Bile acids are steroid hormones that play an important role in the regulation of various metabolic pathways as well as digestive processes. However, high concentrations of bile acids have been linked to many gastrointestinal diseases, including cancers. 12α-hydroxysteroid dehydrogenase (12α-HSDH) is an oxidoreductase that catalyzes the reversible conversion of hydroxyl groups on toxic bile acids to keto groups, resulting in less toxic epimers. Previous research on kinetic parameters have established that 12α-HSDH has a higher affinity for its substrate deoxycholic acid rather than cholic acid. Also, we have hypothesized that 12α-HSDH should have a higher affinity for substrates rather than products to synergically work with other enzymes in the pathway. To test this hypothesis, we have designed the tyrosine fluorescence quenching assays to determine the dissociation constant (KD) values for various ligands. The determination of the dissociation constants will also supplement the kinetic findings of the enzyme. To start with, 12α-HSDH was overexpressed using recombinant plasmid from Methanosphaera stadtmaniae and purified using immobilized metal affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was run to confirm the protein purity ensuring molecular weight of His-tagged protein at 29 kDa. For the tyrosine fluorescence quenching assays, the absorption and emission spectra of the protein was determined to be 246 nm and 492 nm respectively. We are in the process of conducting fluorescence quenching experiments without and with substrates deoxycholic acid, cholic acid, NADP+ and their respective products.

Determination of ligand interactions for 12α-hydroxysteroid dehydrogenase: Critical enzyme in catabolic pathway of bile acids

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