A MECHANISM-BASED FLUOROGENIC PROBE FOR THE CYTOCHROME-P-450 CHOLESTEROL SIDE-CHAIN CLEAVAGE ENZYME

The rate-limiting step of steroid biosynthesis is the enzymatic conversion of cholesterol to pregnenolone by cytochrome P-450scc (side chain cleavage) located in the inner mitochondrial membrane of all steroid producing cells. We report here the synthesis and application of a fluorogenic probe which is a cholene-based steroid with a fluorogenic moiety (resorufin) strategically located at the site of side chain cleavage. Synthesis of the probe required four steps starting from 3-beta-acetoxy-22,23-bisnor-5-cholenic acid and resorufin. Reaction of the probe with P-450scc yields pregnenolone and the highly fluorescent resorufin, thus providing a sensitive fluorescent signal representative of enzyme activity. The fluorescence quantum yield of this probe is approximately 40-fold lower (phi = 0.006) than resorufin (phi = 0.23) and is essentially nonfluorescent at wavelengths used to excite resorufin. The utility of the probe is demonstrated biochemically by incubation with mitochondria known to contain the P-450scc enzyme, and its specificity for this enzyme is shown by regulation of the enzyme activity with inhibitors and through the use of a nonspecific substrate.