Figure 1.
Structural representation of pinostrobin.
(A) Chemical structure and (B) ball-and-stick model.
Figure 2.
Fluorescence quench titration of HSA with increasing PS concentrations.
[HSA] = 3 µM, [PS] = 0–22.5 µM with 1.5 µM intervals (1–16), λex = 280 nm studied in 10 mM Tris-HCl buffer, pH 7.4, 25°C. Arrow depicts the blue shift in the emission maximum of HSA with increasing PS concentrations. Inset shows the decrease in the relative fluorescence intensity of HSA at 336 nm (FI336 nm) with increasing PS/HSA molar ratios.
Figure 3.
Analysis of fluorescence quenching data.
(A) Stern–Volmer and (B) against
plots of PS–HSA system at different temperatures. Inset of (B) shows the van’t Hoff plot for PS–HSA interaction.
Table 1.
Binding and thermodynamic parameters for the interaction between PS and HSA, studied at different temperatures, pH
Figure 4.
Spectral overlap between the fluorescence spectrum of HSA and absorption spectrum of PS.
Both spectra were recorded at 25°C in 10 mM Tris-HCl buffer, pH 7.4. The concentrations of HSA and PS were 3 µM each.
Figure 5.
Synchronous fluorescence spectra of HSA obtained in the absence and presence of increasing PS concentrations.
[HSA] = 3 µM, [PS] = 0–22.5 µM with 1.5 µM intervals (1–16) studied in 10 mM Tris-HCl buffer, pH 7.4, 25°C. The difference between excitation and emission wavelengths (Δλ) was (A) 15 nm and (B) 60 nm. Arrows depict the position of the emission maximum of HSA in presence of increasing PS concentrations.
Figure 6.
3-D fluorescence spectral projections and corresponding contour maps of HSA and various PS–HSA complexes.
(A and A′) Free HSA, (B and B′) 1∶1 PS–HSA, (C and C′) 2∶1 PS–HSA and (D and D′) 3∶1 PS–HSA. The spectra were recorded in 10 mM Tris-HCl buffer, pH 7.4, 25°C using a protein concentration of 3 µM.
Table 2.
Characteristics of three-dimensional fluorescence spectra of native HSA and its complexes with PS at pH 7.4, 25°C.
Figure 7.
Thermal stability profiles of HSA (3 µM) and 5∶1 PS–HSA complex.
MRE values at 222(A) 25–100°C and (B) 25–80°C, obtained in 10 mM Tris-HCl buffer, pH 7.4. The smaller symbols represent the refolding experimental results obtained with the same HSA and PS–HSA systems.
Figure 8.
Displacement of site marker ligands bound to HSA in presence of increasing PS concentrations.
(A) CD spectra of BR–HSA complex (10 µM each) in the absence (1) and presence (2–9) of increasing PS concentrations (10–80 µM with 10 µM intervals). Spectra marked as ‘a’, ‘b’, ‘c’ and ‘d’ refer to 10 µM BR, 10 µM HSA, 50 µM PS and PS–HSA (5∶1) complex, respectively. Inset shows the decrease in the relative CD value at 459 nm (CD459 nm) with increasing PS/HSA molar ratios. (B) Fluorescence spectra of WFN–HSA complex (3 µM each) in the absence (1) and presence (2–9) of increasing PS concentrations (3–24 µM with 3 µM intervals). Spectra labeled as ‘a’, ‘b’, ‘c’ and ‘d’ refer to 3 µM WFN, 3 µM HSA, 15 µM PS and PS–HSA (5∶1) complex, respectively. Inset shows the decrease in the relative fluorescence intensity at 383 nm (FI383 nm) with increasing PS/HSA molar ratios. (C) CD spectra of DZM–HSA complex (20 µM DZM +10 µM HSA) in the absence (1) and presence (2–9) of increasing PS concentrations (10–80 µM with 10 µM intervals). Spectra marked as ‘a’, ‘b’, ‘c’ and ‘d’ represent 20 µM DZM, 10 µM HSA, 50 µM PS and PS–HSA (5∶1) complex, respectively. Inset shows the change in the relative CD value at 259 nm (CD259 nm) with increasing PS/HSA molar ratios. (D) CD spectra of KTN–HSA complex (20 µM KTN +10 µM HSA) in the absence (1) and presence (2–9) of increasing PS concentrations (10–80 µM with 10 µM intervals). Spectra labeled as ‘a’, ‘b’, ‘c’ and ‘d’ refer to 20 µM KTN, 10 µM HSA, 50 µM PS and PS–HSA (5∶1) complex, respectively. Inset shows the change in the relative CD value at 340 nm (CD340 nm) with increasing PS/HSA molar ratios.
Figure 9.
Cluster analysis of PS docking to binding sites I and II of HSA.
The different crystal structures of HSA used in the analysis were (A) 1BM0, (B) 2BXD and (C) 2BXF. A total of 100 runs were performed for each binding locus.
Figure 10.
Model of PS docking to site I at subdomain IIA of HSA.
Domain structure of HSA (I, red; II, blue and III, green), showing the binding orientation of the lowest docking energy conformation of PS. The zoomed-in view of the binding locus shows the ball-and-stick representation of the ligand and the side-chains of the protein residues (yellow) that form hydrogen bonds (green lines) with PS.
Table 3.
Distance of the predicted hydrogen bonds formed between interacting residues of HSA and PS.