Binding constants and in silico analysis of albumin interaction with phenolic acids and flavonoids

Abstract

In this study, fluorescence techniques were utilized to investigate the interactions of selected phenolic acids (PAs) and flavonoids (FLs) with bovine serum albumin (BSA) under physiological conditions. The binding of PAs/FLs with BSA was investigated at three temperatures: 292, 303 and 310 K. From the obtained spectra, the Stern-Volmer constant (Ksv), bimolecular quenching constant (kq), binding constants (Kb), and binding site number (n) constants were calculated. Presented results indicate that fluorescence quenching of BSA in the presence of phenolic acids/flavonoids is a static quenching process. The strongest static binding occurs during the formation of the BSA-pHBA (p-hydroxybenzoic acid) complex (kq = 57.1×1012 M-1 s-1 at 292 K), and BSA-Que (quercetin) complex (kq = 42.8×1012 M-1 s-1 at 292 K). The structure of PAs/FLs was revealed to significantly affect the binding/quenching process and additionaly, fluorescence resonance energy transfer studies confirmed the static nature of this process. The results of synchronous fluorescence spectra suggest changes in the microenvironment of tyrosine. Three-dimensional spectra showed changes related to the backbone structures of the protein chain (caused by the π-π∗ transition of the carbonyl group). Furthermore, thermal denaturation was performed by nano differential scanning fluorimetry (nanoDSF) and transition temperature (Tm) values for BSA complexes with PAs/FLs are slightly lower than Tm for BSA, except Tm for BSA complexes with kaempferol and chrysine. According to in silico analysis, theoretically, caffeic acid and quercetin showed the best binding position with albumin (4F5S).