3.1. Determination of binding affinity and mechanism of the ligand to the protein
The endogeneneous fluorescence shown by protein is due to the presence of tryptophan(Trp), tyrosine(tyr) and phenylalanine(phe). The environmentally sensitive Trp moiety mainly contributes to the changed fluorescence of BSA. When excited at 285 nm, BSA had a characteristic band at around 344 nm.[17, 18, 19] Furthermore, when the concentration of the added Cuminaldehyde increased from 0 to 61×10− 6 M, the fluorescence intensity of BSA decreased significantly, indicating there is a binding interaction of cuminaldehyde with BSA and HSA. The reaction temperatures for Cuminaldehyde-Serum albumins system were maintained at 298 K, 303 K and 308K respectively. The fluorescence quenching data are analyzed by Stern-Volmer equation[20]:
$$\frac{{F}_{0}}{F}=1+{k}_{q}{\tau }_{0}\left[Q\right]=1+{K}_{SV}\left[Q\right] \left(1\right)$$
KSV is the Stern–Volmer quenching constant with the unit being M− 1, and [Q] is the concentration of the quencher. kq is the quenching rate constant of BSA, τ0 is the average fluorescence lifetime of BSA in the excited state without the quencher (the order of magnitude is 10− 8 ). KSV and Kq value of BSA and HSA triggered by Cuminaldehyde at different temperatures can be determined by calculating the slope of the curve, as shown in Fig. 1B and 1D. In this experiment, the fluorescence intensities of BSA (Fig. 1A) and HSA(Fig. 1C) significantly decreased with increasing concentration of ligands, indicating that the ligand weakens the fluorescence intensities by interacting with protein. In other words, the existence of the ligands would have certain influence on fluorescence chromophore of both BSA and HSA. KSV were obtained from the Stern–Volmer equation at three different temperatures 298, 304, and 310 K and presented in Table 1. KSV values decreased as the temperature increases, which indicated that the mechanisms of the two systems were all static quenching.[21, 22] For static quenching, the binding constants (K) can be represented by the double logarithm regression curve
$$\text{l}\text{o}\text{g}({F}_{0}-F)/F=logK+nlog\left[Q\right] \left(2\right)$$
where F0, F, and [Q] are the same as in Eq. (1). K is the binding constant, and n is the number of binding sites per HSA. The results(Table 1) showed that the binding constant of the Cuminaldehyde–BSA system decreased and Cuminaldehyde–HSA system increased with the increase in temperature, which may hint a temperature-sensitive complex formation in the binding reaction.[23] In addition, K values in the Cuminaldehyde–BSA system were bigger than that in the Cuminaldehyde–HSA system, indicating the binding ability of cuminaldehyde with BSA was better than HSA.
Thermodynamic parameters and binding forces
The binding forces between macromolecules and ligands include hydrogen bonds, van der Waals forces, hydrophobic forces, and electrostatic interactions.[24–28] The temperature effect was very small and did not result in structural degradation of BSA and HSA; thus, enthalpy change can be regarded as a constant within a little range of temperature. The binding mode was verified by using the Van’t Hoff equation[29]:
$$lnK=-\varDelta H/RT+ \varDelta S/R \left(3\right)$$
$$\varDelta G=\varDelta H-T\varDelta S \left(4\right)$$
where R is the gas constant, T is the experimental temperature, and K is the binding constant at the corresponding temperature. The calculated results of the thermodynamic parameters were represented in Table 1. The negative value of
ΔG indicates the interaction of cuminaldehyde and both BSA and HSA is spontaneous. The negative value of ΔH and ΔS for Cuminaldehyde-BSA system indicates that vander Waal’s interaction and hydrogen bonding play a major role in binding of the ligand to the protein. On the other hand, positive value of ΔH and ΔS for Cuminaldehyde-HSA system indicates that electrostatic interaction plays vital role in binding of the ligand to the protein.
Table 1
The quenching constants (KSV), binding constants (K) and thermodynamic parameters for the interaction of Cuminaldehyde with BSA and HSA at different temperatures:
| T(K) | KSV×104 (M-1) | Ra | K×104 (Lmol-1) | Rb | ΔG (KJ/mol) | ΔS (J/mol·K) | ΔH (kJ/mol) | Rc |
| 298 | 1.998 | 0.992 | 8.5 | 0.997 | -12.18 | -7.61 | -14.45 | 0.979 |
BSA | 303 | 1.439 | 0.987 | 6.3 | 0.996 | -12.15 | | | |
| 308 | 1.143 | 0.988 | 5.5 | 0.993 | -12.11 | | | |
| 298 | 1.902 | 0.998 | 1.54 | 0.996 | -12.88 | 189.55 | 43.6 | 0.991 |
HSA | 303 | 1.89 | 0.995 | 3.51 | 0.995 | -13.832 | | | |
| 308 | 0.88 | 0.966 | 5.73 | 0.977 | -14.77 | | | |
3.3. Combination of fluorescent probes
BSA and HSA are made up of three linearly arranged structurally homolgous domains(I-III) and each domain in turn is the product of two subdomains(A,B). The two binding sites present on BSA and HSA protein are designated as Site I and Site II, and are present in sub-domains IIA and IIIA, respectively.[31–34] To determine the displacement percentage of fluorescent combination probe, fluorescent markers for distinct binding sites were chosen: ketoprofen for site I and ibuprofen for site-II.[35] The following equation is adopted:
$$Probe displacement=\frac{{F}_{2}}{{F}_{1}}\times 100 \left(5\right)$$
Where F1 and F2 represent the fluorescence intensity of Cuminaldehyde-BSA/HSA system in the absence and presence of the probe respectively.
The increase in ketoprofen concentration results in significant decrease in fluorescence intensity(Fig. 3) for both the system indicating that Cuminaldehyde binds at site I of both BSA and HSA.