Volume 4 Issue 3
Authors: Edith M. Kutorglo; Justice K. Sarfo
Abstract: Ultrafiltration, fluorescence spectroscopy, fluorescence-detected competitive binding, X-ray crystallography and chromatographic methods (HPLC) have all been used to evaluate protein-ligand (drug) interactions. Although these methods provide useful binding information, they are constrained by high costs, low sensitivity, and complicated instrumentation. Accurate, fast and simple methods of determining the affinity of a small molecule for a target protein are needed to speed the discovery of new medications and biological probes. A simplified lab-made equilibrium dialysis setup was coupled with absorption spectrophotometry and tested with protein-ligand interactions using methylene blue (MB) and bovine serum albumin (BSA) as models. The results from the absorption spectroscopic analysis showed a reduction in the absorption of methylene blue after dialysis as a result of MB-BSA complex formation with high association constant. The wavelength for maximum absorption (λmax) of methylene blue was found to be 664 nm, and the time for equilibration was approximately 2 hours. The Kd, Bmax and n were 9.2µM, 0.0143 µM/min and 0.5051, respectively, and the association constant was Ka = 1.087 x 105M-1. The thermodynamic constants ∆G, ∆H and ∆S were found to be 12.392 kCalmol-1, -13.227 kCalmol-1, and -41.63 Calmol-1K-1, respectively, and the activation energy was 56.23 kCalmol-1K-1. This method is simple and cost effective, and can be used to measure the drug-protein binding parameters with the aid of basic laboratory equipment.
Keywords: Home-made Equilibrium Dialysis Setup; Methylene Blue; Bovine Serum Albumin; Absorption Spectrophotometry