Journal of Luminescence 130 (2010) 2476–2486 Contents lists available at ScienceDirect Journal of Luminescence journal homepage: www. elsevier. com/locate/jlumin Spectroscopic and nano-molecular modeling investigation on the binary and ternary bindings of colchicine and lome? oxacin to Human serum albumin with the viewpoint of multi-drug therapy J.
Chamani a,n, A. Asoodeh b, M. Homayoni-Tabrizi a, Z. Amiri Tehranizadeh c, A. Baratian c, M. R. Saberi c, M. Gharanfoli d aDepartment of Biology, Faculty of Sciences, Islamic Azad University-Mashhad Branch, Mashhad, Iran Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran Medical Chemistry Department, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran d Department of Development Biology, Culture and Science University, Tehran, Iran b c a r t i c l e in f o Article history: Received 21 April 2010 Received in revised form 14 August 2010 Accepted 18 August 2010 Available online 26 August 2010 Keywords: HSA Lome? oxacin Colchicine Spectroscopy Molecular dynamic b s t r a c t Combination of several drugs is often necessary especially during long-term therapy.
The competitive binding drugs can cause a decrease in the amount of drug bound to protein and increase the biological active fraction of the drug. The aim of this study is to analyze the interactions of Lome? oxacin (LMF) and Colchicine (COL) with human serum albumin (HSA) and to evaluate the mechanism of simultaneous binding of LMF and COL to protein. Fluorescence analysis was used to estimate the effect of drugs on the protein ? uorescence and to de? ne the binding and quenching properties of drugsHSA complexes.The binding sites for LMF and COL were identi? ed in tertiary structure of HSA with the use of spectro? uorescence analysis.
The analysis of ? uorescence quenching of HSA in the binary and ternary systems show that LMF does not affect the complex formed between COL and HSA. On the contrary, COL decreases the interaction between LMF and HSA. The results of synchronous ? uorescence, resonance light scattering and circular dichroism spectra of binary and ternary systems show that binding of LMF and COL to HSA can induce micro-environmental and conformational changes in HSA.The simultaneous presence of LMF and COL in binding to HSA should be taken into account in the multidrug therapy, and necessity of using a monitoring therapy owning to the possible increase of the uncontrolled toxic effects. Molecular modeling of the possible binding sites of LMF and COL in binary and ternary systems to HSA con? rms the spectroscopic results.
& 2010 Elsevier B. V. All rights reserved. 1. Introduction Human serum albumin (HSA) is the most abundant carrier protein in blood circulation that can bind many endogenous and exogenous compounds e. g. atty acids, bilirubin, prostaglandins, steroids, cholesterol, uric acid, hormones, drugs and contrasting substances reversibly, with dissociation binding constant (KD) in the range of 10 A 3–10 A 8 mol L A 1 [1].
The structure of HSA has been determined crystallographically [2,3] and discovered that it consisted of three structurally homologous, predominantly helical domains (domain I, II and III), which each domain contained two subdomains (A and B). Two principal binding sites were located in sub-domains IIA and IIIA, which are named as site I and site II, respectively.The drugs bound in site I are generally bulky heterocyclic anions with the charge situated in a central position of the molecules and that in site II are aromatic carboxylic acids with n Corresponding author. Tel. : + 98511 8437107; fax: +98511 8424020. E-mail address: Chamani@ibb. ut. ac.
ir (J. Chamani). an extended conformation and the negative charge located at one end of the molecule [4,5]. HSA is a globular protein composed of a single polypeptide chain of 585 amino acid residues with a largely alpha-helical structure.Its amino acid sequence contains a total of 17 disul? de bridges, one free thiol (Cys-34) and a single tryptophan (Trp-214). It is capable of binding a wide variety of drugs and xenobiotics [6–11].
Binding of drugs to plasma proteins is an important pharmacological parameter, since it frequently affects the distribution and elimination of a drug as well as the duration and intensity of its physiological action [12–14]. Drugs, which are administered in multi-drug therapy, may alter each others binding to the transporting protein.Therefore, it is necessary to determine both the location of the binding sites and the possible interactions for each individual drug. Combination of several drugs is often required, especially during long-term therapy. The simultaneous presence of two drugs for the binding sites on HSA may result in decrease in binding and hence increase in the concentration of free biologically active fraction of one or both the drugs.
HSA is a ? exible molecule and binding of a drug often affects the simultaneous binding of other 0022-2313/$ – see front matter & 2010 Elsevier B.V. All rights reserved. doi:10. 1016/j. jlumin. 2010.
08. 016 J. Chamani et al. / Journal of Luminescence 130 (2010) 2476–2486 2477 Fig. 1. (a) Structure of lome? oxacin and (b) structure of colchicine. drugs.
Therefore, study of the interaction between HSA and drugs is imperative and can obtain a lot of information of drug actions. Lome? oxacin (LMF, Fig. 1a) is an antibiotic in a class of drugs called ? uoroquinolones with a wide spectrum of activity against various bacterial infections, such as bronchitis and urinary tract infections [15].Colchicine (COL, Fig. 1b) is used for the treatment of acute or chronic gout [16,17]. Colchicine produces its antiin? ammatory effects by binding to the intracellular protein tubulin, thereby preventing its polymerization into microtubulin and leading to the inhibition of leukocyte migration and phagocytosis [18]. This study is designed to analyze the simultaneous interactions of LMF and COL to HSA under physiological conditions.
