The direct measurement of cytochrome P450 in unfixed tissue sections

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Abstract
Summary A method for the measurement of cytochrome P450 in unfixed cryostat sections is described. The sections are incubated for 10 minutes at room temperature in a buffered solution containing polyvinyl alcohol and sodium dithionite. Two incubations are performed on serial sections, one in nitrogen and the other in carbon monoxide. Readings are taken on a Vickers M85 microdensitometer fitted with a high sensitivity photomultiplier amplifier system, the measurements being made on corresponding fields in the serial sections. Subtraction of the nitrogen values from the carbon monoxide values, after allowing for an absorption shift, gives the absolute spectrum of cytochrome P450. The subtraction corrects for the tissue content of other haem-containing proteins. The cytochrome P450 spectrum shows a sharp maximum at 450 nm, and two other minor components absorbing at 444 nm and 458 nm. The content of cytochrome P450 in animals fed with phenobarbitone was 2.4 times greater than in control animals.