Bovine and human serum albumin interactions with 3-carboxyphenoxathiin studied by fluorescence and circular dichroism spectroscopy

Abstract

The interactions of 3-carboxyphenoxathiin with Bovine Serum Albumin (BSA) and Human Serum Albumin (HSA) have been studied by fluorescence and circular dichroism spectroscopy. The binding of 3-carboxyphenoxathiin quenches the BSA and HSA fluorescence, revealing a 1:1 interaction with a binding constant of about 10(5) M(-1). In addition, according to the synchronous fluorescence spectra of BSA and HSA in presence of 3-carboxyphenoxathiin, the tryptophan residues of the proteins are most perturbed by the binding process. Finally, the distance between the acceptor, 3-carboxyphenoxathiin, and the donor, BSA or HSA, was estimated on the basis of the Förster resonance energy transfer (FRET). The fluorescence results are correlated with those obtained from the circular dichroism spectra, which reveal the change of the albumin conformation during the interaction process.

Figure 1

The structure of 3-carboxyphenoxathiin (I).

Figure 2

Emission spectra of BSA in the presence of different concentrations of I; [BSA] = 3 × 10^-6 M; (a) 1–16: d/p = 0–1; (b) 1–7: d/p = 1–5; λ_ex = 286 nm; pH = 7.4.

Figure 3

Two linear segments in Stern-Volmer plots for the quenching of HSA/BSA with I; [BSA]=[HSA] = 3×10^-6M; λ_ex = 286 nm; λ_em = 345 nm; first segment d/p<1 (r² = 0.999); second segment d/p>1 (r² = 0.997).

Figure 4

Stern-Volmer plots for the systems HSA—I (A) and BSA–I (B); [HSA] = [BSA] = 3×10^-6 (M); A) r² = 0.999; B) r² = 0.999; the plots represent the best fits of the experimental points using Equation (2).

Figure 5

(A) Two linear segments fitting plot for the HSA–I system to Equation (6); [HSA] = 3×10^-6 M; λ_ex = 286 nm; λ_em = 345 nm; inset, a triplicate experiments for d/p ≥ 1.0, fitted to Equation (6). (B) Two linear segments fitting plot for the BSA–I system to Equation (6); [BSA] = 3×10^-6 M; λ_ex = 286 nm; λ_em = 346 nm.

Figure 6

Nonlinear fitting of experimental data for the system (A) HSA–I (r² = 0.998) and (B) BSA-I (r² = 0.996) to Equation (7).

Figure 7

The effect of addition of I on the synchronous fluorescence spectrum of HSA (A) and BSA (B); Δλ = 60 nm.

Figure 8

The overlap of the fluorescence spectrum of HSA (a) and the absorption spectrum of I (b).

Figure 9

CD spectra of the (A) HSA–I system; (B) BSA-I system at pH 7.4; [HSA] =[BSA]= 3×10^-6 M; 1–6: d/p = 0, 0.46, 0.66, 1.16, 2.33, 3.66, respectively.