Besides using ? uorescence spectroscopic, resonance light scattering, circular dichroism (CD) methods and molecular odeling are also applied. The effects of drugs on the HSA are estimated and the binding constant, the number of binding sites and protein conformational changes of drug-HSA complexes are determined. measured using the buffer solutions of drugs in corresponding concentration as reference. Measurements were performed for each studied drug using HSA solution, (1) with a single drug (LMF or COL) of various concentrations and (2) with the two drugs, which were added simultaneously to HSA. A dilution series of drugs were prepared in a 50 mM phosphate buffer.For each data point, 5 ml of the drug solution was added into 2 ml HSA solution. The reaction time has been investigated and the results showed that 3 min was enough for the stabilization.
So the change in ? uorescence emission intensity was measured within 3 min of adding drugs to the HSA and the concentration of oxygen at the experimental conditions is maintained as a constant. The addition of a constant volume of quencher to the protein solution avoided complications due to dilution effects within titration type experiments.Each measurement was repeated in triplicate and the mean and standard deviation were calculated. The ? uorescence quenching data were plotted as relative ? uorescence intensity against drug concentrations. The synchronous ? uorescence spectra were obtained by scanning simultaneously the excitation and emission monochromators. The synchronous ? uorescence spectra only show the tyrosine and tryptophan residues of HSA when the wavelength interval (Dl) is 15 nm and 60 nm, respectively [19].
By scanning both the excitation and emission monochromators of a common spectro? uorometer with Dl ? nm, a resonance light scattering spectrum can be developed [20], which has been proved to be able to investigate the aggregation of small molecules and the longrange assembly of organic dyes on biological templates [20,21]. All RLS spectra were obtained by scanning simultaneously the excitation and emission monochromators (namely Dl ? 0) from 250 to 700 nm with slit widths at 5 nm for the excitation and the emission. 2. 3. Circular dichroism measurements Circular dichroism (CD) measurements were carried out on a Jasco-815 automatic recording spectropolarimeter (Japan) in cell of path length 2 mm at room temperature.The induced ellipticity was obtained by the ellipticity of the drug-HSA mixture subtracting the ellipticity of drug at the same wavelength and is expressed in degrees.
The results are expressed as mean residue ellipticity (MRE) in deg. cm2/dmol, which is de? ned as [MRE yobs (mdeg)/10 nlCp]. The yobs represents the CD in milli-degree, n is the number of amino acid residues (585), l and Cp are the path length of the cell and the mole fraction, respectively. The alphahelical content of HSA was calculated from the MRE value at 208 nm using the equation a% helix? [MRE 208A 4000)/ 33000 A4000] A 100 as described by Khan et al. 22]. 2. 4. Molecular modeling calculations The crystal structure for human serum albumin (HSA) (PDB ID: 1BM0) was employed in the docking calculation performed with autodock4 on an Ubuntu Linux platform, which combines a rapid energy evaluation through pre-calculated grids of af? nity potentials with a variety of search algorithm to ? nd suitable for a ligand on a given macromolecule.
The complexes (Colchicine, Lome? oxacin) used in docking calculations were submitted to a preminimization with the MM+ force ? eld. All compounds were energy minimized by means of AM1 semi-empirical force ? eld.The energy minimized ligands were further processed using Autodock tools program, adding partial atomic charges for each atom, and de? ning the rigid root and rotatable bond for each ligand automatically. A grid map for the whole protein structure ? was generated with the default 0.
375 A spacing by the auto-grid 2. Materials and methods 2. 1. Materials Human serum albumin (fatty acid free, 90%), lome? oxacin and colchicine were purchased from Sigma Chemical Co.
(St. Louis, MO, USA). All reagents were used as supplied without further puri? cation. The protein was dissolved in 50 mM phosphate buffer solutions at pH 7. and the stock solution was kept in the dark at 4 1C. All other reagents were of analytical grade and doubly distilled water used through out all the experiments.
2. 2. Fluorescence measurements Fluorescence spectra were recorded at room temperature with a Eclips carry ? uorimeter (Varian, USA) using 5 nm A 5 nm slit widths, equipped with a thermostatically controlled cell holder in the range of 315–500 nm (at an excitation wavelength of 295 nm) and 300–500 nm (at an excitation of 280 nm) individually, and the emission spectra of the free HSA and HSA-drug mixtures were 478 J. Chamani et al. / Journal of Luminescence 130 (2010) 2476–2486 program.
