Spectroscopic Investigation on the Interaction of Pyrimidine Derivative

ARTICLE pubs.acs.org/IECR

Spectroscopic Investigation on the Interaction of Pyrimidine Derivative, 2-Amino-6-hydroxy-4-(3,4-dimethoxyphenyl)-pyrimidine-5-carbonitrile with Human Serum Albumin: Mechanistic and Conformational Study Vishwas D. Suryawanshi, Prashant V. Anbhule, Anil H. Gore, Shivajirao R. Patil, and Govind B. Kolekar* Fluorescence Spectroscopy Research Laboratory, Department of Chemistry, Shivaji University, Kolhapur-416 004, Maharashtra, India ABSTRACT: In the present study, fluorescence spectroscopy in combination with UVvis absorption spectroscopy and synchronous fluorescence spectroscopy (SFS) was employed to investigate the binding affinity of pyrimidine derivative, 2-amino-6-hydroxy4-(3,4-dimethoxyphenyl)-pyrimidine-5-carbonitrile (AHDMPPC) to human serum albumin (HSA) under the physiological conditions. In the mechanism discussion, it was proved that the fluorescence quenching of HSA by AHDMPPC is a result of the formation of AHDMPPCHSA complex. The quenching mechanism and number of binding sites (n ≈ 1) were obtained by fluorescence titration data. Binding parameters calculated from SternVolmer method showed that the AHDMPPC bind to HSA with the binding affinities of the order 104 L mol1. The thermodynamic parameters studies revealed that the binding was characterized by negative enthalpy and positive entropy changes 13.06 kJ/mol and 51.34 J/mol K1 (from the Van’t Hoff equation) and suggest that the binding reaction was exothermic and hydrophobic interaction is the predominant intermolecular forces stabilizing the complex. The specific binding distance (r = 2.25 nm) between donor HSA and acceptor AHDMPPC was obtained according to fluorescence resonance energy transfer (FRET). Furthermore, the synchronous spectral result, threedimensional fluorescence spectra and circular dichroism (CD) indicates that the secondary structure of HSA was changed in the presence of AHDMPPC.

in vivo and vitro,12,13 (ii) decrease in the free (that is, protein unbound) drug concentration, which is the pharmacologically active form.14 In addition, many promising new drugs are rendered ineffective because of their unusually high affinity for plasma proteins.15 Proteindrug interaction plays an important role in pharmacokinetics (including distribution, metabolism, and elimination) and pharmacodynamics of the drugs.16,17 Because of clinical and pharmaceutical importance, a great deal of attention has been paid to the interaction of HSA with natural or synthetic drugs, including the study of binding constants and binding sites.1820 The measurement of the binding parameters of a drug bound to HSA is of utmost importance for drug discovery and preclinical studies of drug candidates in pharmaceutical research.21 Information on the binding of HSA with drugs can also help us better to understand the drugs pharmacology and pharmacodynamics. Therefore, it has become an important research field in life science, chemistry, and clinical medicine. In a series of study methods concerning the interaction of drugs and protein, fluorescence techniques are great aids in the study of interactions between drugs and plasma proteins in general and serum albumin in particular because of their high sensitivity, rapidity, and ease of implementation.22,23So far, some reports have focused on the determination of drug samples using fluorescence techniques at low concentration with different probes.24,25 In this article, we present a spectroscopic analysis of the interaction of HSA with AHDMPPC (pyrimidine derivative) at

1. INTRODUCTION Human serum albumin (HSA) is the most abundant serum protein, which comprises 5060% of the total plasma protein in humans. It facilitates the disposition and transportation of varieties of exogenous and endogenous ligands. The protein is capable of binding an extraordinarily broad range of pharmaceuticals, including fatty acids, amino acids, steroids, metal ions, etc. It is also responsible for the maintenance of blood pH, the drug disposition, and efficacy, and the contribution of colloid osmotic pressure.1,2 Crystal structure analysis has revealed that HSA is a globular protein composed of a single, largely α-helical polypeptide chain of 585 amino acid residues, contains three structurally domains (I, II, and III), each containing two subdomains (A and B) and stabilized by 17 disulfide bridges. There is a sole tryptophan (Trp) residue (Trp-214) located in subdomain IIA.1,3 Aromatic and heterocyclic ligands were found to bind within two hydrophobic pockets in subdomains IIA and IIIA, site I and site II.4 Despite of very high stability, HSA is a flexible protein with the 3D structure susceptible to environmental factors such as pH, ionic strength, etc.5 AHDMPPC (molecular structure: Figure 1), a pyrimidine derivative, its structural analogues are important biologically active antibacterial compounds effective against grampositive and gram-negative bacteria, which is synthesized in our laboratory. 6 Pyrimidine moiety is an important class of Ncontaining heterocycles widely used as key building blocks for pharmaceutical agents. It exhibits a wide spectrum of pharmacophore as it acts as bactericidal, fungicidal, analgesic, antihypertensive, and antitumor agents.710 Also, preclinical data from literature survey indicate continuing research in polysubstituted pyrimidine as potential antitumor agents.11 Binding of a drug to serum albumin has two important consequences: (i) solubilization of hydrophobic drugs that then helps drug delivery cells r 2011 American Chemical Society

