Comparative study of two highly purified forms of liver microsomal cytochrome P-450: Circular dichroism and other properties

The two major forms of rabbit liver microsomal cytochrome P-450, P-450LM2 and P-450LM4, which were previously shown to differ in their absorption spectra, electrophoretic and immunochemical properties, and substrate specificities, have been further characterized by several methods, (a) The two cytochromes have different CD spectra in the ferric state but similar spectra when reduced. Upon conversion of P-450LM2 to P-420 by treatment with sodium dodecyl sulfate, the CD spectrum is greatly diminished except in the far ultraviolet region, whereas the conversion of P-450LM4 toP-420 with this detergent results in a spectrum with a new positive band in the visible region, (b) Although P-450LM4 has a much higher tryptophan content than P-450LM2, the fluorescence spectra of these proteins are similar in magnitude. Upon denaturation, the fluorescence of P-450LM4 increases, thereby indicating a large quenching effect in the native protein, (c) Studies on the interaction of dilauroylglyceryl-3-phosphorylcholine with the cytochromes showed that P-450LM2 gives a much stronger Type I difference spectrum than does P-450LM4. This phospholipid has no significant effect on the state of aggregation of these cytochromes as judged by calibrated gel filtration. The CD spectra of P-450LM2 and P-450LM4 are unchanged in the visible region but are enhanced in the far ultraviolet region upon the addition of phosphatidylcholine. The results appear to indicate an increase in [alpha]-helical content, particularly with P-450LM4, in the presence of the phospholipid.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23557/1/0000517.pd