The sigmoidal distance-dependent the electric permittivity of Mehler and Solmajer was used for the calculation of the electrostatic grid maps. The lamarkian genetic algorithm method was applied for minimization, using default parameters and random starting positions on the entire protein area, random orientations and torsions were used for ligands. After determining high af? nity area of the protein to the ligands, grid map was restricted to the said area and its amino acids were marked as ? xible with default torsions. (LEU115, MET123, TYR161, ILE142, PHE157, LEU182, ARG186 for colchicine; LEU115, MET123, TYR161, ILE142, PHE157, LEU182, ARG186, TYR138 for lome? oxacin; MET123, TYR161, ILE142, PHE157, LEU182, ARG186, TYR138 for lome? oxacin and colchicine; TYR138, ILE142, PHE157, LEU185, ARG117, PHE149, TYR161 for colchicine and lome? oxacin) Number of GA runs was set to 200 for each set; other parameters were left at their defaults [23–25]. 3. Results and discussion 3.
1. Fluorescence quenching of HSA induced by LMF and COL Protein ? orescence is quenched by a ligand when the distance between the ligand and the ? uorophore in protein is smaller than 10 nm [26]. There is one tryptophan residue in HSA, Trp-214, which is situated in sub-domain IIA [2]. When the excitation wavelength is 295 nm, only the tryptophan residue has a ? uorescence emission at about 350 nm and the other one at about 450 nm. The emission wavelengths of 350 and 450 nm are related to HSA and LMF, respectively.
With increase in the LMF to the HSA solution, the emission peak at 450 nm increase that enhance the LMF concentration in solution.At the excitation wavelength of 280 nm, both tryptophan and tyrosyl amino acid residues have ? uorescence emission. Comparison of ? uorescence quenching of protein excited at 280 nm and 295 nm allows estimating the participation of tryptophan and tyrosine groups in the complex. The quenching take place when the ligand (quencher) is suf? ciently close to the tryptophanyl or/and tyrosyl residues. Then the energy transfer between a ligand and ? uorophore is possible. Fig. 2 shows the ? uorescence quenching spectra of HSA excited at 280 and 295 nm in he presence of LMF and COL. LMF and COL have no ? uorescence emission at the range measured and the background can be subtracted by using the corresponding concentration of the drugs solutions as reference when recording the spectra.
It can be seen from Fig. 2 that the intensities of ? uorescence emission of HSA decreased gradually with the increase in drugs concentrations. The quenching curves of HSA excited at 280 and 295 nm in the presence of LMF (Fig. 3a) overlap below the molar ratio LMF/HSA, 1/1 and do not overlap above it.It seems that when there is less than 1 LMF molecule for 1 HSA molecule, only tryptophan residue takes part in the interaction, whereas above this number of LMF molecules, tryptophan and tyrosine residues participate in it. Moreover the quenching curves of HSA excited at 280 nm and in the presence of COL (Fig. 3b) are slightly higher than that excited at 295 nm. This means that, both tryptophan and tyrosine residues take part in the interaction.
The synchronous ? uorescence spectra were obtained by scanning simultaneously the excitation and emission monochromators. Thus, the synchronous ? orescence applied to the equation of synchronous luminescence [27] F ? kcdEex ? lem ADl? Eem ? lem ? ?1? where F is the relative intensity of synchronous ? uorescence, Dl ? lem A lex ? constant, Eex the excitation function at the given 10000 Fluorescence Intensity 8000 6000 4000 2000 0 300 Fluorescence Intensity 6000 5000 4000 3000 2000 1000 0 300 350 400 450 500 550 600 350 400 450 500 550 Wavelength / nm 600 Wavelength / nm 7000 Fluorescence Intensity Fluorescence Intensity 6000 5000 4000 3000 2000 1000 0 300 350 400 450 500 550 Wavelength / nm 600 4000 3000 2000 1000 0 300 350 400 450 Wavelength / nm 500 Fig. 2.Emission spectra of HSA in the presence of various concentrations of LMF (a), COL (b) at 298 K, pH? 7. 4 and lex ? 280 nm. C (HSA)? 1. 5 A 10 A 6 mol L A 1; C (LMF)/ ( A 10 A 3 mol L A 1), C (COL)/( A 10 A 3 mol L A 1). The b and d parts correspond to the emission spectra of HSA in the presence of LMF and COL at lex ? 295 nm.
Arrows indicate the direction of the change. For the clarity of the spectra at the blue-shift, there are shown with arrows direction. J. Chamani et al. / Journal of Luminescence 130 (2010) 2476–2486 2479 1 16 14 0. 8 Fluorescence Intensity 12 10 8 6 4 2 0.
6 F/F0 0. 4 0. 2 0 260 280 300 Wavelength / nm 320 0 2 4 LMF/HSA 50 Fluorescence Intensity 6 8 10 60 1 40 30 20 10 0. 8 0. 6 F/F0 0.
4 0 250 260 270 280 290 300 Wavelength / nm 0. 2 Fig. 4. Synchronous ? uorescence spectrum of HSA: (a) Dl ? 15 nm; (b) Dl ? 60 nm; C (HSA)? 1. 5 A 10 A 6 mol L A 1; C (LMF)/( A 10 A 3 mol L A 1).
Arrows indicate the direction of the change. 0 0 0. 2 0. 4 0. 6 0. 8 1 COL / HSA Fig.
