PURIFICATION AND FURTHER CHARACTERIZATION OF PEROXISOMAL TRIHYDROXYCOPROSTANOYL-COA OXIDASE FROM RAT-LIVER

The acyl-CoA oxidase, catalysing the peroxisomal desaturation of the C oA-ester of trihydroxycoprostanic acid, a bile acid intermediate, has been purified to homogeneity from rat liver. Its native molecular mass , as determined by gel filtration and native gel electrophoresis, was 120 and 175 kDa respectively, suggesting a homodimeric protein consist ing of 68.6 kDa subunits. If isolated in the presence of FAD, the enzy me showed a typical flavoprotein spectrum and contained most likely 2 mol of FAD per mol of enzyme. The cofactor, however, was loosely bound . The enzyme acted exclusively on 2-methyl-branched compounds, includi ng pristanoyl-CoA and 2-methylhexanoyl-CoA if albumin was present. Imp ortant parameters to obtain a pure and active enzyme were the followin g: (1) using chromatographic separations like hydrophobic interaction and metal affinity, which allow the presence of high salt concentratio ns, conditions which stabilize the oxidase; (2) avoiding dialysis and (NH4)(2)SO4 precipitation; (3) including, when appropriate, FAD, dithi othreitol and a diol-compound in the solvents; and (4) carefully monit oring the removal of other acyl-CoA oxidases which possess the same na tive molecular mass and subunit size.