All chemicals and reagents were of analytical grade and doubly distilled water was used throughout the study. BSA (fatty acid free, fraction V, 96% - 98%), sodium dihydrogen phosphate (NaH₂PO₄), potassium dihydrogen phosphate (KH₂PO₄) were purchased from Sigma Chemical Co., USA., and sildenafil citrate (99.4%) was kind gift from the ACI Ltd., Bangladesh.

All fluorescence spectra were recorded on fluorescence spectrophotometer (Model: F-7000, Hitachi, Japan) equipped with 1.0 cm quartz cell. For different temperatures a thermostat bath (Unitronic Orbital, P-Spectra, Spain) was used.

Five mL of previously prepared 20 × 10⁻⁶ mol∙L⁻¹ BSA in phosphate buffer of pH 7.4 was taken in each of the eight test tubes. Sildenafil citrate was added in different volumes to seven out of eight test tubes to have the following concentrations: (20, 40, 80, 120, 160, 240 and 320) × 10⁻⁶ mol∙L⁻¹, respectively. The ratio of SC and BSA ([SC]/[BSA]) in BSA-SC system of seven test tubes were 1:1, 2:1, 4:1, 6:1, 8:1, 12:1 and 16:1, respectively. The mixture solutions of BSA and SC must be hatched at least 5 min before the spectroscopic measurements.

The fluorescence emission spectra for BSA-SC system were recorded at the two excitation wavelengths of BSA (280 nm and 293 nm) at two different temperatures (298 K and 308 K). The widths of both entrance and exit slit were set to 5 nm. These emission spectra were recorded for three times for each treatment in the range of 320 - 460 nm for BSA at same experimental conditions since there were no emission spectra of SC in this range.

When BSA is excited by appropriate wavelength of light, all of its fluorophores (tryptophan, tyrosine and phenylalanine) can emit fluorescence. When 280 nm excitation wavelength is used, fluorescence of albumin comes from both tryptophan and tyrosine residues, whereas 293 nm wavelength only excites tryptophan residues [16] . It was compared the fluorescence of BSA excited at 280 nm and 293 nm in the presence of SC that would be determined the interactions residues of BSA with SC. The plots F/F_o against [SC]/[BSA] at excitation wavelengths 280 nm and 293 nm were compared at 298 K, respectively. Here, F_o is the fluorescence intensity of BSA, F is the fluorescence intensity of BSA in presence of SC.

Figure 2 indicates that the fluorescence of BSA excited at 280 nm obviously differed from that excited at 293 nm in the presence of SC. This difference between quenching of serum albumin fluorescence showed that the both tyrosine and tryptophan residues participated in the molecular interactions between BSA and SC.

In order to determine the effect of SC with BSA, the fluorescence emission spectra were measured at two excitation wavelengths of BSA (280 nm and 293 nm) at two different temperatures (298 K and 308 K).

Figure 3 shows the fluorescence of BSA gradually decreased with the increasing concentration of SC, indicating that there was a strong interaction and energy transfer between SC and BSA at the both excitation wavelengths of BSA (λEx_max of BSA = 280 nm and 293 nm) at two different temperatures (298 K and 308 K). As a result, there were quenching of intrinsic fluorescence of BSA but no significant shift of the emission maximum wavelength was observed.

Quenching refers to any process which decreases the fluorescence intensity of a given substance (fluorophore)

induced by a variety of molecular interactions with quencher molecule [17] . A variety of processes can result in quenching, such as excited state reactions, energy transfer, complex-formation and collisional quenching. Formation of complex between quencher and the fluorophore refers to static quenching. On the other hand, collision of the quencher and fluorophore during the excitation refers to dynamic quenching [18] . The fluorescence quenching data are usually analyzed by Stern-Volmer equation [7] .

where, F_o and F are the fluorescence intensities in the absence and presence of quencher, [Q] is the quencher concentration and Ksv is the Stern-Volmer quenching constant which indicates the strength of interaction between albumin protein and quencher molecule. Hence, this equation was applied to determine Ksv by linear regression of a plot of F_o/F against [Q]. The static quenching distinguished from dynamic quenching by their differing dependence of temperature [7] . Dynamic quenching depends upon diffusion and higher temperatures result in larger diffusion coefficients. As a result, the Stern-Volmer quenching constants (Ksv) were expected to increase with increasing temperature. In contrast, increased temperature is likely to result in decreasing stability of complexes, and thus lower value of static quenching constants [19] .

