Accepted Manuscript Interaction of sulfanilamide and sulfamethoxazole with bovine serum albumin and adenine: Spectroscopic and molecular docking investigations N. Rajendiran, J. Thulasidhasan PII: DOI: Reference:

S1386-1425(15)00201-2 http://dx.doi.org/10.1016/j.saa.2015.01.127 SAA 13339

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

26 September 2014 9 January 2015 29 January 2015

Please cite this article as: N. Rajendiran, J. Thulasidhasan, Interaction of sulfanilamide and sulfamethoxazole with bovine serum albumin and adenine: Spectroscopic and molecular docking investigations, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.01.127

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Interaction of sulfanilamide and sulfamethoxazole with bovine serum albumin and adenine: Spectroscopic and molecular docking investigations N. Rajendiran* and J. Thulasidhasan Department of Chemistry, Annamalai University, Annamalai Nagar 608 002, Tamilnadu India *Corresponding author: [email protected] Tel.: +91 94866 28800 ABSTRACT Interaction between sulfanilamide (SAM) and sulfamethoxazole (SMO) with BSA and DNA base (adenine) was investigated by UV–visible, fluorescence, cyclic voltammetry and molecular docking studies. Stern–Volmer fluorescence quenching constant (Ka) suggests SMO is more quenched with BSA/adenine than that of SAM. The distance r between donor (BSA/adenine) and acceptor (SAM and SMO) was obtained according to fluorescence resonance energy transfer (FRET). The results showed that hydrophobic forces, electrostatic interactions, and hydrogen bonds played vital roles in the SAM and SMO with BSA/adenine binding interaction. During the interaction, sulfa drugs could insert into the hydrophobic pocket, where the non-radioactive energy transfer from BSA/adenine to sulfa drugs occurred with high possibility. Cyclic voltammetry results suggested that when the drug concentration is increased, the anodic electrode potential deceased. The docking method indicates aniline group is interacted with the BSA molecules.

Keywords: Sulfanilamide; Sulfamethoxazole; Bovine serum albumin; Fluorescence quenching; Cyclic voltammetry; Molecular docking.

Introduction Serum albumin is the major protein constituent of blood plasma which facilitates the disposition and transport of various exogenous and endogenous drugs to the specific targets [1]. The specific delivery of drugs by serum albumin originates from the presence of two major and structurally selective binding sites, namely, site I and site II, which are located in three homologous domains that form a heart-shaped protein [2]. Bovine serum albumin (BSA) is a single-chain 582 amino acid globular non-glycoprotein cross-linked with 17 cystine residues (eight disulfide bonds and one free thiol) [3–5]. BSA has two tryptophans, Trp-134 and Trp- 212, embedded in the first sub-domain IB and sub-domain IIA, respectively [6]. The binding affinity offered by site I is mainly through hydrophobic interactions, whereas site II involves a combination of hydrophobic, hydrogen bonding, and electrostatic interactions [7]. Due to its structural homology (80%) with human serum albumin, water solubility and versatile binding ability, BSA has been used as a model protein for a great variety of biophysical and physicochemical studies. Adenine is one of the purine nucleobases and it is an essential molecule of life and evolution. Adenine has tremendous biological significance since it is one of the nitrogeneous bases found on deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) to make up genetic information. It is a component of adenosine triphosphate (ATP) which is major energy releasing molecule in cells. Adenine is also a part of coenzymes and being a part of nucleic acid, it has a central role in protein synthesis [8]. Among the many and different families of organic and inorganic chemicals being currently investigated because of their applications, sulfonamides and their N-derivatives are one of the outstanding groups. Sulfonamides were the first effective chemotherapeutic agents employed systematically for the prevention and cure of bacterial infections in humans. After the introduction of penicillin and other antibiotics, the popularity of sulfonamides decreased. However, they are still considered useful in certain therapeutic fields, especially in the case of

ophthalmic infections as well as infections in the urinary and gastrointestinal tract. Besides, sulfa drugs are still today among the drugs of first election (together with ampicillin and gentamycin) as chemotherapeutic agents in bacterial infections by Escherichia coli in humans. The sulfanilamides exert their antibacterial action by the competitive inhibition of the

enzyme

dihydropterase

synthetase

towards

the

substrate

p-aminobenzoate.

