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A colorimetric assay method for 3beta-hydroxy-delta5-steroid dehydrogenase.

Shivanandappa, T. and Venkatesh, S. (1997) A colorimetric assay method for 3beta-hydroxy-delta5-steroid dehydrogenase. Analytical Biochemistry, 254 (1). pp. 57-61. ISSN 0003-2697

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Abstract

3beta-Hydroxy-Delta5-steroid dehydrogenase is an important enzyme of steroid hormone biosynthesis present in steroidogenic tissues like adrenal, testis, and ovary of vertebrates. The enzyme is assayed mainly by radiochemical substrates. Spectrophotometric assay is not adequately sensitive to detect the enzyme activity since it is often present in low levels. We have developed a simple colorimetric assay based on formazan formation due to the reduction of the tetrazolium salt. The reaction mixture containing the 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 37 degrees C. 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 two- to threefold more sensitive than the spectrophotometric assay.

Item Type: Article
Uncontrolled Keywords: 3b-Hydroxy-D5-steroid dehydrogenase, steroid hormone biosynthesis
Subjects: 500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 16 Enzyme Chemistry
500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 05 Calorimetric Analysis
Divisions: Food Protectants and Infestation Control
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 24 May 2012 09:42
Last Modified: 24 May 2012 09:42
URI: http://ir.cftri.com/id/eprint/2211

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