A Colorimetric Assay Method for 3β-Hydroxy-Δ5-steroid Dehydrogenase

✍ Scribed by T. Shivanandappa; S. Venkatesh

• Publisher: Elsevier Science
• Year: 1997
• Tongue: English
• Weight: 90 KB
• Volume: 254
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1997.2406

✦ Synopsis

tissues such as adrenal, ovary, testis, and placenta. 3b-Hydroxy-D 5 -steroid dehydrogenase is an im-It 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 ( 340nM) (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 FIG. 1. Comparison of the standard curve of NADH measured by 1 This paper is dedicated to Professor H. B. Devaraj Sarkar. 2 To whom correspondence should be addressed. Fax: 91 821 tetrazolium reduction with that of UV absorbance. Graded concentrations of NADH were reacted with iodonitrotetrazolium in the pres-517233.

3 Abbreviations used: 3b-HSDH, 3b-hydroxy-D 5 -steroid dehydro-ence of PMS in Tris buffer (0.1 M, pH 7.4). The color was measured at 490 nm and plotted with the absorbance of NADH at 340 nm. genase; INT, iodonitrotetrazolium chloride, PMS, phenazene methosulfate; DMF, dimethyl formamide.

Each point is the mean of triplicate values.