Electrochemical and in silico study of the interaction between phenazopyridine and bovine serum albumin

Abstract

In this study, an electrochemical sensor was developed using a simple methodology to investigate for the first time the interaction of phenazopyridine with bovine serum albumin (BSA). The modified sensor was fabricated by electropolymerized diphenylamine-4-sulfonic acid barium salt onto the glassy carbon electrode surface by the cyclic voltammetry technique. Very low detection limit and quantification limit values were obtained by differential pulse voltammetry (DPV) in 0.1 M NaClO₄ as 0.13 µM and 0.43 µM, respectively. In addition, the interference effect was studied, and recovery studies were performed in urine samples achieving dramatic recovery values. Selective and reproducible determinations of phenazopyridine were performed perfectly with the developed sensor. The reduction in BSA oxidation signals as measured by DPV upon incubation with different phenazopyridine concentrations indicated that phenazopyridine was bound to BSA. The currents of the BSA peaks decreased linearly with the phenazopyridine concentration in the linear concentration range (2.00–22.60 µM). The results of the DPV experiments showed the formation of the BSA-Phenazopyridine complex. Binding parameters such as binding constants and binding free energy were calculated with the voltammetric data. Molecular docking and molecular dynamics simulation studies were performed to learn more about the interaction mechanism between phenazopyridine and BSA. Residues in the active gorge of BSA playing an important role in binding were determined by in silico studies. In silico studies showed that phenazopyridine binds to BSA with high affinity and this binding was stable. Eventually, the results of the electrochemical and modeling studies were perfectly matched.