Purification and characterization of liver cytochrome P-446 isolated from protein energy malnourished rats

Abstract

A liver cytochrome P-450 isozyme has been purified to homogeneity from protein-energy malnourished rats induced with β-naphthoflavone (β-NF). The purification steps included chromatography on DEAE-Sephadex-A-25, DEAE-cellulose (DE-53), hydroxylapatite (HA) and carboxymethyl-sephadex (CM) columns. The reduced carbon monoxide difference and absolute spectra showed a Soret peak at 446.5 nm. The wavelength maxima for the oxidized and reduced spectra were at 416 and 408 nm, respectively. Cytochrome P-446 appears to have a predominantly low spin ferric iron, migrates as a single band of molecular weight 56000 in sodium dodecyl sulfate polyacrylamide gels and has a specific content of 14 nmol/mg of protein. P-446 oxidized various substrates at different rates in a reconstituted system with NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine. In this system turnover rates for benzo[α]pyrene, testosterone and benzphetamine oxidation were: 81.10; 1.85 and 1.42 nmoles product/min/nm