HYDRODYNAMIC CHARACTERIZATION OF HIGHLY PURIFIED AND FUNCTIONALLY ACTIVE LIVER MICROSOMAL CYTOCHROME-P-450

The subunit MW of highly purified phenobarbital-treated rat liver microsomal cytochrome P-450 (P-450) was estimated to be 55,000 .+-. 2000 by sedimentation velocity and equilibrium measurements in the presence of sodium dodecyl sulfate. Sedimentation equilibrium studies indicate that, in 0.1 M KCl at its isoelectric pH of 6.5, P-450 undergoes self-association with an apparent weight-average MW (MW) of about 500,000 at high dilution. The dependence of MW on concentration could be described by a model in which 6-9 P-450 monomers associate with high affinity to form a basic aggregate P1 which further self-associates isodesmically. Values for k .apprx. 1011 M-1, where k = [PN+1]/([Pn][P1]) did not change appreciably for P-450 in the presence of the substrate d-benzphetamine, phospholipid monomers, or NADPH-cytochrome P-450 reductase. The MW of the P1 aggregate was decreased by .apprx. 100,000 in the presence of phospholipid monomers and increased about 2-fold in the presence of NADPH-cytochrome P-450 reductase plus phospholipid monomers. The presence of phospholipid micelles raised k about 3-fold, while 6 M guanidine hydrochloride and 0.1% (wt/vol) Triton N-101 lowered k to 1010 and 109 M-1, respectively, and decreased the MW of P1 to .apprx. 150,000. Monomeric P-450 was detected only in the presence of amphipathic detergents. For the isodesmic self-association of the .apprx. 400,000 dalton aggregate of P-450 at pH 6.5 and 20.degree. C, .DELTA.G [standard free energy] = -14.4 .+-. 0.1 kcal mol-1 .DELTA.H [enthalpy] = -4.8 .+-. 1.3 kcal mol-1 and .DELTA.S [entropy] = 33 .+-. 5 eu. The results suggest that hydrophobic interaction is important for the isodesmic association of the P1 aggregate. Several lines of evidence also suggest that P-450 activity is not very dependent upon the state of aggregation of the enzyme. P-450 isolated from rats and rabbits also aggregates in a similar manner.