New assay for pregnenolone and progesterone 17alpha- hydroxylase based on the specific substitution of a tritium situated on carbon 17.

A new assay for the measurement of steroid 17alpha-hydroxylase activity in beef adrenals is described. This method is based on the biochemical mechanism of the enzymic reaction, i.e. the direct and stereospecific substitution of the proton located on the hydroxylated position. Progesterone or pregnenolone specifically labelled on the 17 position are solubilized in the incubation mixture with the help of Tween 80 and incubated under optimal conditions. The tritium enzymically released from the substrate is found in the medium as a molecule of water which is then distilled under reduced pressure and counted by liquid scintillation. The results obtained with this new method are comparable with those obtained with a conventional method using a 14C-labelled substrate.