Multi-spectroscopic investigation of the binding interaction of fosfomycin with bovine serum albumin

Abstract

The interaction between fosfomycin (FOS) and bovine serum albumin (BSA) has been investigated effectively by multi-spectroscopic techniques under physiological pH 7.4. FOS quenched the intrinsic fluorescence of BSA via static quenching. The number of binding sites n and observed binding constant K_A were measured by the fluorescence quenching method. The thermodynamic parameters ΔG⁰, ΔH⁰ and ΔS⁰ were calculated at different temperatures according to the van't Hoff equation. The site of binding of FOS in the protein was proposed to be Sudlow's site I based on displacement experiments using site markers viz. warfarin, ibuprofen and digitoxin. The distance r between the donor (BSA) and acceptor (FOS) molecules was obtained according to the Förster theory. The effect of FOS on the conformation of BSA was analyzed using synchronous fluorescence spectra (SFS), circular dichroism (CD) and 3D fluorescence spectra. A molecular modeling study further confirmed the binding mode obtained by the experimental studies.

Keywords: 3D spectra; Fosfomycin; Serum albumin; Spectroscopic methods; Synchronous fluorescence.

Fig. 1

Fluorescence spectra of BSA (5 μM) in the presence of FOS: 0 (a) to 45 μM (j) at 298 K.

Fig. 2

Synchronous fluorescence spectrum of BSA–FOS (T=298 K, pH 7.40): (A) ∆λ=15 nm and (B) ∆λ=60 nm. (a) [BSA]=5 μM; (b)–(j) [FOS]=5–45 μM.

Fig. 3

Three dimensional fluorescence spectra of (A) BSA and (B) the FOS+BSA system.

Fig. 4

The CD spectra of the FOS–BSA system: (a) free BSA, (b) and (c) BSA+FOS obtained at 298 K and at pH=7.4.

Fig. 5

(A) Details of the interactions between BSA and FOS in the docked model. (B) 2D schematic representation of hydrogen bond interactions. (C) Hydrophobic and (D) hydrophilic amino acids surrounded to FOS. (E) and (F) Binding domain for FOS at BSA chain A.