Binding interaction of phosphorus heterocycles with bovine serum albumin: A biochemical study

Abstract

Interaction between bovine serum albumin (BSA) and phosphorus heterocycles (PHs) was studied using multi-spectroscopic techniques. The results indicated the high binding affinity of PHs to BSA as it quenches the intrinsic fluorescence of BSA. The experimental data suggested the fluorescence quenching mechanism between PHs and BSA as a dynamic quenching. From the UV-vis studies, the apparent association constant (K_app) was found to be 9.25×10², 1.27×10⁴ and 9.01×10² L/mol for the interaction of BSA with PH-1, PH-2 and PH-3 respectively. According to the Förster's non-radiation energy transfer (FRET) theory, the binding distances between BSA and PHs were calculated. The binding distances (r) of PH-1, PH-2 and PH-3 were found to be 2.86, 3.03, and 5.12 nm, respectively, indicating energy transfer occurs between BSA and PHs. The binding constants of the PHs obtained from the fluorescence quenching data were found to be decreased with increase of temperature. The negative values of the thermodynamic parameters ΔH, ΔS and ΔG at different temperatures revealed that the binding process is spontaneous; hydrogen bonds and van der Waals interaction were the main force to stabilize the complex. The microenvironment of the protein-binding site was studied by synchronous fluorescence and circular dichroism (CD) techniques and data indicated that the conformation of BSA changed in the presence of PHs. Finally, we studied the BSA-PHs docking using Autodock and results suggest that PHs is located in the cleft between the domains of BSA.

Keywords: BSA; BSA-PHs docking; Phosphorus heterocycles; Spectroscopy.

Graphical abstract

Scheme 1

Synthesis of phosphorous containing heterocycles by ring closing metathesis (RCM).

Fig. 1

Absorption spectra of BSA (0.1 µM) in presence of PH-1: (0, 5, 10, 15, 20 and 25 µM). The inset shows calculation of K_app of BSA-PH-1 complex; 1/(A_obs–A₀) vs 1/[PH-1] plot.

Fig. 2

Fluorescence quenching spectra of BSA in presence of PH-1, The inset shows the Stern-Volmer plot for PH-1 and BSA at 293, 298, 303, and 313 K, respectively.

Fig. 3

Plots of the pH-1 quenching effect on BSA fluorescence at 293, 298, 303, and 313 K.

Fig. 4

The Van’t Hoff plot for the interaction of BSA and PHs.

Fig. 5

Synchronous fluorescence spectra of BSA in presence of PH-1 using (A) Δλ=15 nm (λ_ex=240 nm, λ_em=255 nm) and (B) Δλ=60 nm (λ_ex=240 nm, λ_em=300 nm).

Fig. 6

UV-CD spectra of PH-1 and PH-1-BSA system at the BSA concentration of 0.01 µM and PH-1 concentrations of 0, 12.5 and 25 µM.

Fig. 7

The overlap plot of the fluorescence emission spectra of BSA (0.1 µM) and the UV absorption spectra of PH-1 (1 mM).

Fig. 8

Docking of PHs with BSA. (A) place of interaction of PH-1 and BSA and (B) the surrounding amino acid residues of BSA within 5 Å from PH-1 (green colored).