3. Comparison of quenching curves of HSA excited at 280 and 295 nm in the presence of LMF (a) and COL (b). C (HSA) ? 1. 5 A 10 A 6 mol L A 1; C (LMF)/ ( A 10 A 3 mol L A 1), C (COL)/( A 10 A 3 mol L A 1). Open symbols are at lex ? 280 nm and ? lled symbols are at lex ? 95 nm. excitation wavelength, Eem the normal emission function at the corresponding emission wavelength, c the analytical concentration, d the thickness of the sample cell and k is the characteristic constant comprising of the ‘‘instrumental geometry factor’’ and related parameters. Since, the relationship of the synchronous ? uorescence intensity (F) and the concentration of LMF and COL should follow the Eq. (1).
F should be indirect proportion to the concentration of drugs. The optimal value of the wavelength intervals (Dl) is important for the correct analysis and interpretation of the binding mechanism.When the wavelength interval (Dl) was ? xed at 60 nm of protein, the synchronous ? uorescence has the same intensity as the emission ? uorescence following excitation at 280 nm, just the emission maximum wavelength and shape of the peaks were changed [28–30]. Therefore the synchronous ? uorescence measurements can deduce the binding mechanism as the emission ? uorescence measurements did. Figs. 4 and 5 show the synchronous ? uorescence at Dl ? 15 and Dl ? 60 of HSA in the absence and presence of LMF and COL, respectively.
The synchronous ? uorescence spectra of HSA with various concentrations of LMF at Dl ? 5 nm (Fig. 4a) and Dl ? 60 nm (Fig. 4b) show that the emission wavelength of the tyrosine and tryptophan residues are red shift with increase in concentration of LMF. It suggests that the tyrosine and tryptophan ? uorescence spectrum may represent that the conformation of HSA is somewhat changed, leading to the hydrophilicity around tyrosine and tryptophan residues strengthened and the polarity increased [31]. Fig. 5 shows the effect of addition of COL on the synchronous ? uorescence spectrum of HSA when Dl ? 15 nm and Dl ? 60 nm. The addition of the COL results in the ? uorescence enhancement at both Dl ? 5 nm and Dl ? 60 nm.
The emission wavelength of the tyrosine residues is red shift but no signi? cant change in wavelength was observed in the tryptophan ? uorescence emission. It means that the interaction of COL with HSA does not affect the conformation of tryptophan microenvironment. It is important to note that COL affect only the tyrosine residues present in HSA.
This is because tyrosine contains one aromatic hydroxyl group unlike tryptophan. In addition, tyrosine can undergo an excited state ionization, resulting in the loss of the proton on the aromatic hydroxyl group.The hydroxyl group can dissociate during the lifetime of its excited state, hence the aromatic hydroxyl group present in the tyrosine residues is possible for the interaction of HSA with COL. On the other hand, the Resonance Light Scattering (RLS) spectra of the system were recorded by synchronous scanning at lem ? lex from 220–720 nm.
The RLS spectra of the HSA-LMF and 2480 J. Chamani et al. / Journal of Luminescence 130 (2010) 2476–2486 45 40 Fluorescence Intensity 35 30 25 20 15 10 5 0 260 280 300 Wavelength / nm 320 IRLS 300 250 200 150 100 50 0 200 300 400 500 Wavelength / nm 00 700 200 60 50 Fluorescence Intensity 40 30 50 IRLS 150 100 20 10 0 250 0 220 320 420 520 Wavelength / nm 620 720 260 270 280 Wavelength / nm 290 300 Fig. 6. Resonance light scattering spectra of HSA in the presence of different concentrations of LMF (a) and COL (b) at 298 K, pH? 7. 4.
C (HSA) ? 1. 5 A 10 A 6 mol L A 1; C (LMF)/( A 10 A 3 mol L A 1), C (COL)/( A 10 A 3 mol L A 1). Fig. 5.
Synchronize ? uorescence spectrum of HSA: (a) Dl ? 15 nm; (b) Dl ? 60 nm; C (HSA) ? 1. 5 A 10 A 6 mol L A 1; C (COL)/( A 10 A 3 mol L A 1). Arrows indicate the direction of the change.HSA-COL systems at physiological condition are shown in Fig. 6(a and b). The RLS intensity of the blank was very small and nearly constant.
When LMF and COL drugs were added to HSA, the RLS intensity was greatly increased. It can be consequently concluded that LMF and COL interacted with HSA and produced a complex of which RLS intensity was much higher than that of HSA, LMF and COL when they existed separately. 3. 2. HSA-LMF and HSA-COL interactions for the binary system Generally speaking, the ? uorescence quenching is the decrease in the quantum yield of ? uorescence from a ? orophore induced by a variety of molecular interactions with quencher molecule, such as excited state reaction, molecules rearrangement, energy transfer, ground state complex formation and collision quenching. As the data shown in Fig. 2, the ? uorescence intensity of HSA decreased regularly with the increasing concentration of LMF and COL. In order to con? rm the quenching mechanism, the ? uorescence quenching data are analyzed by the Lehrar equation [32] F0 =DF ? 1=? Q ? A 1=f A 1=KQ ? 1=f ? 2? ?uorescence; KQ, the quencher constant; and [Q], the quencher concentration.
