In vitro interaction of sildenafil citrate (SC) with bovine serum albumin (BSA) was investigated at two excitation wavelengths of BSA (280 nm and 293 nm) at two different temperatures (298 K and 308 K) by fluorescence emission spectroscopy. The study showed that quenching of BSA fluores-cence by sildenafil citrate was the result of formation BSA-SC complex with probable involvement of both tryptophan and tyrosine residues of BSA. Fluorescence quenching constant was determined from Stern-Volmer equation, and both static quenching and dynamic quenching were showed for BSA by SC at the conditions. Van’t Hoff equation was used to measure the thermodynamic parameters ΔG, ΔH, and ΔS at the temperatures which indicated that the hydrogen bond and the hydrophobic forces played major roles for BSA-SC complexation. The binding number (n) was found to be ≈1 indicating that one mole BSA bound with one mole SC. The binding affinity of SC to BSA was calculated at different temperatures. The binding constant was decreased with increasing temperatures indicating that stability of BSA-SC complex decreased with increasing temperatures.
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 [
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).
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 [
where, Fo 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 Fo/F against [Q]. The static quenching distinguished from dynamic quenching by their differing dependence of temperature [
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/Fo versus concentration of SC based on the fluorescence data (
There are many interaction forces (e.g. hydrophobic force, electrostatic interactions, Vander Waals interactions,
T (K) | Ksv (×103 L∙mol−1) at 280 nm | Ksv (×103 L∙mol−1) at 293 nm |
---|---|---|
298 | 10.2 | 9.7 |
308 | 9.5 | 11.0 |
hydrogen bonds, etc.) between quencher and fluorescence active molecule [
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 [
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 (
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 [
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
T (K) | ∆H (KJ/mol) | ∆S (J/mol) | ∆G (KJ/mol) |
---|---|---|---|
298 | −5.89 | 57.01 | −22.87 |
308 | −23.44 |
T (K) | K (×103 mol∙L−1) at 280 nm | n | K (×103 mol∙L−1) at 293 nm | n |
---|---|---|---|---|
298 | 14.32 | 0.9411 | 6.22 | 1.064 |
308 | 12.37 | 5.62 |
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.
Drug-drug or drug-protein interactions produce an increase or a decrease in the therapeutic action, or produce various adverse effects that are not normally associated with the drugs [