Ligand interaction with hemoprotein P-450. 3. The use of n-octylamine and ethyl isocyanide difference spectroscopy in the quantitative determination of high- and low-spin P-450.

A simple metinod for tine determinations, by ligannd binndinug to octylaminie, of tine absolute amounts of inigharid loss--spins formsss of huemoproteini P-450 of rabbit liver microsomcs is described. A correlationn betus-eenn octvlrsminse bimsdinsg to tine two forms of oxidized P-450 and the binsdinsg of ethyl isocyanide to reduced P-450 inas beeni observed, and advantages of tine aminc-binidinig method over tine use of etiuyl isocyanside are pointed out. In particular, octylaminne binsds to each species of P-450 to produce a distinict differensce spectral minimum. We have derived a relationishnip for tine calculation of tine proportions of each form of P-450. When tine ansimie-binndinng method uvas used to determinne tine amounts of highand lous--spims forms of P-450, insteresting quantitative cluaniges uvere observed. Variations in tue contenuts of tisc tuso forms iii rabbit liver microsonses us-crc rioted following maintenance of rabbits ons different control diets ; dietary oxytetrrscyclinse, connmonlv used ins commercial feeds, further increased tine proportions of tine buy-spin forns. Microsonsal P-450 of bovine adrenal cortex is almost enstirchy ins tine buy-spins fornn, usinile adreisal mitochnonudnirs comntainu both forms ins nsearlv equal proportions. Applications of botin tine octylamimue amid etinyh isocyannide nnethiods provides complemenstary data about P-450 inn either oxidations state. A distinuctiomn betuveenn the tus-o forms of oxidized P-450 in liver microsonnes is mainstainned quanstitatively durimug comnversions to tine reduced state.