FLASH PHOTOLYSIS APPARATUS

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This instrument is used to measure the kinetics of CO binding to cytochromes P450 in liver microsomes from rats treated with different 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. The sample is pulsed with a laser flash at 530 nm to disrupt the photolabile bond between CO and the heme iron of the P450. Transmitted light at 450 nm is detected by the photomultiplier. Several optical filters and lenses select wavelength and focus the light. The photomultiplier voltage output is amplified, filtered, input to an analog-to-digital converter, and stored on the IBM AT. The data are analyzed by least squares fit to multiexponential models. Two kinetic phases were observed; these disparate observations indicate that there are two distinct types of P450. When substrates were present, the reaction rate changed.