A Comparative Study on the Interaction of Sulfonamide and Nanosulfonamide with Human Serum Albumin

Binding parameters of theN-phenyl benzene sulfonyl hydrazide, sulfonamide, andnanosulfonamide interactionwith human serum albumin were determined by calorimetry method. e obtained binding parameters indicated that sulfonamide in the second binding sites has higher affinity for binding than the �rst binding sites.e binding process of sulfonamide to HSA is both enthalpy and entropy driven. e associated equilibrium constants con�rm that sulfonamide binds to HSA with high affinity (2.2 × 10 and 3.8610M for �rst and second sets of binding sites, resp.). e obtained results indicate that sulfonamide increases the HSA antioxidant property. Nanosulfonamide has much more affinity for HSA (3.6 × 10 M) than sulfonamide.


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.e pharmacokinetic parameters of therapeutic drugs against the diseases show limitations in their efficacy.e 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.e advances that nanotechnology-based drug delivery systems have made in improving the pharmacokinetics and efficacy of therapeutic drugs [1][2][3][4].
Sulfonamides were the �rst 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].ey 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 SO 2 NH 2 moiety directly attached to a benzene ring.e term "sulfa allergy" is oen 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].e 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 in�uence 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

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 puri�cation.Sulfonamide derivative (N-phenyl benzene sulfonyl hydrazide) was synthesized.e isothermal titration microcalorimetric experiments were performed with the four-channel commercial microcalorimetric system.Sulfonamide and nanosulfonamide solutions (1612.9M) were injected by the use of a Hamilton syringe into the calorimetric titration vessel, which contained 1.8 mL HSA (60.22 M).Injection of sulfonamide solution into the perfusion vessel was repeated 29 times, with 10 L per injection.e calorimetric signal was measured by a digital voltmeter that was part of a computerized recording system.e heats of each injection was calculated by the "ermometric Digitam 3" soware program.e heat of dilution of the sulfonamide and nanosulfonamide solutions were measured as described above except HSA was excluded.e microcalorimeter was frequently calibrated electrically during the course of the study.

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][10][11][12][13][14]: where  are the heats of sulfonamide + HSA or nanosulfonamide + HSA interactions, and  max represents the heat value upon saturation of all HSA.e parameters    and    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.   or    indicate positive or negative cooperativity of a macromolecule for binding with a ligand, respectively;    indicates that the binding is noncooperative. ′  can be expressed as follows: where  ′  is the fraction of bound sulfonamide or nanosulfonamide to HSA, and  ′   − ′  is the fraction of unbound sulfonamide or nanosulfonamide.We can express   fractions, as the sulfonamide concentrations divided by the maximum concentration of the sulfonamide or nanosulfonamide upon saturation of all HSA as follows: where [sulfonamide] is the concentration of sulfonamide aer every injection, and [sulfonamide] max is the maximum concentration of the sulfonamide upon saturation of all HSA.  and   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,  dilut , as follows: e heats of sulfonamide + HSA interactions, , were �tted to (1) across the entire sulfonamide or nanosulfonamide compositions.In the �tting procedure,  was changed until the best agreement between the experimental and calculated data was approached (Figures 1 and 2).e high  2 value (0.999) supports the method.e binding parameters for sulfonamide + HSA interactions recovered from (1) were listed in Tables 1 and 2. e agreement between the calculated and experimental results (Figures 1 and 2) gives considerable support to the use of (1).   and    values for sulfonamide + HSA interactions are positive, indicating that in the low and high concentrations of the sulfonamide, the HSA structure is stabilized.ese results suggest that the antioxidant property of HSA increased.   indicates that the binding is noncooperative.
For a set of identical and independent binding sites, a plot of (Δ max ) [HSA] versus (Δ) [sulfon] should be a linear plot by a slope of  and the vertical-intercept of T 1: Binding parameters for HAS + sulfonamide interaction.e interaction is both enthalpy and entropy driven, but the electrostatic interactions are more important than hydrophobic forces.  values show that sulfonamide in the second class of binding sites has higher affinity for binding than the �rst class of binding sites.e positive values of    and    indicate that the antioxidant property of HSA increased as a result of its interaction with sulfonamide.

Parameters
First binding sites Second binding sites   /, through which  and   can be obtained [15][16][17][18][19] as follows: where  is the number of binding sites,   is the dissociation equilibrium constant, [HSA] and [sulfon] are the concentrations of HSA and sulfonamide or nanosulfonamide, respectively, Δ =  max − ,  represents the heat value at a certain ligand concentration and  max represents the heat value upon saturation of all HSA.If  and  max are calculated per mole of biomacromolecule, then the molar enthalpy of binding for each binding site (Δ) will be Δ =  max /.e best linear plots with the correlation coefficient value of 0.999 were obtained using amounts of −2670 and −5400 J (equal to −24.63, −49.81 kJ mol −1 ) for  max in the �rst and second binding sites, respectively.Dividing the  max amounts of −24.63 kJ mol To compare all thermodynamic parameters in metal binding process for HSA, the change in standard Gibbs free energy (Δ ∘ ) should be calculated according to (6), whose value can be used in (7) for calculating the change in standard entropy (Δ ∘ ) of binding process: where   is the association binding constant (the inverse of the dissociation binding constant,   ).e   values are obtained as 22.1 × 10 5 ± 250 and 3.86 × 10 5 ± 250 M −1 for the �rst and second binding sites, respectively.e results show that there are two sets of binding sites for sulfonamide.e 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 �rst class of binding sites and 4 sites in the second class of binding sites.  values show that sulfonamide in the second binding sites has higher affinity for binding than the �rst binding sites.
Energy of binding (Δ = −36.43kJ mol −1 ) for nanosulfonamide with HSA is more negative than that of sulfonamide.erefore, the energetic interaction between nanosulfonamide and HSA has become more favorable.e affinity of nanosulfonamide is roughly twice of sulfonamide, therefore reduces the drug dosage frequency, treatment time, and side effects.  values show that nanosulfonamide has higher affinity for binding with HSA than sulfonamide.e more effectiveness of nanosulfonamide can be attributed to its small size which result in reducing drug toxicity, controlling time release of the drug and modi�cation of drug pharmacokinetics and biological distribution.e positive    value (Table 2) shows that nanosulfonamide (in around 30 M of nanosulfonamide) stabilizes HSA structure and increases the anti-oxidant property of HSA.e negative    value indicates that nanosulfonamide dampened the anti-oxidant property of HSA in the high concentration domain (around 250 M of nanosulfonamide).

�on��ct of �nterests
ere is no con�ict of interest for any authors with ermometric Digitam 3 soware.
−1 by  = 1, and −49.81 kJ mol −1 by  = 4, T 2: Binding parameters for HAS + nanosulfonamide interactions.einteraction is both enthalpy and entropy driven indicating that the electrostatic interactions are dominant.valuesshow that nanosulfonamide has high affinity for binding to HSA. e positive value of    indicates that the antioxidant property of HSA increased as a result of its interaction with nanosulfonamide.enegative value proves that nanosulfonamide dampened the anti-oxidant property of HSA in the high concentration of nanosulfonamide. Δ = −24.63 for the �rst binding sites and Δ = −12.45kJ mol −1 for the second binding sites.