The original Stern–Volmer equation allows describing ligand movement within the ? uorophore microenvironment when dynamic quenching occurs [33] F0 =F ? 1? KQ ? Q ? ?3? where F0 and DF are relative ? uorescence intensity of protein in the absence and present of quencher [Q], respectively; DF, the difference between F0 and F; f, the fractional accessible protein where F and F0 are ? uorescence intensities of protein in the absence and presence of quencher [Q]; [Q], quencher concentration and KQ the quenching constant. Analysis of the Lehrer equation allowed us to determine the quenching constants KQ and f values for HSA-LMF and HSA-COL (Fig. (a and b)). The value of KQ allows us to estimate the ? uorophore availability for LMF and COL. The constant KQ is a mean value of the quenching constants characterizing all binding sites of the HSA. Table 1 shows the KSV and n for HSA-LMF and HSA-COL excited at 280 and 295 nm. The KSV value of the quenching effect allows for the assessment of the af? nity of LMF and COL to the HSA.
The dependence of F0/F on concentration of the drug allows us to determine the kind of quenching. If dependence forms a straight line then it points to dynamic quenching. A positive deviation points to both static and dynamic quenching.As can be shown from Figs. 7 and 8, dynamic quenching in the sub-domain where ? uorophores occurs below the concentrations of LMF (0. 0112 mM), COL (0.
0108 mM) excited at 280 nm and LMF (0. 0165 mM), COL (0. 021 mM) excited at 295 nm for HSA-LMF and HSA-COL complexes, respectively. Above these concentrations, the positive deviation from the J.
Chamani et al. / Journal of Luminescence 130 (2010) 2476–2486 2481 7 6 5 F0/(F0-F) F0/F 18 15 12 9 6 3 0 4 3 2 1 0 0 1 2 3 1/LMF x 10-2 4 5 6 0 0. 005 0. 01 0. 015 [LM] / mM 0. 02 0. 025 0. 03 16 14 12 F0/(F0-F) 10 F0/F 6 5 4 3 2 1 8 6 4 2 0 0 2 4 6 1/COL x 10-2 8 10 12 0 0 0.
005 0. 01 0. 015 0. 2 [COL] / mM 0. 025 0. 03 0.
035 Fig. 7. The modi? ed Stern–Volmer plots of the HSA-LMF (a) and HSA-COL (b) at pH? 7. 4. Open symbols are at lex ? 280 nm and ? lled symbols are at lex ? 295 nm. Fig. 8. The Stern–Volmer plots of the HSA-LMF (a) and HSA-COL (b) at pH? 7.
4. Open symbols are at lex ? 280 nm and ? lled symbols are at lex ? 295 nm. straight line indicates that both the static and dynamic quenching is observed. A similar effect was observed for the ASA-SA system [34]. Table 1 shows that the values of quenching constants KSV are higher for HSA-COL than for HSA-LMF system. This means that COL has a higher af? nity to HSA than LMF.The highest values are determined at protein excitation of lex 280 and 295 nm for all complexes.
Fluorescence quenching data of HSA were analyzed to obtain various binding parameters. The binding constant (Ka) and the number of binding sites (n) can be calculated according to the equation [35] log?? F0 AF? =F? ? log Ka ? n log? Q ? ?4? where F0 and F are the ? uorescence intensity without and with the drug, respectively. A plot of log [(F0 A F)/F] vs. log [Q] gave a straight line using least squares analysis whose slope was equal to n (binding sites) and the intercept on Y-axis to log Ka (Ka equal to the binding constant).Fig. 9 shows the plots of log [(F0 A F)/F] vs.
log [Q] for the HSA-LMF and HSA-COL systems obtained from the ? urometric titration. In Table 1, the binding constants and binding sites listed for HSA-LMF and HSA-COL binary systems. From Eq. (4), the values of Ka and n at 298 K were obtained to be 2. 37 A 10 A 4 Lmol A 1 and 0. 98 for HSA-LMF and 4. 97 A 10 A 4 Lmol A 1 and 1.
14 for HSA-COL, respectively, which implied that COL bounds strongly to HSA than LMF and there were one independent class of binding sites for both LMF and COL towards HSA. The linear coef? cient R (0. 973 and 0.
9987) indicated that the assumptions underlying the deviation of Eq. (4) were satisfactory [36]. To con? rm how the protein structure is changed upon drug binding, changes in chirality are a good indication of ongoing folding changes and especially of tertiary and secondary structure modi? cations. Thus LMF, COL-induced conformational transitions of HSA were monitored by far UV-CD spectroscopy at different drug concentrations. The CD spectrum of HSA exhibits two negative bands about 208 and 222 nm (Fig.