Received: September 3, 2011 Accepted: December 4, 2011 Revised: November 14, 2011 Published: December 05, 2011 95

dx.doi.org/10.1021/ie202005c | Ind. Eng. Chem. Res. 2012, 51, 95–102

Industrial & Engineering Chemistry Research

ARTICLE

Figure 1. Molecular structure of 2-amino-6-hydroxy-4-(3,4-dimethoxy phenyl)-pyrimidine-5-carbonitrile (AHDMPPC).

three temperatures 303, 313, and 323 K under physiological conditions, using constant protein concentration and various AHDMPPC composition. The aim of the present investigation was to study the affinity of AHDMPPC for HSA to understand the carrier role of serum albumin for such compound in blood under physiological conditions. The interaction information between HSA and AHDMPPC concerning quenching mechanism, binding parameters and thermodynamic parameters were discussed. Moreover binding modes, high-affinity binding site, intermolecular distance and conformation investigation was reported herein.

Figure 2. Effect of AHDMPPC on the UVvis absorption of HSA, CHSA = 2.0 x106 mol L1; CAHDMPPC/(106 mol L1): 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 (T = 303 K, pH = 7.40).

above, 1.0 mL Tris-HCl buffer solution, 1.0 mL NaCl solution, appropriate amounts of HSA (2.0 mL), a known volume of standard AHDMPPC solution were added to 10.0 mL standard flask and diluted up to the mark with double distilled water. Fluorescence quenching spectra of HSA were obtained at excitation wavelength (λex = 290 nm) and emission wavelength (λem = 300450 nm). All measurements were recorded after 5 min of equilibration at the appropriate temperature 303, 313, and 323 K. In addition, the UVvis absorption spectroscopy was performed at RT in the range 220400 nm using a quartz cuvette with 1.0 cm path length. For synchronous fluorescence measurements, the excitation range was 280360 nm, Δλ was set at 15 and 60 nm. The titration method was applied as described above by successive addition of 1.0 mL increments of AHDMPPC stock solution at room temperature.

2. MATERIAL AND METHODS 2.1. Materials and Apparatus. Human serum albumin purchased from Fluka Chemical Company was directly dissolved in water to prepare stock solution at final concentration of (1.0 105 M). HSA stock solution was kept in the dark at 273277 K. TrisHCl (0.1 M) buffer solution containing NaCl (0.1 M) was used to keep the pH of the solution at 7.4. Stock solution (1.0 105 M) of AHDMPPC (synthesized) was prepared in (5:95) ethanol: water mixture. Dissolution of compound was enhanced by sonication in an ultrasonic bath (Spectra lab Model UCB-40). All chemicals were of analytical reagent grade and were used without further purification. Double distilled water was used throughout. The UVvisible absorption spectra were measured on a, UVvis NIR spectrophotometer (Shimadzu UV3600) and fluorescence emission spectra on PC-based spectrofluorometer (JASCO Japan FP-750), respectively, equipped with a Xenon lamp. A 1.0 cm quartz cell was used for measurements. Excitation and emission slit width was fixed to 10 nm. Circular dichroism(CD) spectra were measured with a Jasco J-815 Spectropolarimeter (Jasco, Tokyo, Japan) at room temperature over wavelength range of 200300 nm, using a 1 cm quartz cell. All pH values were measured by a digital pH meter with magnetic stirrer (Equip-Tronics EQ-614 A). 2.2. Optimization of Experimental Conditions. To get the best results, the optimal conditions were investigated. Various experimental parameters including medium, pH, addition order, reaction temperature and Δλ were studied with AHDMPPC concentration being 1.0 105 M in all conditions. The experimental results shown us that 0.1 M TrisHCl buffer solution of pH 7.4 was chosen as the supporting media; the Tris-HCl + NaCl + HSA + AHDMPPC was selected in this work. Room temperature was suggested as the preferable reaction temperature, Δλ = 15 and 60 nm were selected while scanning synchronous fluorescence spectroscopy. 2.3. Measurements of Spectrum. Some preliminary investigations were carried out to select optimum protein and AHDMPPC concentrations. On the basis of these experiments, under the optimum physiological conditions described

3. RESULT AND DISCUSSION 3.1. UVvis Absorption Spectra Study. UVvis absorption measurement is a very simple method and applicable to explore the structural change and to know the complex formation.26,27 The UV absorption spectra (Figure 2) shows the effect of AHDMPPC on the HSA absorption spectrum. As shown in figure, two absorption peaks were observed at 278 and 338 nm and the peak intensity increased with the addition of AHDMPPC. Furthermore, the formation of AHDMPPCHSA complex resulted in a slight shift (from 278 to 286 nm) of spectrum toward longer wavelength indicating the interaction between AHDMPPC and HSA. The obvious enhancement of absorbance intensity also indicated the formation of a new complex between AHDMPPC and HSA. In addition, the structure of HSA changed upon interaction with AHDMPPC. The results can be explained by that the environment of Trp residue was changed, and the hydrophobicity of the microenvironment of Trp residue was increased.28,29 3.2. Fluorescence Quenching Mechanism. Fluorescence is the process of photon emission as a result of the return of an electron in a higher energy orbital back to a lower orbital, a variety of molecular interactions can result in quenching, including excitedstate reactions, molecular rearrangements, energy transfer, groundstate complex formation, and collisional quenching. Such decrease in intensity of fluorescence is called fluorescence quenching. For macromolecules, the fluorescence measurements 96

dx.doi.org/10.1021/ie202005c |Ind. Eng. Chem. Res. 2012, 51, 95–102


ARTICLE

Figure 4. SternVolmer plots describing HSA quenching caused by AHDMPPC at three different temperatures. CHSA and CAHDMPPC are the same as those in Figure 3.

