The water was prepared using a Milli-Q system (EMD Millipore, Billerica, MA, USA). HSA, warfarin, ibuprofen and Tris were purchased from Sigma-Aldrich (Sigma-Aldrich; Merck KgaG, Darmstadt, Germany). The other reagents were of analytical grade.

Tris was dissolved in water to a concentration of 0.05 M, and was calibrated to pH 7.4 with hydrochloric acid. Next, HSA was added into Tris-HCl solution to make a solution at a concentration of 1.0×10⁻⁵ M. Warfarin and ibuprofen were dissolved in dimethyl sulfoxide (DMSO) to obtain 5.0×10⁻³ M solutions, respectively. In addition, KR solution was prepared with DMSO at 3.0×10⁻³ M.

In this experiment, KR solution was gradually added to the HSA solution to increase its concentration from 0 to 3.0×10⁻⁵ M at increments of 0.3×10⁻⁵ M. The fluorescence spectra were recorded using an L55 fluorescence spectrometer (PerkinElmer, Inc., Waltham, MA, USA) at 298, 304 and 310 K. Furthermore, the excitation wavelength was 290 nm and the emission wavelength was between 300 and 450 nm.

To identify the binding site of KR with HSA, a competitive experiment with site markers has been implemented. In the mixed solution with equal quantities of HSA and site maker, the KR solution was gradually added at increments of 0.3×10⁻⁵ M. In addition, the fluorescence intensities were determined.

To study the characteristics of intrinsic Tyr and tryptophan residues, synchronous fluorescence spectroscopy was employed. The excitation and emission slit widths were set at 5 and 10 nm, respectively. Furthermore, the concentrations of HSA were adjusted to 1.5×10⁻⁶ and 0.15×10⁻⁶ M according to the varied scanning intervals (Δλ) at 15 and 60 nm. Next, the spectra of HSA were recorded upon the titration of KR from 0 to 1.8×10⁻⁵ M.

The excitation and emission slit widths were set at 10 and 5 nm, respectively. The emission spectra were recorded from 260 to 450 nm with an excitation wavelength range between 220 and 450 nm, and with a 5-nm interval. Furthermore, the concentration of HSA was 0.6×10⁻⁶ M, and the ratios of KR and HSA were 0:1 and 10:1, respectively.

The structure of KR was generated as the ligand using SYBYL software, version X2.1 (Certara USA, Inc., Princeton, NJ, USA) on a Windows 7 workstation and minimized using a Tripos force field. Furthermore, the HSA crystal file was obtained from the RCSB Protein Data Bank (Protein Data Bank code: 2BXD). After the water was removed from the crystal structure, hydrogens and charges were added. Next, docking was performed by the Surflex-Dock module.

As shown in Fig. 1, the HSA can emit fluorescence without KR. The fluorescence intensity decreased gradually following the addition of KR solution, which indicated that fluorescence quenching occurred. At the same time, the maximum emission wavelength showed a slight ‘blue shift’ from 339 to 337 nm (demonstrated when the peak moves towards the short wave length). The quenching can be analyzed by the Stern-Volmer equation (i) at different temperatures: F₀/F=1+K_sv[Q]; in which F₀ and F are the fluorescence intensity with or without quencher. K_sv represents the quenching constant and [Q] is the concentration of quencher. Hence, K_sv can be derived from the linear regression of (F₀-F)/F vs. [Q].

Fluorescence quenching can be classified as static and dynamic quenching based on the response to temperature. As the K_sv value decreases following the increasing temperature, the quenching between KR and HSA is static.

For the static quenching, the ligand will be bound to HSA to form a complex. Furthermore, the binding site can be identified using the double logarithm equation (ii): log(F₀-F)/F=logK+nlog[Q]; where F₀ and F are the fluorescence intensities of protein in the absence and presence of ligand, respectively. K is the binding constant; n is the number of binding sites and [Q] is the concentration of quencher. As shown in Table I, the values of n are close to 1 at different temperatures. Additionally, K values decrease following the increasing temperature, which is in accordance with the K_sv values.

To further confirm the binding site of KR, a competitive experiment was implemented with specific markers, warfarin and ibuprofen. After the addition of warfarin, the maximum emission wavelength presented an evident ‘red shift’ from 339 to 359.5 nm (demonstrated when the peak moves towards the long wave length) and the fluorescence intensity of HSA descended (Fig. 2A). However, the fluorescence quenching was weaker than ibuprofen when KR was added. In the presence of ibuprofen, the fluorescence of HSA was not affected and the quenching was evident after the addition of KR (Fig. 2B).

To elucidate the binding forces between KR and HSA, the Van't Hoff equation (iii) was employed in thermodynamic analysis: lnK=−ΔH/RT+ΔS/R; where K is the binding constant at a respective temperature, and R is the gas constant. ΔH and ΔS are the changes of enthalpy and entropy, respectively. The thermodynamic parameters of the KR-HSA system were calculated from the Van't Hoff plot of lnK against 1/T. In addition, the driven force can be expressed by the Gibbs free energy change (ΔG) and obtained from the following equation (iv): ΔG=ΔH-TΔS.

As shown in Table I, ΔH, ΔS and ΔG values are negative, which indicates that the interaction is exothermic and spontaneous. In addition, the values of ΔG decrease according to the increasing temperature, which demonstrates the binding interaction weakens accordingly.

Synchronous fluorescence spectroscopy can describe the microenvironment of Tyr and Trp as the wavelength differences (Δλ) between the excitation and emission wavelength were fixed at 15 and 60 nm, respectively. As in Fig. 3, when the addition of KR was initialized, fluorescence quenching appeared but the maximum wavelength (λ_m) remained at 289 and 284 nm respectively.

3D fluorescence spectroscopy is a tool used to investigate the conformational changes of proteins. As shown in Fig. 4, there was a weak peak (λ_ex=λ_em) caused by Rayleigh scattering. In the presence of KR at the molar ratio of 10:1, the 3D fluorescence spectrum of HSA changed evidently. The intensity of peaks I and II dropped greatly following the fluorescence quenching, particularly of peak I.

Molecular modeling can visualize the detailed interaction between KR and HSA. KR can enter the cavity of subdomain IIA and interact through hydrogen bonds with Glu153 (1.9 Å), Lys199 (2.2, 2.8 Å), Arg257 (1.9, 2.1, 2.1 Å), His288 (2.6 Å) and Glu292 (2.1 Å). The total score of KR (7.49) is similar to warfarin (7.41). KR in site I is near the Trp-214 and affects the microenvironment around the fluorescence quenching (Fig. 5A). A 2D interaction diagram shows there are van der Waals forces, Pi and electrostatic interactions besides the hydrogen bonds (Fig. 5B). In detail KR can interact with the residues of HSA, including Tyr150, Phe156, Phe157, Glu188, Ser192, Trp214, Arg222, Leu219, Phe223, Leu238, Leu260, Ala258, Ala261, Ser287 and Val241 through van der Waals forces. Furthermore, Pi interaction occurs between the 3-phenylchromone scaffold of KR and both the His242 and Ala291 residues. In addition, an electrostatic interaction can be found between KR and the Glu153, Lys195, Lys199, Arg257, His288 and Glu292 residues.