10), which are characteristic of a high alpha-helical content.The values of the negative peaks at [y]208 and [y]222 for HSA-LMF slightly decrease which denotes that the secondary structure of HSA is not greatly affected by LMF (Fig. 10a). The molar ellipticity values of HSA-COL interactions show a more sever decrease, which arises from an unfolding process undergone by the protein in the presence of COL molecules. The results suggest that the conformational transition probably results in the exposure of the hydrophobic cavities and a perturbation of microenvironments around the deprotonated aromatic amino acid residues, which are favourable for the HSA adsorption onto the surface of COL. 3. 3.HSA-LMF-COL and HSA-COL-LMF interactions for the ternary system RLS spectra of HSA-LMF-COL and HSA-COL-LMF systems are shown in Fig.
11. It can be seen that, when COL and LMF were added, respectively, into the solution containing HSA-LMF and HSA-COL, the RLS intensity of the solution was enhanced. The most important experimental phenomenon is that RLS intensity of HSA-LMF and HSA-COL were enhanced in presence of COL and LMF, respectively. COL and LMF can interact with HSA-LMF and HSA-COL binary systems to form kinds of three-component complex (HSA-LMF-COL and HSA-COL-LMF) by hydrophobic and electrostatic interactions, which are the ain reason of enhanced RLS intensity. 2482 J. Chamani et al.
/ Journal of Luminescence 130 (2010) 2476–2486 Table 1 Stern–Volmer quenching and binding constants of binary and ternary interaction of LMF and COL with HSA at pH ? 7. 4. System KSV A 104 M A 1 KSV A 104 M A 1 lex ? 295 nm 1. 647 0.
22 1. 447 0. 21 0. 3117 0. 05 1. 367 0. 18 Ra Ka A 104 M A 1 lex ? 280 nm 2.
37 7 0. 19 4. 97 7 0. 21 0. 373 7 0. 04 4.
88 7 0. 22 Ka A 104 M A 1 lex ? 295 nm 1. 55 7 0. 13 1. 17 7 0. 24 0. 293 7 0. 05 1.
11 7 0. 08 n R lex ? 280 nm HSA-LMF HSA-COL HSA-COL-LMF HSA-LMF-COL a 2. 86 7 0. 21 5. 18 7 0. 17 0. 413 7 0. 05 5.
4 7 0. 19 0. 9981 0. 9975 0. 9931 0. 9976 0.
98 1. 14 0. 93 1. 09 0.
9973 0. 9987 0. 9954 0. 9985 R is the correlation coef? cient. -1. 4 0 -25 -1. 6 log [(F0-F)/F] -50 CD (mdeg) -2.
5 -2. 3 -2. 1 log [LMF] -1. 9 -1. 7 -1. 5 -75 -100 -125 -150 -175 -1. 8 -2 -2. 2 -2.
7 -200 200 210 220 230 Wavelength / nm 240 250 0. 7 0 0. 6 0. 5 log [(F0-F)/F] 0. 4 0. 3 0.
2 0. 1 0 -2 -1. 9 -1. 8 -1. 7 log [Col] -1. 6 -1.
5 -30 -60 -90 -120 -150 -180 200 CD (mdeg) 210 220 230 Wavelength / nm 240 250 Fig. 10. Far UV-CD spectra of the HSA-LMF (a) and HSA-COL (b) systems.
Arrows indicate the direction of the change. Fig. 9.Plots of log [(F0 A F)/F] vs.
log [Q] at pH? 7. 4, lex ? 280 nm, HSA-LMF (a) and HSA-COL (b). Fig. 12 shows the quenching curves of ternary (HSA-LMF-COL and HSA-COL-LMF) systems in comparison with the binary systems at both 280 and 295 nm. The quenching curves of HSALMF and HSA excited at 280 nm in the presence of COL overlap. It means that only tryptophanyl residue participates in the both HSA-LMF-COL and HSA-COL interactions (Fig. 12a).
This may suggest that COL does not affect or only weakly contribute to the quenching of HSA ? uorescence in the presence of LMF in ternary system (HSA-LMF-COL). Fig. 12b shows the ? orescence quenching of HSA-COL in the presence of LMF at 280 and 295 nm do not overlap. It shows that in the interaction of LMF with HSA-COL, both tryptophanyl and tyrosyl groups take part. On the contrary, LMF in? uences the quenching of HSA by COL. In the presence of COL, the HSA ? uorescence is signi? cantly less quenched by LMF than when COL is absent. The presence of COL probably inhibits the formation of the HSA-LMF complex or makes the HSA-LMF interaction more dif? cult.
COL reduces the af? nity of LMF to HSA, displaces the LMF molecules and hinders the transfer energy due to the hydrophobic interactions stabilizing the complex.COL is bound to HSA with a low af? nity. Therefore we should not exclude reciprocal interactions of both drugs within the subdomain, which is common for this kind of binary of LMF and COL to HSA. This phenomenon can be caused by tyrosyl residues involved in forming the HSA-COL complex in sub-domain IIA J. Chamani et al.
/ Journal of Luminescence 130 (2010) 2476–2486 2483 120 100 80 IRLS 60 F/ F0 1. 2 F/ F0 1 0. 8 0. 6 0.
4 1. 2 1 0. 8 0. 6 0.
4 0. 2 0 0 0. 2 0. 4 0.