Sulphonamides belong to the group of antibacterial drugs which are used for human and animal therapy, to cure infectious diseases of digestive and respiratory systems, infections of the skin (in the form of ointments) and for prevention or therapy of coccidiosis of small domestic animals [9]. Quality control of sulphonamide formulations and their quick systematic monitoring in body fluids are important analytical tasks. A number of articles have been published concerning the determination of sulphonamides by different analytical methods. Sulfamethoxazole (SMO) is widely used as an antibacterial, mainly in combination with trimethroprim. This is a well-recognized preparation, as the combination of a sulphonamide with an inhibitor of the dihydropholate reductase increases the bacteriostatic effect of the sulphonamide, by blocking the metabolic pathways of the microorganisms at two different points. Different methods have been described for determining the sulphonamides, but, in general, they are based on separation methods using different detection types. Its determination in urine has been made by micellar liquid chromatography and by supported liquid membrane with high pressure liquid chromatography-electrospray mass spectrometry detection [10]. The fluorescence characteristics of different sulphonamides have been studied [11] and proposed for the determination of several of these compounds. Thus, sulphanilamide can be determined by reaction with homophthaldehyde. The analysis of sulfa drugs [12, 13] has been performed in foods and pharmaceuticals using the fluorescamine reaction. The reaction of 9-cloroacridine with sulphonamides produces a fluorescence quenching which allows the determination of sulphonamides [14]. Sulfa drugs were determined in milk and

pharmaceutical preparations by photochemically-induced fluorometry [15]. Fluorescence has also been used as HPLC detection for determining sulfa drugs in milk and eggs [16] and recently, fluorescence after pre-column derivatization with fluorescamine has been applied as a detection technique [16]. In our previous studies, the sequences of sulfonamide derivatives with cyclodextrins were investigated through experimental [17-23] and theoretical methods [24]. In the view of increasing attention directed toward the importance of investigating interaction between proteins and drugs, herein we report the interaction behavior of sulfanilamide (SAM, 4aminobenzene sulfonamide) and sulfamethoxazole (SMO, N1-(5-methyl-3-isoxazolyl) sulfanilamide) (Fig. 1) with BSA and adenine. We utilized absorption and fluorescence spectral techniques to determine the interaction stoichiometry, quenching mechanism, binding constant (Ka) and number of binding sites (n) of SAM/SMO-BSA/adenine complex. We report combined results of experimental and molecular docking methods on the protein – drugs complexes of SAM/SMO- BSA/adenine. Sulfa drugs are an easily available drug and have been a strong research field in life sciences, chemistry and clinical medicine. Hence, we are interested in investigating the interaction mechanism of sulfa drugs with BSA/adenine in solution and in computational methods. Experimental Section Materials Sulfanilamide (SAM), sulfamethoxazole (SMO), adenine and bovine serum albumin (BSA) were obtained from Sigma-Aldrich Chemical Company, USA and used without further purification. All other reagents were of analytical grade. The purity of the compound was checked by similar fluorescence spectra when excited with different wavelengths. Triply distilled water was used for the preparation of aqueous solutions. BSA solutions were prepared in 2 × 10-5 M Tris HCl buffered at pH 7.4. The concentration of the sulfa drug solutions was varied from 2 × 10-4 to 1×10-5 M. 0.2 mL of sulfa drug in methanol solution

was used for all binding experiments. All solutions were stored in a refrigerator at 4 °C in the dark. Apparatus Absorption

spectral

measurements

were

carried

out

with

a

UV-visible

spectrophotometer (model-UV-2600 Shimadzu, Japan) and fluorescence measurements were performed on a spectrofluorophotometer (model-RF-5301PC, Shimadzu, Japan) equipped with 1.0 cm quartz cells. The excitation wavelength for all the sulfa drugs is 280 nm. Cyclic voltammetry measurements were performed through an electrochemical work station (modelCHI 620D, CH Instruments, USA) with a three electrode system: surface area 0.1963 cm2 glassy carbon electrode as working electrode, saturated silver electrode as reference electrode and a platinum coil as counter electrode. Prior to use, the working electrode was polished with 0.05 µm alumina and thoroughly washed in an ultrasonic bath for 5 min. Before experiments, the solution within a single-compartment cell was deaerated by purging with pure N2 gas for 5 min. The pH values in the range 2.5-11.5 were measured on Elico pH meter model

LI-120.

Docking

calculations

were

carried

out

using

Docking

server

(http://www.dockingserver.com) [25]. The MMFF94 force field was used for energy minimization of drug molecule (SAM and SMO) using Docking Server [26]. Gasteiger partial charges were added to the drug atoms. Non-polar hydrogen atoms were merged, and rotatable bonds were defined. Docking calculations were carried out on 4F5S protein model. Essential hydrogen atoms, Kollman united atom type charges, and solvation parameters were added with the aid of Auto Dock tools [27]. Affinity (grid) maps of 20×20×20 Å grid points and 0.375 Å spacing were generated using the Auto grid program [27]. Auto Dock parameter set- and distance-dependent dielectric functions were used in the calculation of the van der Waals and the electrostatic terms, respectively. Docking simulations were performed using the Lamarckian genetic algorithm (LGA) and the Solis & Wets local search method. Initial position, orientation, and torsions of the ligand molecules were set randomly. Each docking