Figure 3. Fluorescence quenching spectra of HSA in the presence of AHDMPPC. CHSA = 2.0 106 mol L1; CAHDMPPC /(106 mol L1) ah: 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 (T = 303 K, pH = 7.40, λex= 290 nm).

Table 1. SternVolmer Quenching Constants and Bimolecular Quenching Rate Constants for the Interaction of AHDMPPC with HSA at Various Temperatures

can give some information of the binding of small molecule substances to protein, such as the binding mechanism, binding mode, binding constants, binding sites, and intermolecular distances.30 The fluorescence spectra of HSA in presence of different amounts of AHDMPPC were recorded in the range of 300450 nm upon excitation at 290 nm (Figure 3). AHDMPPC causes a concentration dependent quenching of the intrinsic fluorescence of HSA with changing the emission maximum and shape of the peaks. These results indicated that there were interactions between AHDMPPC and HSA and the binding reactions resulted in nonfluorescent complexes. The interactions of AHDMPPC and HSA were further confirmed by synchronous and three-dimensional fluorescence techniques. The addition of AHDMPPC caused a gradual decrease in the fluorescence emission intensity of HSA with a conspicuous change in the emission spectra. The shift of the maximum of emission wavelength from 346 to 336 nm was consistent with the fact that the change in the environment of the Trp residues were occurring and an increase of hydrophobicity in the vicinity of this residue takes place. These results indicated that the binding of AHDMPPC to HSA quenches the intrinsic fluorescence of the single Trp in HSA (Trp-214). Fluorescence quenching can occur by different mechanisms, usually classified as either dynamic or static quenching, which can be distinguished by their differing dependence on temperature and viscosity.31 Since higher temperatures result in large diffusion coefficients for dynamic quenching, and the quenching constants is expected to increase with increasing temperature. In contrast, a higher temperature may bring about the decrease in the stability of the complexes, resulting in lower quenching constant for the static quenching.32 The SternVolmer equation is often applied to elucidate the quenching mechanism,33 F0 =F ¼ 1 þ kq τ0 ½Q ¼ 1 þ KSV ½Q

pH 7.40

a

105 Ksv /(L mol1)

1013 kq/(L mol1 s1)

Ra

303

1.95

1.95

0.9984

313

1.30

1.30

0.9696

323

1.071

1.071

0.9950

R is the correlation coefficient.

The SternVolmer curves of F0/F versus [Q ] at different temperatures were shown in (Figure 4) and the calculated KSV and kq values were presented in Table 1. The plots showed that within the investigated concentration, the results exhibited a good linear relationship. Table 1 shows that KSV values were inversely correlated with temperatures, which suggested that the fluorescence quenching of HSA was initiated by the formation of ground-state complex.35 Furthermore, kqvalues were much greater than the maximum scatter collision quenching constant (2.0 1010 M1 S1) for various quenchers with biomolecule,36 so it was implied that the static quenching was dominant in drug HSA interaction. 3.3. Binding Constant and Binding Sites. When small molecules bind independently to a set of equivalent sites on a macromolecule, the equilibrium between free and bound molecules is given by the equation:37 log

ðF0 FÞ ¼ log K þ n log½Q F

ð3Þ

where K and n are the binding constant and the number of binding sites respectively. Thus, a plot of log[(F0 F)/F] versus log [Q ] can be used to determine K, as well as n (Figure 5). The results obtained are shown in Table 2. It was found that the binding constant decreased with an increase in temperature, resulting in a reduction of the stability of the AHDMPPCHSA complex. Meanwhile, from the data of n it may be inferred that there was one independent class of binding sites on HSA for AHDMPPC. The value of n approximately equal to one indicate the existence of just a single binding site in HSA for AHDMPPC. Hence AHDMPPC more likely binds to the hydrophobic pocket located in sub domain IIA, that is to say, Trp214 is near or within the binding site. The results in Table 2 also show that the binding between HSA and AHDMPPC is remarkable and the

ð1Þ

where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively. kq is the bimolecular quenching rate constant, τ0 is the lifetime of the fluorophore in the absence of quencher and the fluorescence lifetime of the biopolymer is 108 s,34 KSV is the SternVolmer quenching constant, and [Q] is the concentration of quencher. The formation of complex was further confirmed from the values of quenching rate constants, kq which are evaluated using the equation: kq ¼ KSV =τ0

T (K)

ð2Þ 97



ARTICLE

Figure 5. Plots of log (F0 F)/F versus log [Q ] at three different temperatures. CHSA and CAHDMPPC are the same as those in Figure 3.

Figure 6. Van’t Hoff plot for the binding of HSA to AHDMPPC.

Table 3. Thermodynamic Parameters of AHDMPPCHSA Interaction at pH 7.40

Table 2. Binding Constants (K) and Number of Binding Sites (n) of Competitive Experiment of AHDMPPCHSA System

a

T (K)

ΔH (kJ mol1)

303

13.061

ΔG (kJ mol1)

ΔS (J mol1 K1)

R

28.6194

51.3472

0.9999

T (K)

104 K/(L mol1)

n

Ra

303

8.5408

0.9504

0.9988

313

29.1329

313

7.2677

0.9607

0.9961

323

29.6464

323

6.2230

1.1176

0.9949

R is the correlation coefficient.

effect of temperature is not significant. Thus AHDMPPC can be stored and carried by this protein in the body.38 3.4. Thermodynamic Parameters and Nature of Binding Forces. Generally, there are essentially four types of non covalent interactions that could play a key role in ligand binding to proteins. These are hydrogen bonds, van der Waals forces, electrostatic and hydrophobic bonds interactions.39,40 The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) of binding reaction are the main evidence for confirming binding modes. To obtain such information, the implications of the present result have been discussed in conjunction with thermodynamic characteristics obtained for AHDMPPC binding, and the thermodynamic parameters were calculated from the Van’t Hoff equation ln KT ¼

ΔH ΔS þ RT R

Figure 7. Bar diagram showing thermodynamic parameters of the interaction of HSA with AHDMPPC at 303 K.

is frequently taken as a typical evidence for hydrophobic interaction from the point of view of water structure. The negative (ΔH) value (13.06 kJ mol1) observed cannot be mainly attributed to electrostatic interactions since for electrostatic interactions ΔH is very small, almost zero.42 The negative ΔH and positive ΔS values, in other words enthalpy change ΔH < 0 and entropy change ΔS > 0 obtained in this case therefore indicates that the hydrogen bonding and hydrophobic interactions played a role in the binding of AHDMPPC to HSA.43,44 3.5. Energy Transfer Between HSA and AHDMPPC. There is a spectral overlap between the fluorescence emission spectra of free HSA and absorption spectrum of AHDMPPC (Figure 8). According to F€orster’s nonradiative energy transfer theory (FRET),45 energy transfer is likely to happen under the following conditions: (1) the relative orientation of the donor and acceptor dipoles, (2) the extent of overlap of fluorescence emission spectrum of the donor with the absorption spectrum of the acceptor, and (3) the distance between the donor and the acceptor is less than 7 nm. The distance from the Trp residue (donor) to the bound drug (acceptor) in HSA can be calculated according to the F€orster’s theory. The efficiency of energy transfer (E) is related to the distance R between donor and acceptor by

ð4Þ

KT is the binding constant at temperature T and R is gas constant. The enthalpy change (ΔH) is calculated from the slope of the Van’t Hoff relationship. The free energy change (ΔG) is estimated from the following relationship: ΔG ¼ ΔH TΔS

ð5Þ

The temperatures chosen were 303, 313, and 323 K at which HSA did not undergo any structural degradation. According to the binding constants at the three temperatures, the thermodynamic parameters were determined from linear Van’t Hoff plot (Figure 6) and were presented in Table 3. (The plot of ln K versus 1/T gave a straight line according to the Van’t Hoff equation). Ross and Subramanian41 have characterized the sign and magnitude of the thermodynamic parameter associated with various individual kinds of interaction that may take place in protein association processes. Accordingly, as shown in (Figure 7), the negative values of enthalpy (ΔH) of the interaction of AHDMPPC and HSA indicated that the binding was mainly enthalpy-driven and involves an exothermic reaction, the entropy (ΔS) value was unfavorable for it. A positive (ΔS) value

E ¼ 1 98

F R6 ¼ 6 0 6 F0 R0 þ r0

ð6Þ



ARTICLE

Figure 8. Spectral overlap of AHDMPPC excitation (curve A) with HSA emission (curve F); T = 303 K, CHSA = CAHDMPPC = 2.0 106 mol L1.

where r is the binding distance between donor and receptor and R0 is the critical distance when the efficiency of excitation energy transferred to the acceptor is 50%. It can be calculated from donor emission and acceptor absorption spectra using the F€oster’s formula: R06 ¼ 8:79 1025 K 2 n4 ϕJ

ð7Þ

where K2 is the spatial orientation factor of the dipole, n is the refractive index of the medium, ϕ is the fluorescence quantum yield of the donor, and J is the overlap integral of the fluorescence emission spectrum of the donor with the absorption spectrum of the acceptor, which can be calculated by the following equation: Z ∞ FðλÞεðλÞλ4 dλ 0 Z ð8Þ J ¼ ∞ FðλÞ dλ

Figure 9. Synchronous fluorescence spectrum of HSA (T = 303 K, pH = 7.40), CHSA = 2.0 106 mol L1; CAHDMPPC /(106 mol L1): 0, 1.0, 2.0, 3.0, 4.0 (a) Δλ = 15 nm and (b) Δλ = 60 nm.