6 COL/ HSA 0. 8 1 40 20 0 200 300 400 500 600 700 0. 2 0 0 0. 2 0.
4 0. 6 0. 8 1 COL/ HSA 1. 2 Wavelength / nm 600 500 400 1. 2 0. 8 F/ F0 0.
6 0. 4 0. 8 F/ F0 0. 2 0 0 0. 2 0. 4 0. 6 LMF/ HSA 0. 8 1 IRLS 300 1 200 100 0 200 0.
6 0. 4 0. 2 0 0 0. 2 300 400 500 600 700 Wavelength / nm Fig. 11.
(a) Resonance light scattering spectra of HSA-LMF in the presence of different concentrations of COL at pH 7. 4 and 298 K. (b) Resonance light scattering spectra of HSA-COL in the presence of different concentrations of LMF at pH? 7.
4 and 298 K. C (HSA) ? 1. 5 A 10 A 6 mol L A 1; C (LMF)/( A 10 A 3 mol L A 1), C (COL)/ ( A 10 A 3 mol L A 1). 0. 4 0. 6 LMF/ HSA 0. 8 1 and/or IIIB/IIIA, where Tyrs are located.
Fig. 12(b) con? rms the Tyrs affect in the formation of HSA-LMF in the presence of COL. The ? uorescence quenching of HSA-COL in the presence of LMF at 280 and 295 nm shows that both tryptophanyl and tyrosyl groups participate in the interaction between HSA-COL in the presence of LMF. The Stern–Volmer dependences of the ternary system for HSA excited at 280 and 295 nm is similar to binary system. Fig. 13 shows a positive derivation from the straight line for COL in the presence of LMF and for LMF in the presence of COL; indicate that both static and dynamic quenching is observed.
Table 1 shows the quenching constants of binary and ternary systems determined on the basis of Stern–Volmer equation. It can be shown that Stern–Volmer constant for the ternary system HSA-LMF (constant)-COL is almost similar to the binary system of HSA-COL. It means that the presence of LMF in the system is not effective to the formation of HSA-COL complex.
On the contrary, it can be observed from Table 1 that Stern–Volmer constant for the HSA-COL (constant)-LMF in a ternary system is lower than for the binary system of HSA-LMF.It can be concluded that the presence of COL in the system makes the formation of HSA-LMF complex dif? cult. These results indicate that a precaution is required in the treatment when the combination of LMF and COL is used at the same time, because each of them interferes with the binary of Fig.
12. (a) Comparison of quenching curves of HSA and HSA-LMF excited at 280 and 295 nm in the presence of various concentrations of COL. (b) Comparison of quenching curves of HSA and HSA-COL excited at 280 and 295 nm in the presence of various concentrations of LMF.Open symbols are at lex ? 280 nm and ? lled symbols are at lex ? 295 nm. the other one to HSA.
Table 1 shows the binding constants and binding sites for the ternary systems of HSA-LMF (constant)-COL and HSA-COL (constant)-LMF. The values of Ka and n at 298 K were obtained to be 4. 88 A 10 A 4 Lmol A 1 and 1. 09 for HSA-LMF (constant)-COL and 3. 73 A 10 A 5 Lmol A 1 and 0. 93 for HSA-COL (constant)-LMF, respectively, which implied that LMF dose not effect in the HSA-COL interaction, on the contrary, COL causes decrease in HSA-LMF interaction.
The KSV values in Table 1 con? m our results. To prove the possible in? uence of LMF and COL binding on the secondary structure of HSA-COL and HSA-LMF, the CD spectroscopy of ternary systems were measured. The fraction contents of different secondary structures for HSA-COL and HSA-LMF in the absence and presence of LMF and COL are shown in Table 2, respectively.
A decrease in tendency of the alpha-helixes content and an increase in tendency of beta-sheet, turn and unordered structure contents were observed with the increase in concentration of LMF to the HSA-COL complex 2484 J. Chamani et al. Journal of Luminescence 130 (2010) 2476–2486 4 Table 3 Docking of the COL and LMF into the whole rigid HSA. Ki Binding energy Intermolecular energy A 3. 91 A 6.
49 Electrostatic energy A 0. 22 A 0. 92 3 Colchicine in whole1H9Z Lome? oxacin in whole 1H9Z 11.
81 mM A 2. 63 67. 83 mM A 5.
69 F0/ F 2 1 All these phenomena and analyses revealed that the binding of LMF and COL to HSA in the binary and ternary systems induced some conformational and micro-environmental changes in HSA. 3. 4. Molecule modeling 0 0. 002 0. 004 [LMF] / mM 0.
006 0. 008 0 6 5 4 F0/ F 3 2 1 0 0 0. 01 0. 2 [COL] / mM Fig. 13.
(a) The Stern–Volmer plot of the HSA-COL in the presence of various concentrations of LMF at pH? 7. 4. (b) The Stern–Volmer plot of the HSA-LMF in the presence of various concentrations of COL at pH? 7. 4.
Open symbols are at lex ? 280 nm and ? lled symbols are at lex ? 295 nm. 0. 03 0. 04 Table 2 Fractions of secondary structure of HSA in the absence and presence of LMF and COL in binary and ternary systems at pH? 7. 40.