experiment was derived from 10 different runs that were set to terminate after a maximum of 250000 energy evaluations. The population size was set to 150. During the search, a translational step of 0.2 Å, and quaternion and torsion steps of 5 were applied. Results and Discussion UV-vis absorption spectra studies UV-visible absorption spectroscopy technique can be used to explore the structural changes of protein and to investigate protein-drug interaction [28, 29] Table S1, Fig. 2 and Fig. S1 (supporting file) depicts the absorption maxima, log ε and fluorescence maxima of BSA and adenine with different concentrations of SMO and SAM. The insert in the Fig. 2 and Fig. S1 depict the changes of the absorbance and fluorescence intensities were observed as a function of the drug concentrations. With on increasing the drug concentrations, absorbance of the BSA and adenine was decreased at the same wavelength. The spectral shifts of both drugs with adenine/ BSA are similar to other. When the drug concentrations increased, the absorbance of BSA and adenine was decreased at the same absorption maxima. The decrease in absorbance is due to the interaction of both drug molecules into the BSA and adenine. The absorption maximum of the pure adenine appears at 260 nm. BSA has weak absorption maximum appears at 279 nm to be due to the aromatic amino acids (Trp, Tyr, and Phe). These findings were in accordance with those of other studies [30–33]. With gradual addition of SAM and SMO in to the adenine/BSA solution, the absorbance of the adenine (~ 260 nm) and BSA (~ 279 nm) decreased at the same wavelength. A clear isosbestic point observed in the sulfa drug molecules and the changes that observed in the absorbance are very small. The results indicate that the interaction between SAM / SMO with adenine/ BSA leads to the loosening and unfolding of the protein skeleton and increases the hydrophobicity of the microenvironment of the aromatic amino acid residues. Collectively, the fluorescence quenching of BSA by drug was caused by the static quenching, which supported the BSA absorption spectral shifts [34].

Fluorescence spectra of BSA solutions with SAM and SMO Fluorescence spectra were recorded in the wavelength range of 290–500 nm setting the excitation at 280 nm, and excitation and emission bandwidths at 5 nm. The fluorescence spectra of adenine and BSA with different concentrations of sulfa drugs were determined and have been shown in Fig. 2 and Fig. S1. Both adenine and BSA fluorescence intensities decreased remarkably as the sulfa drugs concentrations increased and no significant spectral shift observed upon adenine-sulfa drug and BSA-sulfa drugs interaction. These spectral changes indicate the formation of sulfa drugs–adenine/BSA complex. It is well known that most of protein molecules naturally fluoresce around 350 nm (under 290 nm wavelength excitation), which mainly attributes to their fluorescent Trp residues [34]. In Fig 2 and Fig. S1, the fluorescence intensity of adenine/BSA was gradually decreased and the fluorescence maxima showed by increasing the sulfa drugs concentration. Apparently, this quenching is owing to the interaction of SAM and SMO with adenine/BSA molecules. Thus the microenvironment of fluorescence chromospheres and conformation of BSA was changed. Quenching mechanism analysis There are two main quenching types in characterizing the binding of drug with protein: static and dynamic quenching. Static quenching refers to the formation of a non-fluorescent fluorophore– quencher complex. Dynamic quenching refers to the quencher diffused to the fluorophore during the lifetime of the exited state and upon contact, the fluorophore returns to ground state, without emission of a photon [35]. The fluorescence quenching behavior of the protein and the Stern–Volmer analysis of the relative fluorescence intensity (F0/F) as a function of quencher concentration [Q] at different drugs were employed to elucidate the quenching mechanism and rates. As a hypothetical dynamic quenching process, the quenching rate constants were determined by using the Stern–Volmer equation [36]: F0/F= 1 + KSV [Q] =1+ kqτ0[Q]

(1)

where F and F0 are the fluorescence intensity in the presence and absence of sulfa drugs, respectively, kq the quenching rate constant, KSV the Stern–Volmer constant, τ0 the

fluorescence lifetime of BSA in the absence of sulfa drugs and [Q] is the concentration of quencher (SAM and SMO). KSV = kqτ0

(2)

Figs. 3 to 5 and Figs. S2 and S3 display the Stern–Volmer plots of adenine/BSA solutions in the presence of sulfa drugs with various concentrations. As can be seen from Fig S2, S3 and Figs. 3 to 5, the Stern–Volmer plots are linear and the slope decreases with SMO drug, which indicates that the quenching process is static. In general, the fluorescence lifetime τ0 of BSA was reported approximately as 10-8 s [37] and the KSV value obtained from the slope of the Stern–Volmer plot. By using Eq. (2), the quenching constants kq at sulfa drugs was determined as shown in Table 1. The obtained kq values are in the range of 1012 L mol-1 s-1, which far exceed the diffusion controlled rate constant in aqueous solution, i.e. 2 x 1010 L mol-1 s-1 [38], confirming that quenching does not involve the dynamic diffusion process but occurs statically in the complex. It can be found that both plots exhibit a comparatively good linear relationship. It indicates that the dominating quenching mechanism is not dynamic but static. Binding constants and number of binding sites For the static quenching interaction, when small molecules bind independently to a set of equivalent sites on a macromolecule, the binding constant (Ka) and the number of binding sites (n) can be determined [39] by the equation (3) log (F0 –F) / F = log Ka + n log [Q]

(3)

where F0, F, and [Q] are the same as in (Eq 1.) According to (Eq 2), values of n and Ka (Table 1) at physiological pH ~ 7.4 were calculated. The number of binding sites n approximately equals 1, indicating that there is one binding site in BSA for SAM or SMO during their interaction.