AHDMPPC was closer to tryptophan residues than to tyrosine residues. The addition of the drug led to a dramatic decrease in the fluorescence intensity with a slight shift of emission to a shorter wavelength from 341 to 338 nm (blue shift). It might be referred to a change in the conformation of Trp microregion caused by the interaction of HSA with AHDMPPC. This also reveals that AHDMPPC bound to hydrophobic cavity of HSA results in the loose structure of HSA and the polarity around Trp residues was decreased and the hydrophobicity was increased.50 To further verify the structural change of HSA when exposed to AHDMPPC, we measured the absorbance spectra of HSA with various amounts of AHDMPPC. As can be seen in Figure 6, the absorbance of HSA increased with the addition of AHDMPPC, this indicated that the peptide strands of protein molecules extended more upon the addition of AHDMPPC to HSA,51 and the addition resulted in the shift toward a longer wavelength, which give an original evidence that there was interaction between AHDMPPC and HSA. To obtain more information on the binding of AHDMPPC to HSA, three-dimensional spectroscopy were investigated on HSA and HSAAHDMPPC complex. The three-dimensional fluorescence spectra are a rising analysis technique in recent years. It is well-known that three-dimensional fluorescence spectrum can provide more detailed information about the configuration of proteins. The three-dimensional fluorescence spectra and contour ones are shown in (Figure 10), with the corresponding parameters shown in Table 4. The contour map displayed a bird’s eye view of the fluorescence spectra. Peak 1 is the Rayleigh scattering peak (λex = λem), where as the strong peak 2 mainly reveals the spectral characteristic of tryptophan and tyrosine residues.

0

where F(λ) is the fluorescence intensity of the fluorescent donor at wavelength λ to λ + Δλ; and ε(λ) is the molar absorption coefficient of the acceptor at wavelength λ. In the present case, K2 = 2/3, n = 1.336, ϕ = 0.118.46,47 Hence, from eqs 6 to 8, we could calculate that R0 = 2.052 nm; E = 0.36 and r = 2.25 nm, r < 7 nm. The values for R0 and r are on the 2 8 nm scale and 0.5R0 < r < 1.5R0,48 indicating an interaction between AHDMPPC and HSA (Trp-214). These data suggested that the energy transfer from HSA to AHDMPPC could occur with high probability. In accordance with prediction by F€orster’s nonradiative energy transfer theory, these results indicated again a static quenching interaction between AHDMPPC and HSA.49 3.6. Conformation Investigation. To explore the structural change of HSA by addition of AHDMPPC, we measured synchronous fluorescence spectra of HSA with various amounts of AHDMPPC. The synchronous fluorescence spectroscopy is a common method to provide information about the conformational changes of protein. They have several advantages like spectral simplification, spectral bandwidth reduction, and avoiding different perturbing effects. The spectrum characteristic of tyrosine and tryptophan residues in protein was observed when Δλ was set as 15 and 60 nm, respectively. Synchronous fluorescence spectral changes of HSA upon addition of AHDMPPC with varied concentration were displayed in Figure 9. The emission intensity underwent change when Δλ was 60 nm indicating 99



ARTICLE

Figure 10. The three-dimensional projections and the corresponding excitationemission matrix fluorescence diagrams of HSA before (A) and after AHDMPPC addition (B).CHSA= CAHDMPPC = 2.0 106 mol L1.

Table 4. Three-Dimensional Fluorescence Spectral Characteristics of HSA and HSAAHDMPPC System HSA

HSAAHDMPPC

peak position

Stokes

instensity

peak position

Stokes

instensity

peaks

λex/λem (nm/nm)

Δλ (nm)

F

λex/λem (nm/nm)

Δλ (nm)

F

Rayleigh scattering peaks fluorescence peaks

280/280 280/341

0 61

75.86 147.61

280/280 280/341

0 63

54.06 82.75

The results showed that threedimensional fluorescence map of HSA and AHDMPPCHSA was different obviously. Analyzing from the intensity changes of peak 1 and peak 2, they decreased obviously but to different degree. The decrease of the fluorescence intensity of the two peaks in combination with the synchronous fluorescence spectra results, we can conclude that the interaction of AHDMPPC with HSA induced the slight unfolding of the polypeptides of protein, which resulted in a conformational changes that increased the exposure of some hydrophobic regions which were previously buried.52,53 The above phenomenon and analysis of fluorescence characteristic of the peak

revealed that a complex between HSA and AHDMPPC has formed and binding induced some microenvironmental and conformational changes in human serum albumin. To ascertain the possible influence of AHDMPPC binding on the secondary structure of HSA, we have performed circular dichroism (CD) studies in the range of 200300 nm in presence of AHDMPPC. The far-UV CD spectra of HSA exhibit two negative bands in the ultraviolet region at 209 (π f π*) and 222 nm (n f π*), which are characteristic of an α-helical structure of protein.54 Figure 11 shows the CD spectra of the free HSA and its AHDMPPC-HSA complex obtained at pH = 7.4 100



ARTICLE

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Fax: +91 0231 2692333.

