Use of laser-excited fluorescence to measure mixed-function oxidase activity.
The microsomal mixed-function oxidase (MFO) system is involved in the metabolism of various chemical compounds. Polycyclic aromatic hydrocarbons are metabolized by the cytochrome P-448 enzyme system, which contains MFOs. Induction of this MFO activity may be useful as an indicator of the toxicity of the inducer material. We have successfully used a portable centrifugal analyzer equipped with an argonion laser light source to quantify cytochrome P-448 activity induced in mouse-liver microsomes by exposing the animal's skin to different doses of liquids derived from a coal-liquefaction process. The MFO activity was determined kinetically by measuring the rate at which the highly fluorescent compound, resorufin, produced by the oxidation of 7-ethoxyresorufin substrate, was formed. The 514.5-nm laser excitation beam was directed with a fiber-optic bundle from the laser to the cuvets of a specially designed rotor; emitted fluorescence was monitored at 90 degrees to the incident beam through a 560-nm cut-on secondary filter. Use of the laser excitation source allowed very low MFO activities to be measured: picomole quantities of resorufin could be determined. We anticipate that the increased sensitivity of the method described here will allow MFO activities to be determined in body fluids and skin.
D000818:Animals; D003031:Coal; D004789:Enzyme Activation; D005453:Fluorescence; D005470:Fluorometry; D007700:Kinetics; D007834:Lasers; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000032:analysis* / Q000378:metabolism; D010078:Oxazines / Q000032:analysis*; D010088:Oxidoreductases / Q000032:analysis*
D003031:Coal; D010078:Oxazines; C014180:resorufin; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases