Spectrofluorimetric Determination of Kinetic Parameters of Bile Acids With 3α-Hydroxysteroid Dehydrogenase from Mutant Pseudomonas Testosteroni

Abstract

The spectrofluorimetric determination of kinetic parameters (K_m, V and k_obs) of conjugated bile acids with 3α-hydroxysteroid dehydrogenase from mutant Ps. Testosteroni is described. Under optimal pH conditions and pseudo- first order kinetics([β-NAD⁺)20 K_mNAD+) the kinetic parameters of bile acids in different buffers are determined. The bile acids exhibit larger kinetic differences in glycine and Tris buffer compared to those measured in pvrophosphate buffer. From these data it is concluded that under certain conditions the differential kinetic determination of one bile acid in the presence of others is possible without prior separation.

Key Words:

• 3α-hydroxysteroid dehydrogenase
• bile acids
• kinetic parameters
• fluorescence