Ross and Subramanian [40] have used the sign and magnitude of the thermodynamic parameters to characterize the major interaction working in a variety of host - guest systems. Hence, the free energy change (∆G0) can be derived from Equation: ∆G0 = - RT ln K

(4)

where, K is the binding constant at a corresponding sulfa drugs; R is the gas constant; and T is the absolute temperature. Meanwhile, the negative value of ∆G0 indicated the spontaneity of the binding between sulfa drugs and BSA. Energy transfer between SAM and SMO with BSA According to the Forster’s dipole-dipole non-radiative energy transfer theory [41], energy transfer from one molecule (donor) to another molecule (acceptor) will happen under the following conditions: (a) the energy donor can produce fluorescence; (b) the absorption spectrum of the receptor sufficiently overlaps with the donor’s fluorescence emission spectrum; (c) the distance between the donor and the acceptor is < 8 nm [42, 43]. The following equation can be used to calculate the efficiency (E) of energy transfer between the donor and acceptor. E = 1- F / F0 = R0 / (R06 + r6)

(5)

In (Eq 5), r is the distance between the donor and acceptor and R0 is the critical distance when the transfer efficiency is 50%, which can be calculated by the equation R06 = 8.79 x10-25 K2 n-4 φ J

(6)

where K2 is the orientation factor related to the geometry of the donor-acceptor dipole, n is the refractive index of the medium, φ is the fluorescence quantum yield of the donor, and J expresses the degree of spectral overlap between the donor emission and the acceptor absorption, which can be calculated by the equation (7)

J =

∫o∞ F(λ) ε(λ) (λ)4 dλ

∫o∞ F(λ) dλ

(7)

where F(λ) is the fluorescence intensity of the donor at wavelength range λ and ε(λ) is the molar absorption coefficient of the acceptor at wavelength λ. Here the donor and acceptor were BSA and sulfa drugs, respectively. J can be evaluated by integrating the spectra according to (Eq 6). For BSA, K2 = 2/3, n = 1.336, and Φ = 0.15 (5). According to Eqs (5-7), the following parameters are obtained for BSA – SAM and SMO respectively: J = 2.09 and 1.68 x10-14 cm3 L mol-1, R0 = 0.88 and 0.68 nm, E = 0.24 and 0.22, r =0.63 and 0.73 nm. (Table S1) The donor (tryptophan residues of the BSA) to acceptor (SAM and SMO) distance was < 8 nm, indicating that the energy transfer from BSA to sulfa drugs occurred with high probability. However, the most important aspect that should be noted is that the distance calculated here is actually the average value between the bound sulfa drugs and the two tryptophan residues in BSA [42]. The results were in accordance with conditions of Forster theory of nonradioactive energy transfer and indicated again a static quenching between SAM and SMO with adenine/BSA. Cyclic voltammetry studies Table 2 and Fig S4 depict the cyclic voltammetry data and anodic and cathodic peaks of adenine and BSA with varying concentration of SAM and SMO. BSA did not exhibit any observable reduction peak at a glassy carbon electrode (pH 7.4) [44]. When both sulfa drugs (SAM or SMO) were added to BSA/adenine solution, a reduction peak appeared and the redox peaks shifted towards high and low potentials, respectively, and a decrease of oxidation current was observed. The electrochemical reduction process is due to disulfide (S-S) bonds in BSA reduced to -SH groups and the tertiary structure of the molecule completely collapse. The oxidation peak shifted towards lower potentials and an increasing oxidation current was observed. This shift may be attributed to the changes of the molecular environment around the SAM or SMO molecules as a result of its interaction with BSA or adenine [45]. This observation is consistent with the view that the drug–BSA or drug–adenine interaction occurred between the most hydrophobic segments of the drug molecule [46] and the

hydrophobic region of the BSA cavity. These results indicated the interaction between SAM or SMO and BSA occurred and a new electrochemically inactive SAM or SMO–BSA complex formation lowered free SAM or SMO concentration, and thus decreased the peak current. The diffusion coefficient of the sulfa drugs with adenine and BSA was calculated using Randles – Seveik equation Ip = 2.69 × 105 n3/2 A C*0 D0 1/2 ν1/2

