Binding parameters of the N-phenyl benzene sulfonyl hydrazide, sulfonamide, and nanosulfonamide interaction with human serum albumin were determined by calorimetry method. The obtained binding parameters indicated that sulfonamide in the second binding sites has higher affinity for binding than the first binding sites. The binding process of sulfonamide to HSA is both enthalpy and entropy driven. The associated equilibrium constants confirm that sulfonamide binds to HSA with high affinity (2.2×106
and 3.86105 M−1 for first and second sets of binding sites, resp.). The obtained results indicate that sulfonamide increases the HSA antioxidant property. Nanosulfonamide has much more affinity for HSA (3.6×106 M−1) than sulfonamide.
1. Introduction
Physicochemical properties of nanoparticles such as their small size, large surface area, surface charge, and ability to make them potential delivery systems for effective treatments. The pharmacokinetic parameters of therapeutic drugs against the diseases show limitations in their efficacy. The poor bioavailability, side effects due to the high doses administered, long treatment, and the emergence of drug resistant strains are the disadvantages of ordinary drugs. The advances that nanotechnology-based drug delivery systems have made in improving the pharmacokinetics and efficacy of therapeutic drugs [1–4].
Sulfonamides were the first chemical substances systematically used to treat and prevent bacterial infections in humans. Sulfonamides are bacteriostatic drugs; they work by inhibiting the growth and multiplication of bacteria without killing them. Currently, their most common use in humans is treating urinary tract infections [5]. They are estimated to be 16–21% of annual antibiotic usage, making them the most important group of antibiotics consumed by humans [6]. Sulfonamides are compounds that contain sulfur in a SO2NH2 moiety directly attached to a benzene ring. The term “sulfa allergy” is often incorrectly applied to all adverse reactions that occur with sulfonamide-containing medications and not just to those due to hypersensitivity mechanisms. Patients who experience side effects such as nausea and vomiting may interpret this as an allergy and subsequently report that they are allergic to sulfas [7]. The binding of the sulfonamides to serum albumins, an important factor of the pharmacokinetic of these drugs, has been extensively studied by several workers, especially regarding the extent of binding, the stoichiometry, and the influence of the chemical structure on the binding. But only little information is available on the mechanism of the binding and on the nature of the sulfonamide-albumin complex. Some workers have shown a correlation between the partition coefficients of the sulfonamides and the extent of the binding and concluded that the binding is mainly hydrophobic [8]. In this work, we compared the most comprehensive study on the interactions of sulfonamide and nanosulfonamide (N-phenyl benzene sulfonyl hydrazide) with HSA for further understanding of their effects on the stability and the structural changes of the HSA molecules.
2. Materials and Method
Human serum albumin (HSA; MW = 66411 gr/mol) and Tris buffer used were of analytical grade with the highest purity available without any purification. Sulfonamide derivative (N-phenyl benzene sulfonyl hydrazide) was synthesized. The isothermal titration microcalorimetric experiments were performed with the four-channel commercial microcalorimetric system. Sulfonamide and nanosulfonamide solutions (1612.9 μM) were injected by the use of a Hamilton syringe into the calorimetric titration vessel, which contained 1.8 mL HSA (60.22 μM). Injection of sulfonamide solution into the perfusion vessel was repeated 29 times, with 10 μL per injection. The calorimetric signal was measured by a digital voltmeter that was part of a computerized recording system. The heats of each injection was calculated by the “Thermometric Digitam 3” software program. The heat of dilution of the sulfonamide and nanosulfonamide solutions were measured as described above except HSA was excluded. The microcalorimeter was frequently calibrated electrically during the course of the study.
3. Results and Discussion
We have shown previously that the heats of the ligand + HSA interactions in the aqueous solvent mixtures, can be calculated via the following equation [9–14]:
(1)q=qmaxxB′-δAθ(xA′LA+xB′LB)-(δBθ-δAθ)(xA′LA+xB′LB)xB′,
where q are the heats of sulfonamide + HSA or nanosulfonamide + HSA interactions, and qmax represents the heat value upon saturation of all HSA. The parameters δAθ and δBθ are the indexes of HSA stability in the low and high sulfonamide concentrations, respectively. Cooperative binding requires that the macromolecule has more than one binding site, since cooperativity results from the interactions between identical binding sites with the same ligand. If the binding of a ligand at one site increases the affinity for that ligand at another site, then the macromolecule exhibits positive cooperativity. Conversely, if the binding of a ligand at one site lowers the affinity for that ligand at another site, then the enzyme exhibits negative cooperativity. If the ligand binds at each site independently, then the binding is noncooperative. p>1 or p<1 indicate positive or negative cooperativity of a macromolecule for binding with a ligand, respectively; p=1 indicates that the binding is noncooperative. x′B can be expressed as follows:
(2)xB′=pxBxA+pxB,
where xB′ is the fraction of bound sulfonamide or nanosulfonamide to HSA, and xA′=1-xB′ is the fraction of unbound sulfonamide or nanosulfonamide. We can express xB fractions, as the sulfonamide concentrations divided by the maximum concentration of the sulfonamide or nanosulfonamide upon saturation of all HSA as follows:
(3)xB=[sulfonamide][sulfonamide]max,xA=1-xB,
where [sulfonamide] is the concentration of sulfonamide after every injection, and [sulfonamide]max is the maximum concentration of the sulfonamide upon saturation of all HSA. LA and LB are the relative contributions of unbound and bound sulfonamide in the heats of dilution in the absence of HSA and can be calculated from the heats of dilution of sulfonamide or nanosulfonamide in buffer, qdilut, as follows:
(4)LA=qdilut+xB(∂qdilut∂xB),LB=qdilut-xA(∂qdilut∂xB).