’ ACKNOWLEDGMENT The authors (VDS) gratefully acknowledges receiving a fellowship from UGC, New Delhi [University Grant Commission, XIth plan (Faculty Improvement Programme)] and thanks to DST and UGC for providing funds to department under FIST and SAP programme. ’ REFERENCES (1) He, X. M.; Carter, D. C. Atomic structure and chemistry of human serum albumin. Nature 1992, 358, 209–215. (2) Colmenarejo, G. In silico prediction of drug-binding strengths to human serum albumin. Med. Res. Rev. 2003, 23, 275–301. (3) Curry, S.; Brick, P.; Frank, N. P. Fatty acid binding to human serum albumin: New insights from crystallographic studies. Biochim. Biophys. Acta 1999, 1441, 131–140. (4) Li, Y.; He, W. Y.; Liu, J. Q.; Sheng, F. L.; Hu, Z. D.; Chen, X. G. Binding of the bioactive component jatrorrhizine to human serum albumin. Biochim. Biophys. Acta: Bioenerg. 2005, 1722, 15–21. (5) Cui, F. L.; Qin, L. X.; Zhang, G. S.; Yao, X. J.; Du, J. Binding of daunorubicin to human serum albumin using molecular modeling and its analytical application. Int. J. Biol. Macromol. 2008, 42, 221–228. (6) Deshmukh, M. B.; Salunkhe, S. M.; Patil, D. R.; Anbhule, P. V. A novel and efficient one step synthesis of 2-amino-5-cyano-6-hydroxy-4aryl pyrimidines and their anti-bacterial activity. Eur. J. Med. Chem. 2009, 44, 2651–2656. (7) Pershin, N. G.; Sherbakova, L. I.; Zykova, T. N.; Sakolova, V. N. Antibacterial activity of pyrimidine and pyrrolo-(3,2-d)-pyrimidine derivatives. Farmakol. Taksikol. World Rev. Pest. Contr. 1972, 35, 466–471. (8) Regnier, G.; Canevar, L.; Le, R. J.; Douarec, J. C.; Halstop, S.; Daussy, J. J. Triphenylpropylpiperazine derivatives as new potent analgetic substances. Med. Chem. 1972, 15, 295–298. (9) Winter, C. A.; Fisley, E. A. R.; Nuss, G. W. Carrageenin-induced edema in hind paw of the rat as an assay anti-inflammatory drugs. Proc. Soc. Exp. Biol. Med. 1962, 111, 544–547. (10) Sugiura, K.; Schmid, A. F.; Schmid, M. M.; Brown, F. G. Effect of compounds on a spectrum of rat tumors. Cancer Chemother. Rep., Part 2 1973, 3, 231–233. (11) Maquoi, E.; Sounni, N. E.; Devy, L.; Olivier, F.; Frankenne, F.; Krell, H.-W.; Grams, F.; Foidart, J.-M.; No€el, A. Anti-invasive, antitumoral, and antiangiogenic efficacy of a pyrimidine-2,4,6-trione derivative, an orally active and selective matrix metalloproteinases inhibitor. Clin. Cancer Res. 2004, 10, 4038–4047. (12) Kragh-Hansen, U. Molecular aspects of ligand binding to serum albumin. Pharmacol. Rev. 1981, 33, 17–53. (13) Kamat, B. P. Study of the interaction between fluoroquinolones and bovine serum albumin. J. Pharm. Biomed. Anal. 2005, 39, 1046–1050. (14) Kurono, M.; Fujii, A.; Murata, M.; Fujatani, B.; Negoro, T. Stereospecific recognition of a spirosuccinimide type aldose reductase inhibitor (AS-3201) by plasma proteins: A significant role of specific binding by serum albumin in the improved potency and stability. Biochem. Pharmacol. 2006, 71, 338–353. (15) Carter, D. C.; Ho, J. X. Structure of serum albumin. Adv. Protein Chem. 1994, 45, 153–203. (16) Kratochwil, N. A.; Huber, W.; Muller, F.; Kansy, M.; Gerber, P. R. Predicting plasma protein binding of drugs: A new approach. Biochem. Pharmacol. 2002, 64, 1355–1374. (17) Ahmad, B.; Parveen, S.; Khan, R. H. Effect of albumin conformation on the binding of ciprofloxacin to human serum albumin: A novel approach directly assigning binding site. Biomacromolecules 2006, 7, 1350–1356.

Figure 11. Circular dichroism (CD) spectra of free HSA and its AHDMPPCHSA complex. CHSA = 3.0 106 mol L1, CAHDMPPC = 3.0 106 mol L1, pH = 7.4, T = 303K.

and room temperature. As expected for a protein that is predominately α-helical, the CD spectrum of free HSA and its AHDMPPC complex exhibited a characteristic negative bands (elipticity) at 209 and 219 nm. The binding of AHDMPPC to HSA caused a decrease in band intensity at all wavelength of the far- UV CD without any significant shift of the peaks, indicating the decrease of the α-helical content in protein, which means the peptide strand unfolding even more. However, the CD spectra of HSA in presence and absence of AHDMPPC are similar in shape, indicating that the structure of HSA is also predominantly α-helical. From the above results, it is apparent that the effect of AHDMPPC on HSA causes a conformational change of the protein, with the loss of α-helical stability. The calculating results exhibited a reduction of α-helix structures from 56.63% to 52.33% at molar ratio AHDMPPC/HSA of 1:1.

