A Colorimetric Assay Method for 3-Hydroxy-5-steroid Dehydrogenase

tissues such as adrenal, ovary, testis, and placenta. 3b-Hydroxy-D-steroid dehydrogenase is an imIt is a microsomal NAD-dependent enzyme that catportant enzyme of steroid hormone biosynthesis alyzes the conversion of pregnenolone to progesterpresent in steroidogenic tissues like adrenal, testis, one, a rate-limiting step in the biosynthesis of horand ovary of vertebrates. The enzyme is assayed monal steroids (4). HSDH activity has been assayed mainly by radiochemical substrates. Spectrophotomainly by the reduction of NAD spectrophotometrimetric assay is not adequately sensitive to detect the cally (340 nM) (11) or fluorometrically (465 nM) and enzyme activity since it is often present in low levels. by the quantitation of the product by extraction and We have developed a simple colorimetric assay based TLC (5–8). In recent years, a radiochemical assay on formazan formation due to the reduction of the for HSDH has been available (9, 10). The spectrotetrazolium salt. The reaction mixture containing the photometric method suffers from poor sensitivity substrate, pregnenolone or dehydroepiandrosterone, NAD and iodonitrotetrazolium in 0.1 M Tris–HCl buffer (pH 7.8), and the enzyme extract is incubated for 1 h at 377C. Absorbance at 490 nm is read in a spectrophotometer. The enzyme activity was linear with time and protein concentration. The assay works well with adrenal tissue extract, whereas in the case of testis, Leydig cell preparation may be required. We have assayed the enzyme activity in the adrenal of rat, mouse, and gerbil. The method is twoto threefold more sensitive than the spectrophotometric assay. q 1997 Academic Press