The pattern of quenching of BSA fluorescence by SC was determined by measuring the value of Stern- Volmer quenching constant (Ksv) at the excitation wavelength of BSA (280 nm and 293 nm) at two different temperatures (298 K and 308 K). Ksv was calculated from the slope of the plot of F/F_o versus concentration of SC based on the fluorescence data (Figure 4) at the conditions.

Figure 4 displays the Stern-Volmer plots of the quenching of BSA fluorescence by SC at two excitation wavelength of BSA (280 nm and 293 nm) at two different temperatures (298 K and 308 K). The plots showed that within the experimental concentrations, the results were good agreement with the Stern-Volmer equation. The plots were linear and Stern-Volmer quenching constants were obtained from the slopes at two different temperatures; these are mentioned in Table 1. The Stern-Volmer quenching constant decreased with increasing temperature for static quenching while for dynamic quenching the reverse effect was observed [20] . It was seen from the Table 1 that the Ksv decreased by increasing temperature at 280 nm but increased by increasing temperature at 293 nm. So it was observed that both dynamic and static quenching were present of BSA by SC at two different temperatures.

There are many interaction forces (e.g. hydrophobic force, electrostatic interactions, Vander Waals interactions,

hydrogen bonds, etc.) between quencher and fluorescence active molecule [10] . The thermodynamic parameters were calculated in order to elucidate the interaction between the drug and BSA, which can be determined from the Van’t Hoff equation:

where, ∆S = entropy change, ∆H = enthalpy change, R = universal gas constant and Ka = analogous to the Stern-Volmer quenching constants Ksv at the corresponding temperature [21] .

The enthalpy change (ΔH) and the entropy change (ΔS) can be determined from the slope and intercept of the fitted curve of lnKsv against 1/T, respectively (Figure 5). The free energy, ∆G can be estimated from the following relationship:

Table 2 shows that ΔS was a positive value, and ΔH was a small negative value. The negative value of ΔH reveals that the formation of BSA-SC complex was an exothermic reaction. Moreover, the negative sign for ΔG indicates the spontaneity of the binding process of SC with BSA. According to the views of Ross and Subramanian [22] , the model of interaction between drug and biomolecule can be summarized as follows: 1) the positive ΔS value is frequently regarded as the evidence for a hydrophobic interaction [23] because the water molecules arranged in an orderly fashion around the drug and protein establish a more random configuration; 2) the negative value of ΔH can be obtained whenever there is a possibility of hydrogen bonding [22] . Thus both hydrogen bonding and hydrophobic interactions were present in the SC-BSA binding at 280 nm at both temperatures.

When sildenafil citrate binds independently to a set of equivalent sites on BSA, the equilibrium between free and bound sildenafil citrate is given by the following equation [24]

where, K = binding constant to site of albumin, n = number of binding sites for drug per albumin.

The values of K and n are calculated from the values of intercept and slope of the plot of versus log[Q].

Table 3 contains the values of binding constant (K) and binding number (n), at two excitation wavelength of BSA (280 nm and 293 nm) which were obtained from the intercept and slope of Figure 6. It was observed that

the binding constant decreases with the increase in temperature of the BSA-SC complex resulting in the reduction of stability of the complex. The values of n were found to be ≈1 at both excitation wavelength of BSA at two different temperatures. The molar ratio of the BSA-SC system at 280 nm and 293 nm was 1:1 indicated that one mole SC bound with 1 mole of BSA.