’ CONCLUSIONS In this work, we used different approaches to explore the interactions between AHDMPPC and HSA under physiological conditions. The interactions were confirmed through alterations in the UVvis absorption spectra and quenching of the intrinsic fluorescence of the protein. The results indicated that the probable mechanism of interaction is a static quenching process. The binding process was exothermic, enthalpy driven and spontaneous, as indicated by the thermodynamic parameters analyzed, and the major part of the action force is hydrophobic interactions. Such interactions seem to slightly induce microenvironmental changes and alteration in protein conformation as shown by synchronous fluorescence, three-dimensional and circular dichroism studies. On the efficiency of energy transfer between the donor and acceptor, the distance between the fluorophore (Trp-214) of HSA and the drug AHDMPPC was estimated to be r = 2.25 nm. The significance of this work was evident since HSA serves as a carrier molecule for multiple drugs and the interaction of pyrimidine derivative AHDMPPC and HSA was not characterized so far. Hence, the report had a great importance in pharmacology and clinical medicine, as well as methodology. Therefore, this is also expected to open the door to new avenues in the screening and design of appropriate pyrimidine-based drugs that will be of important in modern medical research. 101



ARTICLE

(18) Suzkowska, A.; Macia) z_ ek-Jurczyk, M.; Bojko, B.; Rownicka, J.; Zubik-Skupien, I.; Temba, E.; Pentak, D.; Suzkowski, W. W. Competitive binding of phenylbutazone and colchicine to serum albumin in multidrug therapy: A spectroscopic study. J. Mol. Struct. 2008, 881, 97–106. (19) Bojko, B.; Suzkowska, A.; Macia) z_ ek-Jurczyk, M.; Rownicka, J.; Suzkowski, W. W. Fluorescence analysis of competition of phenylbutazone and methotrexate in binding to serum albumin in combination treatment in rheumatology. J. Mol. Struct. 2009, 924926, 332–337. (20) Shaikh, S. M. T.; Seetharamappa, J.; Kandagal, P. B.; Ashoka, S. Binding of the bioactive component isothipendyl hydrochloride with bovine serum albumin. J. Mol. Struct. 2006, 786, 46–52. (21) Oravcova, J.; B€ohs, B.; Lindner, W. Drugprotein binding studies: New trends in analytical and experimental methodology. J. Chromatogr., B 1996, 677, 1–28. (22) Epps, D. E.; Raub, T. J.; Caiolfla, V.; Chiari, A.; Zamai, M. Determination of the affinity of drugs toward serum albumin by measurement of the quenching of the intrinsic tryptophan fluorescence of the protein. J. Pharm. Pharmacol. 1999, 51, 41–48. (23) Bian, Q. Q.; Liu, J. Q.; Tian, J. N.; Hu, Z. D. Binding of genistein to human serum albumin demonstrated using tryptophan fluorescence quenching. Int. J. Biol. Macromol. 2004, 34, 275–279. (24) More, V. R.; Mote, U. S.; Patil, S. R.; Kolekar, G. B. Spectroscopic studies on the interaction between norfloxacin and p-amino benzoic acid: Analytical application on determination of norfloxacin. Spectrochem. Acta, Part A 2009, 74, 771–775. (25) Gore, A. H.; Mote, U. S.; Tele, S. S.; Anbhule, P. V.; Rath, M. C.; Patil, S. R.; Kolekar, G. B. A novel method for ranitidine hydrochloride determination in aqueous solution based on fluorescence quenching of functionalised CdS QDs through photoinduced charge transfer process: spectroscopic approach. Analyst 2011, 136, 2606–2612. (26) Bi, S. Y.; Song, D. Q.; Tian, Y.; Zhou, X.; Liu, Z. Y.; Zhang, H. Q. Molecular spectroscopic study on the interaction of tetracyclines with serum albumins. Spectrochim. Acta, Part A 2005, 61, 629–636. (27) Kandagal, P. B.; Ashoka, S.; Seetharamappa, J.; Vanb, V.; Shaikh, S. M. T. Study of the interaction between doxepin and human serum albumins by spectroscopic methods. J. Photochem. Photobiol., A 2006, 179, 161–166. (28) Zhou, H.; Sun, Z. Y.; Hoshi, T.; Kashiwagi, Y.; Anzai, J. I.; Li, G. X. Electrochemical studies of danthron and the DNAdanthron interaction. Biophys. Chem. 2005, 114, 21–26. (29) Qi, Z. D.; Zhou, B.; Xiao, Q.; Shi, C.; Liu, Y.; Dai, J. Interaction of rofecoxib with human serum albumin: determination of binding constants and the binding site by spectroscopic methods. J. Photochem. Photobiol., A 2008, 193, 81–88. (30) Kandagal, P. B.; Ashoka, S.; Seetharamappa, J.; Shaikh, S. M. T.; Jadegoud, Y.; Ijare, O. B. Study of the interaction of an anticancer drug with human and bovine serum albumin: Spectroscopic approach. J. Pharm. Biomed. Anal. 2006, 41, 393–399. (31) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, 2006. (32) Ding, F.; Zhao, G. Y.; Chen, S. C.; Liu, F.; Sun, Y.; Zhang, L. Chloramphenicol binding to human serum albumin: Determination of binding constants and binding sites by steady-state fluorescence. J. Mol. Struct. 2009, 929, 159–166. (33) Stern, O.; Volmer, M. On the quenching-time of fluorescence. Phys. Z. 1919, 20, 183–188. (34) Lakowicz, J. R.; Weber, G. Quenching of fluorescence by oxygen. A probe for structural fluctuations in macromolecules. Biochemistry 1973, 12, 4161–4170. (35) Kathiravan, A.; Chandramohan, M.; Renganathan, R.; Sekar, S. Spectroscopic studies on the interaction between phycocyanin and bovine serum albumin. J. Mol. Struct. 2009, 919, 210–214. (36) Ware, W. R. Oxygen quenching of fluorescence in solution— An experimental study of diffusion process. J. Phys. Chem. 1962, 66, 455–458. (37) Gao, H.; Lei, L.; Liu, J.; Qin, K.; Chen, X.; Hu, Z. The study on the interaction between human serum albumin and a new reagent with antitumour activity by spectrophotometric methods. J. Photochem. Photobiol., A 2004, 167, 213–221.

