Document Type

Honors Project

First Advisor

Dr. Rippa Sehgal

Degree Award Date

Spring 2024

Keywords

Ligand Binding, Protein, Oxidoreductase, Dissociation Constant, Fluorescence Quenching

Disciplines

Biochemistry

Abstract

12α-hydroxysteroid dehydrogenase (12α-HSDH) catalyzes the conversion of cholesterol into bile acids and the interconversion of deoxycholic acid into 12-oxolithocholic acid, making it an important enzyme of the human gut microbiome. Using fluorescence quenching, emission and excitation spectra for the enzyme and variable concentrations of multiple ligands – to include NADP+, NAD+, FAD, deoxycholic acid (DCA), and cholic acid (CA) – were collected and used to create corresponding protein binding saturation curves. Using these curves, the dissociation constants (Kd) for each of the ligands was calculated, with NADP+’s being 3.480 x 10-3 mM, NAD+’s 9.285 x 10-4 mM, FAD’s 3.123 x 10-2 mM, DCA’s 2.008 x 10-5 mM, and CA’s 3.775 x 10-10 mM. As a lower value indicates higher binding affinity, cholic acid appears to have the best affinity for the enzyme, but more research needs to be done to validate the results. Future work will also need to be done to fully compare these values with the inhibition constant (Ki) and Michaelis constant (Km) for each ligand, as they were unable to be ascertained due to time restrictions and technical difficulties.

Recommended Citation

Hillegass, Sabrina. "Determination and Comparison of Dissociation and Inhibition Constants of 12α-Hydroxysteroid Dehydrogenase with Inhibitors and Substrates." Senior Honors Projects, Bridgewater College, 2024.

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