This instrument is used to measure the kinetics of carbon monoxide (CO) binding to cytochrome P450 in liver microsomes from rats treated with various drugs and carcinogens. When CO is added directly to rat liver microsomes, the absorbance change at 450 nm occurs too rapidly to follow on a standard spectrophotometer. In order to observe this rapid reaction, a continuous dye-laser flash photolysis apparatus was constructed to monitor the kinetics of the absorbance change. Recombinant data generally were fit to a three-exponential kinetic model. However, each exponent included the contribution of several P450 forms. In order to determine the kinetic parameter for individual P450s, a difference kinetic method was used that employs the difference between the kinetic profiles in the absence and presence of a specific P450 effector. This new approach successfully yielded kinetic parameters for microsomal P450.