(8)

where Ip is the peak current (in ampere), A is the surface area of the electrode (in cm 2) C0 is the bulk concentration of the electroactive species Do is the diffusion coefficient (in cm2 s–1) and ν is the scan rate (in Vs–1). The current charge (whether increase or decrease) is due to interaction of sulfa drugs with adenine or BSA. Hence the electrochemical reaction (oxidation or reduction) of the drug with adenine or drug-BSA drug–adenine was more difficult than pure sulfa drugs. The anodic peak potential (Epa) of SAM (1323 mV) and SMO (1341 mV) was changed with adenine/BSA (Table 2) the separation of anodic and cathodic peak potential (∆Ep) and the ratio Ipa/Ipc was significantly changed, indicating that this was a quasireversible, two-electron redox process. Epa was shifted to more positive potential revealed that electrochemical oxidation of sulfa drugs with adenine/BSA was more easy on increasing sulfa drugs concentration from 3 x 10 -5 to 1 x 10 -4, the difference between the oxidation and reduction potential (∆Ep) is changed in the SAM and SMO, ∆Ep is shifted that the electron transfer process moves towards reversible due to adenine/BSA interaction with SAM and SMO. Table 2 and Fig. S4 show the CV of sulfa drugs in absence and presence of adenine / BSA at glassy carbon electrode in Tris - HCl buffer (pH ~7). On the addition of adenine/BSA a decrease on anodic peak potential with significant shift in peak potential was observed. Two factors may be considered to decreasing of the oxidation peak potential. First one is the competitive adsorption between the sulfa drugs and adenine/BSA on GCE and second is the

formation of electroactive complex without changes of electrochemical parameters. The peak current of sulfa drugs did not disappear completely with the addition of adenine/BSA, which was not the character of competitive adsorption. The interaction of biomolecules like hemoglobin, BSA, HSA and DNA etc., showed that in such lower concentration of protein and shorter accumulation time, the coverage of electrode surface was only accounted for about 10% of the total area of electrode, so the competitive absorption between small molecules with protein can hardly exist [47, 48]. Thus the decrease or increase in the peak current without any changes in electrochemical parameters is a good indication of sulfa drugs with adenine and BSA. The CV of sulfa drugs with adenine/BSA in Tris- HCl buffer (pH ~7) solution at the glassy carbon electrode indicated that the anodic peak current decreases with increasing concentration of drugs, which means that the peak current had adsorption behavior. According to the method of Qu et al. [49], it is assumed that SAM and SMO with adenine/BSA only produced a single complex. The reaction scheme: BSA + m drug

BSA: drugm

(9)

f = [BSA: drugm] / BSA: drugsm] max = [drug] m / [(drug) m + Kdm)

(10)

and Kdm = [drug] m (1− f) / f

(11)

where [BSA: drugs m] max - is the maximum concentration of complexed binding sites, [drug] - is the concentration of free sulfa drugs, Kd - is the dissociation equilibrium constant and m is the Hill coefficient. Note that Kd = [drugs]0.5, i.e., at half occupation. The association constant Ka - is given by the reciprocity: Ka = Kd−1. It is not advisable to use the overall constant K = (Ka)m, which would have the physically meaningless dimension of M−m. The interaction partners for the sulfa drugs are the binding sites of BSA. Mass conservation dictates that: [BSA] = [BSA: drugm]max− [BSA: drugm]

(12)

[drug] = [drug]0 − m [BSA: drugm]

(13)

Where [drug]0 is the total concentration of sulfa drugs. According to the Ilkovic equation of irreversible electrode process [50]: I = k [drug]

(14)

where k is a constant. In line with this relationship the current difference ∆I is defined as: ∆I = k ([drug0] – [drug])

(15)

Insertion of Eq. (14) and (15) into Eq. (16) yields: ∆I = k [drug0] − [drug] = k m [BSA: drugm]

(16)

∆I max = k: m [BSA: drugm] max

(17)

From Eq. (10), we obtain that: log [f / (1− f ) = m log (Ka / M−1) + m log [drug] / M)

(18)

Insertion of Eq. (16) and (17) into Eq. (19) yields: log [∆I / [∆I max − ∆I] = log βs + m log [drug]

(19)

where ∆I is the peak current difference between the presence and absence of BSA and ∆Imax corresponds to the obtained value when the concentration of the drug is extremely higher than that of BSA. CBSA, [BSA], [BSA– mdrug] are corresponding to the total, free and bound concentration of protein in the solution, respectively. From Eq. (19), the relation of log [∆I/ (∆Imax-∆I)] with log [drug] was calculated and plotted in Fig. 6. From the intercept and slope m and βs were deduced, which indicated that SAM and SMO binding to BSA/adenine formed the complex of BSA/adenine–drug. 3.7. Docking analysis Molecular docking was employed to understand the interaction of BSA with sulfa drugs and to ultimately elucidate the interaction mechanism. The MMFF94 force field was used for energy minimization of drug molecules (SAM and SMO) using Docking Server. Gasteiger partial charges were added to the ligand atoms. Non-polar hydrogen atoms were merged, and rotatable bonds were defined. Docking calculations were carried out on 4F5S