The heats of sulfonamide + HSA interactions, q, were fitted to (1) across the entire sulfonamide or nanosulfonamide compositions. In the fitting procedure, p was changed until the best agreement between the experimental and calculated data was approached (Figures 1 and 2). The high r2 value (0.999) supports the method. The binding parameters for sulfonamide + HSA interactions recovered from (1) were listed in Tables 1 and 2. The agreement between the calculated and experimental results (Figures 1 and 2) gives considerable support to the use of (1). δAθ and δBθ values for sulfonamide + HSA interactions are positive, indicating that in the low and high concentrations of the sulfonamide, the HSA structure is stabilized. These results suggest that the antioxidant property of HSA increased. p=1 indicates that the binding is noncooperative.
Binding parameters for HAS + sulfonamide interaction. The interaction is both enthalpy and entropy driven, but the electrostatic interactions are more important than hydrophobic forces. Ka values show that sulfonamide in the second class of binding sites has higher affinity for binding than the first class of binding sites. The positive values of δAθ and δBθ indicate that the antioxidant property of HSA increased as a result of its interaction with sulfonamide.
Parameters
First binding sites
Second binding sites
p
1
1
g
1
4
Ka/L·mol−1
2.2×106±250
3.86×105±750
ΔH/kJ mol-1
-24.63±0.08
-12.45±0.06
ΔG/kJ mol-1
-30.57±0.08
-32.09±0.11
ΔS/kJ mol-1K-1
0.02±0.003
0.06±0.005
δAθ
4.86
δBθ
4.76
Binding parameters for HAS + nanosulfonamide interactions. The interaction is both enthalpy and entropy driven indicating that the electrostatic interactions are dominant. Ka values show that nanosulfonamide has high affinity for binding to HSA. The positive value of δAθ indicates that the antioxidant property of HSA increased as a result of its interaction with nanosulfonamide. The negative δBθ value proves that nanosulfonamide dampened the anti-oxidant property of HSA in the high concentration of nanosulfonamide.
Parameters
p
1
g
1
Ka/L·mol−1
3.6×106±650
ΔH/kJ mol-1
-36.43±0.12
ΔG/kJ mol-1
-37.63±0.15
ΔS/kJ mol-1K-1
0.004±0.001
δAθ
2.65±0.06
δBθ
-38.14±0.09
Comparison between the experimental heats (▲) at 300 K, for (nanosulfonamide + HSA) interactions and the calculated data (lines) via (1).
Comparison between the experimental heats (∆) at 300 K, for (sulfonamide + HSA) interactions and the calculated data (lines) via (1).
For a set of identical and independent binding sites, a plot of (Δq/qmax) [HSA] versus (Δq/q) [sulfon] should be a linear plot by a slope of 1/g and the vertical-intercept of Kd/g, through which g and Kd can be obtained [15–19] as follows:
(5)Δqqmax[HSA]=(Δqq)[sulfon]1g-Kdg,
where g is the number of binding sites, Kd is the dissociation equilibrium constant, [HSA] and [sulfon] are the concentrations of HSA and sulfonamide or nanosulfonamide, respectively, Δq=qmax-q, q represents the heat value at a certain ligand concentration and qmax represents the heat value upon saturation of all HSA. If q and qmax are calculated per mole of biomacromolecule, then the molar enthalpy of binding for each binding site (ΔH) will be ΔH=qmax/g. The best linear plots with the correlation coefficient value of 0.999 were obtained using amounts of −2670 and −5400 μJ (equal to −24.63, −49.81 kJ mol−1) for qmax in the first and second binding sites, respectively. Dividing the qmax amounts of −24.63 k kJ mol−1 by g=1, and −49.81 k kJ mol−1 by g=4, thus, gives ΔH=-24.63 for the first binding sites and ΔH=-12.45 k kJ mol−1 for the second binding sites.
To compare all thermodynamic parameters in metal binding process for HSA, the change in standard Gibbs free energy (ΔG°) should be calculated according to (6), whose value can be used in (7) for calculating the change in standard entropy (ΔS°) of binding process:
(6)ΔG°=-RTlnKa(7)ΔG°=ΔH°-TΔS°,
where Ka is the association binding constant (the inverse of the dissociation binding constant, Kd). The Ka values are obtained as 22.1×105±250and3.86×105±250M-1 for the first and second binding sites, respectively.
The results show that there are two sets of binding sites for sulfonamide. The interaction is both enthalpy and entropy driven, but the electrostatic interactions are more important than hydrophobic forces. It was found that there is 1 site in the first class of binding sites and 4 sites in the second class of binding sites. Ka values show that sulfonamide in the second binding sites has higher affinity for binding than the first binding sites.
Energy of binding (ΔH=-36.43kJmol-1) for nanosulfonamide with HSA is more negative than that of sulfonamide. Therefore, the energetic interaction between nanosulfonamide and HSA has become more favorable. The affinity of nanosulfonamide is roughly twice of sulfonamide, therefore reduces the drug dosage frequency, treatment time, and side effects. Ka values show that nanosulfonamide has higher affinity for binding with HSA than sulfonamide. The more effectiveness of nanosulfonamide can be attributed to its small size which result in reducing drug toxicity, controlling time release of the drug and modification of drug pharmacokinetics and biological distribution. The positive δAθ value (Table 2) shows that nanosulfonamide (in around 30 μM of nanosulfonamide) stabilizes HSA structure and increases the anti-oxidant property of HSA. The negative δBθ value indicates that nanosulfonamide dampened the anti-oxidant property of HSA in the high concentration domain (around 250 μM of nanosulfonamide).
Conflict of Interests
There is no conflict of interest for any authors with Thermometric Digitam 3 software.
Acknowledgment
The financial support of Imam Khomeini International University is gratefully acknowledged.
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