Multi-spectroscopic characterization of bovine serum albumin upon interaction with atomoxetine

Abstract

The quenching interaction of atomoxetine (ATX) with bovine serum albumin (BSA) was studied in vitro under optimal physiological condition (pH=7.4) by multi-spectroscopic techniques. The mechanism of ATX-BSA system was a dynamic quenching process and was confirmed by the fluorescence spectra and lifetime measurements. The number of binding sites, binding constants and other binding characteristics were computed. Thermodynamic parameters ∆H° and ∆S° indicated that intermolecular hydrophobic forces predominantly stabilized the drug-protein system. The average binding distance between BSA and ATX was studied by Försters theory. UV-absorption, Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD), synchronous spectra and three-dimensional (3D) fluorescence spectral results revealed the changes in micro-environment of secondary structure of protein upon the interaction with ATX. Displacement of site probes and the effects of some common metal ions on the binding of ATX with BSA interaction were also studied.

Keywords: 3D fluorescence spectra; Atomoxetine; Bovine serum albumin; Energy transfer; FT-IR; Lifetime measurement.

Fig. 1

(A) Pictorial structure of bovine serum albumin and (B) chemical structure of atomoxetine.

Fig. 2

Fluorescence quenching spectra of BSA–ATX system with increased concentration of ATX (a) 0 µM, (b) 5 µM, (c)10 µM, (d) 15 µM, (e) 20 µM, (f) 25 µM, (g) 30 µM, (h) 35 µM, (i) 40 µM, and (j) 45 µM while BSA concentration was fixed at 5 µM in physiological pH 7.4 PBS buffer at 298 K.

Fig. 3

The Stern-Volmer curves for quenching of ATX with BSA at different temperatures. λ_ex =296 nm; λ_em =355 nm and [BSA] =5 µM.

Fig. 4

Time-resolved fluorescence decay for lifetime measurement spectra of (A) BSA and (B) ATX-BSA system, (pH=7.4, 298 K).

Fig. 5

The plots of log (F₀–F)/F versus log [Q] for binding ATX to BSA at different temperatures.

Fig. 6

van't Hoff plot logK vs 1/T for binding of ATX with BSA.

Fig. 7

Absorbance spectra of BSA–ATX system with increasing concentration of ATX (a) 0 µM, (b) 5 µM, (c)10 µM, (d) 15 µM, (e) 20 µM, (f) 25 µM, (g) 30 µM, (h) 35 µM, (i) 40 µM, and (j) 45 µM while BSA concentration was fixed at 5 µM in physiological pH 7.4 PBS buffer at 298 K. ((x) pH 7.4 PBS and (y) PBS+ATX (5 µM)).

Fig. 8

The overlap of (a) UV-absorption spectra of ATX (5 µM) with (b) fluorescence spectra of BSA (5 µM).

Fig. 9

Synchronous fluorescence spectra of BSA-ATX system with increased concentration of ATX (0–45 µM) and BSA concentration was fixed (5 µM) when (A) ∆λ=15 nm, and (B) ∆λ=60 nm, at pH 7.4 and 298 K.

Fig. 10

CD spectra of (a) BSA (5 µM) (b) BSA 5 µM + ATX 5 µM, (c) BSA 5 µM + ATX 10 µM and (d) BSA 5 µM + ATX 15 µM.

Fig. 11

Three-dimensional fluorescence spectra of (A) BSA and (B) BSA–ATX system (pH 7.4, 298 K).

Fig. 12

FT–IR spectra of ATX, BSA, and BSA–ATX at physiological pH 7.4 (298 K). Concentration of BSA and ATX was fixed at 5 µM.