protein model. The analysis showed that in SAM-BSA interaction the PHE506, PHE508, LYS524, ALA527, LEU528, LEU531, VAL546, PHE550, LEU574 and THR578 were the most vital residues present at the active site. Similarly in the SMO-BSA interaction the THR190, SER192, ALA193, ARG196, ARG435, TYR451, LEU454 and ARG458 THR578 were the most vital residues present at the active site. The possible interacting model and the main residues involved in the interaction have been depicted in Fig. 7. The drug core contact in with the protein was anchored in the binding site by H-bonds. The two nitrogen atoms at the N1, N2 and O2 positions form three H-bonds with the OH of THR190 (–N1…OH, 2.58 Å), of LEU454 (–N2…OH, 2.98 Å), and NH of ARG458 (–O2…NH, 3.37 Å) respectively (Fig. 8). Meanwhile, the sulfonyl oxygen atoms at the O1 and O3 position of sulfa drugs act as hydrogen acceptor to form H–bond with the group of ARG458 (–O…..HC, 2.04 Å, 105.7°). We found that the arene of the sulfa drugs formed an arene-cation through contact with the – NH2 of the LUE454 and ILE455. However, a series of hydrophobic residues, Ala341, Pro446, and Ala290, around the peripheral region of the molecule interacted with sulfa drugs through hydrophobic interactions. The human serum albumin (HSA) is homologous to BSA. The study of HSA inhibitor thyroxine indicated that its binding position was approximately the same as that of sulfa drugs. From the docking analysis, we could presume that the hydrophobic interactions and the polar contacts collectively constituted the primary force for the binding of the molecule. Conclusions The interaction between SAM and SMO with BSA/adenine was investigated in vitro using spectroscopic and molecular docking methods under physiological conditions. The results showed that hydrophobic forces, electrostatic interactions, and hydrogen bonds played vital roles in the SAM and SMO with BSA/adenine binding interaction, which was spontaneous. During the interaction, sulfa drugs could insert into the hydrophobic pocket, where the non-radioactive energy transfer from BSA/adenine to sulfa drugs occurred with

high possibility. The binding distance (r) between sulfa drugs and the BSA/adenine was calculated as ~1.64 nm (308 K). The results of cyclic voltammetry suggested that the drugs – BSA complex is not electroactive. The docking method provided a means to estimate the participation and interactions of specific chemical groups in the process of complex stabilization at the molecular level. In summary, this study may provide valuable insight into the binding mechanism of SAM and SMO with BSA/adenine, and could provide a better understanding of the drugs effect on protein function during the course of its transportation and distribution in the blood. Acknowledgments This work was supported by the Council of Scientific Industrial Research [No. 01(2549)/12/ EMR-II] and the University Grants Commission [F. No. 41-351/2012 (SR)], New Delhi, India. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at References [1] P. Qin, R. Liu, X. Pan, X. Fang, Y. Mou, J. Agric. Food Chem. 58 (2010) 5561–5567. [2] M. Dockal, D. C. Carter, F. J. Ruker, Biol. Chem. 274 (1999) 29303-29310. [3] D.C. Carter, J.X. Ho, Adv. Protein Chem. 45 (1994) 153–203. [4] K.F. Brown, M.J. Crooks, Biochem. Pharmacol. 25 (1976) 1175–1178. [5] K. Hirayama, S. Akashi, M. Furuya, K. Fukuhara, Biochem. Biophys. Res. Commun. 173 (1990) 639–646. [6] U.K. Hansen, Pharmacol. Rev. 33 (1981) 17–53. [7] M. C. Jimenez, M. A. Miranda, I. Vaya, J. Am. Chem. Soc. 127 (2005) 10134–10135. [8] Goyal Rajendra N, B. sunita, S. Rakesh K, Ind. J. Chem., 50A (2011) 1026-1034. [9] J. Marek, J. Marek, Pharmacotherapy of Internal Diseases, Grada Publishing, Prague (1998) 159. [10] T.A.M. Msagati, M.M. Nindi, Multi residue. Talanta, 64 (2004) 87–100.

