CHARACTERISTICS OF THE LIGAND BINDING SITE OF ALDEHYDE DEHYDROGENASE FOR SANGUINARINE

Abstract

Sanguinarine, a quaternary benzophenanthridine alkaloid molecular interaction with ALDH was investigated by steady state fluorescence and UV-Visible spectroscopy techniques under simulated physiological conditions. Fluorescence spectra and data showed that sanguinarine has a strong ability to quench the intrinsic fluorescence of ALDH through a static quenching procedure, and the effective quenching constant (KSV) was 4.70×104 M. The Scatchard plot indicated that Aldehyde dehydrogenase has one binding site for sanguinarine with a binding constant (Ka) of 23.1 ×103 L. mol-1 and a dissociation constant of 2.069 ×105 L. mol-1 at 298K and pH 7.4 showing a strong bonding interaction of ALDH-sanguinarine. The thermodynamic data suggest that sanguinarine binding to ALDH was not spontaneous. Hydrophobic bond played a major role in the binding process. UV-Visible spectra of the ALDH-sanguinarine complex showed unperturbed secondary structure but perturbed tertiary structure of ALDH to the ligand. There was no entropy-enthalpy compensation of ALDH-sanguinarine interaction. The percentage binding rate of sanguinarine increases upon binding to ALDH site both at low and high concentrations within the temperatures of study. However, at certain point the response become saturated and reaches plateau. The synchronous fluorescence of ALDH-sanguinarine indicated conformational change near the Tryptohan residue micro-environment rather than tyrosine. According to Forster non-radiation energy transfer theory, the binding of sanguinarine to ALDH was of great probability less than 8nm. This work established a clear relationship between sanguinarine and ALDH has helped in understanding of the ligand binding properties of the enzyme and the dynamic and thermodynamics of their interactions for drug discovery, design and development