Surface Plasmon Resonance Binding Study on the Interaction of Acetazolamide and Bovine Serum Albumin

Abstract

Objective: Serum albumins are major plasma proteins in systemic blood circulation and act as transport proteins for endogenous and exogenous compounds such as drugs. In pharmaceutical applications, it is essential to characterize how drugs bind to serum albumin in the evaluation of drug candidates. Surface plasmon resonance (SPR) is fast, real-time, label-free optical based detection technique that offers the monitoring of molecular interactions, analyzing binding reactions and determining the affinity constants with real-time and high sensitivity. Acetazolamide (AZA) is used in the treatment of epilepsy and glaucoma. Methods: To determine the binding kinetics of AZA-Bovine serum albumin (BSA) interaction, (i) SPR gold sensor surface was functionalized, (ii) amine coupling procedure was applied to activate the surface group and BSA was immobilized on functionalized sensor surface, (iii) the concentration series of AZA (10, 25, 50, 75, 100, 150, 200 and 250 µM) was injected to SPR system and (iv) kinetic values were measured using the software of SPR system. Results: 5 mM MUA was coated for surface functionalization. 250 µg/mL BSA as ligand, 30 µL/min flow rate, 1X PBS buffer (pH 7.4) and 10 mM acetate buffer (pH 5.2) as running and coupling buffers, respectively, were performed for SPR binding study. According to result, equilibrium constant (KD) of AZA-BSA was determined as 67.72 µM. Conclusion: In this study, we investigated the AZA-BSA binding interaction using SPR system based on Kretchmann configuration. The study designed with fast, label-free and real-time approach will provide valuable knowledge for pharmaceutical and clinical applications.