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[29] B. Valeur, J.C. Brochon, New Trends in Fluorescence Spectroscopy, 6th ed., Springer Press, Berlin, Germany (1999) 25–28. [30] P. Ju, H. Fan, T. Liu, L. Cui, S.Y. Ai, J. Lumin. 131 (2011) 1724–1730. [31] X.H. Liu, P.X. Xi, F.J. Chen, Z.H. Xu, Z.Z. Zeng, J. Photochem. Photobiol. B 92 (2008) 98–102. [32] Y.Q. Wang, B.P. Tang, H.M. Zhang, Q.H. Zhou, G.C. Zhang, J. Photochem. Photobiol. B 94 (2009) 183–190. [33] Y.J. Hu, Y. Liu, J.B. Wang, X.H. Xiao, S.S. Qu, J. Pharm. Biomed. Anal. 36 (2004) 915–919. [34] B.K. Sahoo, K.S. Ghosh, S. Dasgupta, Biopolymers, 91 (2009) 108–119. [35] A. Sulkowska, J. Mol. Struct. 614 (2002) 227-232. [36] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed., Plenum Press, New York, (1999). 237 – 265. [37] A.M. Bordbar, A.K. Safaei, E. Ghasemi, J. Mol. Struct. 705 (2004) 41-47. [38] P.B. Kandagal, J. Seetharamappa, S.M.T. Shaikh, D.H. Manjunatha, J. Photochem. Photobiol. A 185 (2007) 239-244. [39] Z. Wang, D.J. Li, J. Jin, Spectrochim. Acta A 70 (2008) 866–870. [40] P.D. Ross, S. Subramanian, Biochem. 20 (1981) 3096–3102. [41] P. Wu, L. Brand, Anal. Biochem. 218 (1994) 1–13. [42] D.J. Li, J.F. Zhu, J. Jin, X.J. Yao, J. Mol. Struct. 846 (2007) 34–41. [43] Hongliang Xu, Nannan Yao, Haoran Xu, Tianshi, Guiying Li, Zhengqiang Li, Int. J. Mol. Sci. 14 (2013) 14185-14203. [44] Y.H. Wu, X.B. Ji, S.S. Hu, Bioelectrochem. 64 (2004) 91–97. [45] Y.N. Ni, X. Zhang, S. Kokot, Spectrochim. Acta A 71 (2009) 1865–1872. [46] Z.W. Zhu, C. Li, N.Q. Li, Microchem. J. 71 (2002) 57–63. [47] Q. Feng, N.Q. Li, Y.Y. Jiang, Anal. Chim. Acta 344 (1997) 97–104.

[48] W. Sun, J. Han, Y. Ren, K. Jiao, J. Braz. Chem. Soc. 17 (2006) 510–517. [49] F. Qu, N.Q. Li, Y.Y. Jiang, Talanta 45 (1998) 787–793. [50] A.J. Bard, L.R. Faulkner, Electrochemical Methods Fundamentals and Applications, 2nd ed., John Wiley and Sons, New York, 2001.

(SAM)

(SMO)

Fig. 1. Chemical structures of sulfanilamide (SAM) and sulfamethoxazole (SMO). Colour of the atoms: blue- nitrogen, red –oxygen, yellow –sulphur, grey- carbon, white – Hydrogen.

1.00

1.00

0.8

SAM

0.8

SMO

278nm

Abs

Abs

278nm 0.6

0.4

Absorbance

0.4 0

0.50

0.6

1

4 8 [SAM] × 10-5 M

0

12

0.50

4 8 [SMO] × 10-5 M

12

1 7 7 0 240 300

300

350 320

0 240 300

SAM

280nm

320

SMO

280nm

Fluorescence intensity

IF 180

IF 180

0

0 0

150

350

300

4

8

12

[SAM] × 10-5 M

0

1

4

8

12

[SMO] × 10-5 M

150 1

7 7 0

0 290

400 Wave length (nm)

500

290

400 Wave length (nm)

500

Fig. 2. Absorbance and Fluorescence spectra of BSA with different concentrations of SAM and SMO (x 10-5 M): 1) 0, 2) 1, 3) 3, 4) 5, 5) 7 and 6) 9, 7) 10; Inset fig.: Absorbance and fluorescence intensity vs. BSA.

Fig. 3. Stern-Volmer plots for quenching of BSA with SAM and SMO at 300 K; CBSA = 2.0 × 10−5 M; pH 7.4; λex = 280 nm, λem = 290–500 nm.

Fig. 4. The modified Stern-Volmer plots of BSA at 300 K; CBSA = 2.0 × 10−5 M; λex = 280 nm, λem = 290–500 nm.

22

Fig. 5. Plots of log [(F0 − F)/F] vs. log [Q] for BSA with SAM and SMO at 300 K; CBSA = 2.0 × 10−5 M; pH 7.4; λex = 280 nm, λem = 290–500 nm

23

(b)

(c)

(d)

log ∆i/ (∆imax-∆i)

(a)

Fig. 6. Linear plot of log [drug] vs log ∆i/ (∆imax-∆i)]. (a) SAMBSA, (b) SMOBSA, (c) SAMadenine and (d) SMOadenine. (BSA and Adenine Concentration – 2 x 10 -5).

24

(b)

(a) 600

600

500

500

400

400

300

300

200

200

100

100

0

100

200

300

(c)

400

500

600 0

100

200

300

400

500

600

(d)

Fig. 7. The best binding mode between BSA with (a) SAM and (b) SMO. The important residues of BSA are represented using lines and the ligand structure is represented using a “Ball and Stick” format. The Hydrogen bonding plots between sulfa drugs (c) SAM and (d) SMO with BSA. The BSA residues are represented using black dots and the hydrogen bonding interactions are represented using red dots. Colour of the atoms: skeleton structure – BSA, blue- nitrogen, red –oxygen, yellow –sulphur, green- carbon.

