High pressure liquid chromatographic separation of multiple forms of cytochrome P-450

The major form of cytochrome P-450 isolated and purified from the hepatic microsomes of phenobarbital pretreated rats by sequential chromatography on n-octylamino-Sepharose 4B and DEAE-cellulose columns was found to be homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. However, this cytochrome P-450 was resolved into three bands by high pressure liquid chromatography on an Anpac ion-exchange column. High pressure liquid chromatography isolated forms had similar molecular weights of 55,000 with lambda max of the CO-reduced difference spectrum at 450 nm and were found to be in the low spin state. The results demonstrate the effectiveness of high pressure liquid chromatography in the resolution of cytochrome P-450s of similar molecular weights but different net charges.