disperse phasphatidylcholine and to facilitate incorporation of cytochrome P-450 into the phospholipid vesicles. Our studies reveal that such treatment in the presence of dissolved gas and glycerol, which is used to prevent conversion of cytochrome P-450 to cytochrome P-420, produced carbon monoxide that may then inhibit the enzymatic activities of the system. This phenomenon is prevented by degassing the medium completely. A method is proposed to perform the reconstitution of cytochrome P-450 metabolizing systems.
KeywordsDifference Spectrum Phospholipid Vesicle Sonic Treatment Reconstituted System Hantzsch Reaction
Unable to display preview. Download preview PDF.
- Franklin, M.R., 1974, The formation of a 455 nm Complex during Cytochrome P-450-dependent N-Hydroxyamphetamine Metabolism, Mol. Pharmacol., 10: 975.Google Scholar
- Luu-The, V., Vandresse,Ch., Cumps, J., and Dumont, P., 1979, Purification of cytochrome P-450 by double chromatography on substituted Sepharose. Effect of type I and type II ligands, Arch. Internat. Physiol. Biochim., 84: 1059.Google Scholar
- Nash,T., 1953, The colorimetric estimation of formaldehyde by means of the Hantzsch Reaction, Biochem. J., 55: 416.Google Scholar
- Omura,T., and Sato, R., 1964, The Carbon Monoxide-Binding Pigment of liver microsomes. I. Evidence for its hemoprotein nature, J. Biol. Chem. 239: 2370.Google Scholar
- Ryan, D.E., Thomas, P.E., Korgeniouski, P., and Levin, W., 1979, Separation and Characterization of Highly Purified Forms of Liver Microsomal Cytochrome P-450 from Rats treated with polychlorinated Biphenyls, Phenobarbital, and 3-Methylcholanthrene J. Biol. Chem., 254: 1365.Google Scholar
- Yasukochi,Y., and Masters, B.S.S., 1976, Some properties of a Deter gent-Solubized NADPH-Cytochrome P-450 Reductase Purified by Biospecific Affinity Chromatography, J. Biol. Chem. 251: 5337Google Scholar