25

PHE 508(A)

THR 578(A) PHE 506(A)

LEU 574(A)

3.14

VAL 546(A) LEU 531(A)

ALA 527(A) PHE 550(A)

2.86

LEU 528(A)

LYS 524(A)

BSA - SAM

ARG 196(A) 3.37 ARG 458(A)

ALA 193(A) SER 192(A) 2.98 LEU 454(A)

ILE 455(A)

2.58 THR 190(A) ARG 435(A) TYR 451(A)

BSA - SMO Fig. 8. Two-dimensional schematic representation of hydrogen bonding interactions of BSA with SAM and SMO. Hydrogen bond depicted in dashed line. Hydrogen bond depicted in dashed line; colour of the atoms: blue- nitrogen, red –oxygen, yellow –sulphur, black- carbon.

26

Table 1 Stern-Volmer quenching constant (Ksv), Modified Stern-Volmer association constant Ka and bimolecular quenching rate constant (Kq) of adenine and BSA with SAM and SMO at 300 K;

S-V quenching

Adenine BSA

Modified S-V quenching

Adenine

BSA

Drugs

Ksv (104 M-1) Kq (1012 M-1s-1)

Ra

SD

SAM

0.45

0.45

0.992

0.327

SMO

0.59

0.59

0.999

0.312

SAM

0.51

0.76

0.995

0.377

SMO

0.76

0.51

0.997

0.272

4

-1

Drugs

Ka (10 M )

∆G0 (kJ/mol)

n

Ra

SAM

0.588

-18.69

0.92

0.9996

SMO

0.602

-18.85

0.95

0.9998

SAM

0.618

-19.42

0.99

0.9998

SMO

0.911

-20.08

1.03

0.9999

λex = 280 nm, λem = 290–500 nm, (pH – 7.4) Ra- linear correlation coefficient, SD – Standard deviation, n - number of binding site.

27

Table 2 CV for adenine and BSA with SAM and SMO (scan rate, 100 mV s-1 , concentration of BSA 2×10-6 M; SAM and SMO concentrations,( 0, 3, 7 and 10 ×10-4M).

BSA SAM

BSA SMO

Adenine SAM

Adenine SMO

Drug concentration (× 10-4) 0

Epa

Ipa

Epc

Ipc

Epa-Epc/2

Ipa/Ipc

978

0.587

-

-

-680

0

3

1323

1.967

-730.7

-1.367

-1026.85

-0.6949

7

1190

2.233

-750.4

-1.57

-970.2

-0.7030

10

1112

2.49

-796.3

-1.66

-954.15

-0.6666

0

984

0.576

-

-

-677

0

3

1341

1.456

-913.6

-1.108

-1127.3

-0.7609

7

1177

1.853

-828.8

-1.166

-1002.9

-0.6292

10

1099

1.935

-776.5

-1.549

-937.75

-0.8005

0

987.6

0.578

217.1

-0.526

-320.25

-0.9100

3

903.5

0.726

223.5

-0.342

-340

-0.4710

7

948.8

0.732

245.6

-0.2935

-351.6

-0.4009

10

1014.4

0.749

269.5

-0.2736

-372.45

-0.3652

0

981

0.597

-1090.1

-0.8635

-1163.05

-2.0574

3

1262

0.4716

-1194.7

-0.8368

-1228.35

-1.7743

7

1243

0.4797

-1181.9

-0.744

-1212.45

-1.5509

10

1053

0.6224

-1214

-0.6056

-1133.5

-0.9730

29

Table 3 Estimated free energy, Inhibition constant, Electrostatic energy and Total intermolecular energy of the BSA with SAM and SMO. Drug

Est. Free Energy of Binding kcal/mol

Est. Inhibition Constant, Ki µM

Electrostatic Energy kcal/mol

Total Frequency Intermolec Energy kcal/mol

Intert. Surfce

177.55

vdW + H-bond + desolv Energy kcal/mol -6.00

SAM

-5.12

-0.01

-6.01

100%

471.9

SMO

-5.87

49.61

-6.80

-0.29

-7.09

30%

628.3

30

Research highlights
Interactions between SAM and SMO with BSA and adenine was investigated
Both adenine and BSA fluorescence intensities decreased as the drug concentrations increased
Hydrophobic forces, electrostatic interactions and hydrogen bonds played vital roles in the binding interactions
Docking method provided the interactions of specific chemical groups in the complex stabilization.

31

Graphical abstract

(a)

(b)

Fig. The best binding mode between BSA with (a) SAM. The important residues of BSA are represented using lines and the ligand structure is represented using a “Ball and Stick” format. The Hydrogen bonding plots between (b) SAM with BSA. The BSA residues are represented using black dots and the hydrogen bonding interactions are represented using red dots.