(38) Hu, Y. J.; Yang, Y. O.; Dai, C. M.; Liu, Y.; Xiao, X. H. Siteselective binding of human serum albumin by palmatine: Spectroscopic approach. Biomacromolecules 2010, 11, 106–112. (39) Mote, U. S.; Bhattar, S. L.; Patil, S. R.; Kolekar, G. B. Interaction between felodipine and bovine serum albumin: Fluorescence quenching study. Luminescence 2010, 25, 1–8. (40) Mote, U. S.; Patil, S. R.; Bhosale, S. H.; Han, S. H.; Kolekar, G. B. Fluorescence resonance energy transfer from tryptophan to folic acid in micellar media and deionised water. Photochem. Photobiol. B 2011, 103, 16–21. (41) Ross, P. D.; Subramanian, S. Thermodynamics of protein association reactions: Forces contributing stability. Biochemistry 1981, 20, 3096–3102. (42) Rahman, M. H.; Maruyama, T.; Okada, T.; Yamasaki, K.; Otagiri, M. Study of interaction of carprofen and its enantiomers with human serum albumin-I. Mechanism of binding studied by dialysis and spectroscopic methods. Biochem. Pharmacol. 1993, 46, 1721–1731. (43) Seedher, N.; Singh, B.; Singh, P. Mode of interaction of metronidazole with bovine serum albumin. Indian J. Pharm. Sci. 1999, 51, 143–148. (44) Yue, Y.; Chen, X.; Qin, J.; Yao, X. Characterization of the mangiferinhuman serum albumin complex by spectroscopic and molecular modeling approaches. J. Pharm. Biomed. Anal. 2009, 49, 753–759. (45) F€orster, T. In Modern Quantum Chemistry, Vol. 3; Sinanoglu, O., Eds.; Academic Press: New York, 1996; p 93. (46) Yue, Y.; Chen, X.; Qin, J.; Yao, X. Spectroscopic investigation on the binding of antineoplastic drug oxaliplatin to human serum albumin and molecular modeling. Colloids Surf., B 2009, 69, 51–57. (47) Cyril, L.; Earl, J. K.; Sperry, W. M. Biochemists’ Handbook; E. &F. N. Spon: London, 1961. (48) Valeur, B. Molecular Fluorescence: Principles and Applications; Wiley Press: New York, 2001. (49) Cui, F. L.; Fan, J.; Li, W.; Fan, Y.; Hu, Z. Fluorescence Spectroscopy Studies on 5-Aminosalicylic Acid and Zinc 5-Aminosalylicylate Interaction with Human Serum Albumin. J. Pharm. Biomed. Anal. 2004, 34, 189–197. (50) Ibrahim, N.; Ibrahim, H.; Kim, S.; Nallet, J. P.; Nepveu, F. Interactions between antimalarial indolone-N-oxide derivatives and human serum albumin. Biomacromolecules 2010, 11, 3341–3351. (51) Hu, Y. J.; Liu, J.; Wang, J. B.; Xiao, X. H.; Qu, S. S. Study of the interaction between monoammonium glycyrrhizinate and bovine serum albumin. J. Pharm. Biomed. Anal. 2004, 36, 915–919. (52) Tian, J. N.; Liu, J. Q.; Hu, Z. D.; Chen, X. G. Interaction of wogonin with bovine serum albumin. Bioorg. Med .Chem. 2005, 13, 4124–4129. (53) Pan, X.; Qin, P.; Liu, R.; Wang, J. Characterizing the interaction between tartrazine and two serum albumins by a hybrid spectroscopic approach. J. Agric. Food Chem. 2011, 59, 6650–6656. (54) Gore, M. G. Spectrophotometry and Spectrofluorimetry: A Practical Approach; Oxford University Press: New York, 2000; pp 123125.

102


Spectroscopic Investigation on the Interaction of Pyrimidine Derivative

Recommend Documents