System HSA HSA-LMF HSA-COL HSA-COL-LMF HSA-LMF-COL ea 53. 94 50. 43 48. 27 48.
03 48. 17 eb 18. 33 19. 53 19.
65 19. 11 19. 68 et 13. 4 14.
34 14. 37 14. 51 14.
05 eu 14. 19 15. 70 17. 71 18.
35 18. 10 Results of docking of the ligands into the whole protein (protein set to rigid) are shown in Table 3. COL shows less af? nity to the protein than lome? oxacin. The determined high af? nity area is shown in Fig. 14.
Results of ? exible dockings are shown in Table 4. It can again be implied from the results that binding af? nity of LMF is far more than that of COL. It can also be concluded that previous attachment of COL reduces the af? nity of LMF to HSA. However, the initial binding of LMF enhances the af? ity of COL to the protein. COL or LMF when solely docked into the protein occupy the same pocket. COL forms a hydrogen bond with TYR161, for LMF though its orientation brings a nitrogen close to OH of the TYR161, it is a bit far away to form a stable hydrogen bond; however, it forms a hydrogen bond with LEU115 on the other end.
COL’s electrophile groups (oxygen containing groups) are surrounded by positively charged residues (ARG145, 196, 197, 117). Nitrogen of LMF’s pyridine stands close to HIS146’s aromatic group to form an electrostatic interaction.ARG186 is also close to the dihydroxyl portion of LMF though it is yet too far to form a hydrogen bond.
LMF is also stabilized in the active site by formation of a hydrogen bond (Fig. 14a and b). In two-step dockings, the ? rst ligand again ? lls the same pocket and the second ligand does not ? nd the opportunity to get inside and binds super? cially to the protein. COL when docked as secondary ligand is able to form a hydrogen bond with TYR138; however, there are no other noticeable interactions with the protein. This may describe lower af? nity of COL as a secondary ligand (Fig. 14c).The story is different for LMF as a secondary ligand; it forms a good hydrogen bond with PHE134. The aromatic group of PHE134 stands to dihydroxyl portion of LMF.
On the other end the NH of the pyrimidine group is close to the acidic side chain of GLU141, but there is a bit far to form a hydrogen bond 4. Conclusion The quenching of relative ? uorescence of human serum albumin proves to be an important tool for a better understanding regarding the crucial but speci? c changes occurring in the molecule. We studied the in? uence of an additional drug (LMF and COL) on changes in the af? nity of HSA transport.On the basis of binding and quenching constants determined from Stern– Volmer equation it has been stated that an af? nity to HSA is higher for COL than for LMF. The conclusions about the effect of one drug on the complex of the other with HSA have been presented by comparing the quenching of HSA ? uorescence in the binary and ternary systems and by analyzing the binding constant values. It has been shown that COL induces the release or displacement of LMF from its binding sites in HSA molecule.
ea, eb, et and eu are the fractions of alpha-helix, beta-sheet, turn and unordered coil. Table 2). As known, the secondary structure contents are related close to the biological activity of protein, thus a decrease in alphahelical content from 50. 43% to 48. 03% meant the loss of the biological activity of HSA upon interaction with LMF in the presence of COL bound with the amino acid residues of the main polypeptide chain of protein and destroyed their hydrogen bonding networks [37], making the HSA adopt a more incompact conformation state and its exposure to the hydrophobic cavities.
J. Chamani et al. / Journal of Luminescence 130 (2010) 2476–2486 2485 ? Fig. 14.Ternary interaction model between COL and HSA-LMF. (A,B) The binary interaction model between LMF and HSA. Only residues around 6. 5 A of LMF are displayed. The residues of HSA are represented using lines and the LMF structure is represented using a stick model. (C,D) The interaction model between COL and HSA-LMF. Protein atoms are shown in CPK representation and the LMF and COL structures are represented using a stick model. Table 4 Flexible docking of the COL and LMF into the whole HSA. Ki Binding energy A 17. 04 A 20. 59 A 24. 69 Intermolecular energy A 5. 1 A 7. 08 A 6. 2 Electrostatic energy A 0. 59 0. 06 A 0. 2 References [1] D. C. Carter, J. X. Ho, Adv. Protein Chem. 45 (1994) 153. [2] X. M. He, D. C. Carter, Nature 358 (1992) 209. [3] T. Wang, B. Xiang, Y. Wang, C. Chen, Y. Dong, H. Fang, M. Wang, Colloids Surf. B: Biointerfaces 65 (2008) 113. [4] M. Dockal, M. Chang, D. C. Carter, F. Ruker, Protein Sci. 9 (2000) 1455. [5] T. J. Peters, in: All About Albumin. Biochemistry, Genetics and Medical Applications, Academic Press, Inc. , New York, 1996. [6] P. J. Sadler, J. H. Viles, Inorg. Chem. 35 (1996) 4490. [7] I. Stanculescu, C. Mandravel, F.Delattre, D. Landy, P. Woisel, G. Surpateanu, J. Photochem. Photobiol. A: Chemistry 161 (2003) 79. [8] U. Kragh-Hansen, Pharmacol. Rev. 33 (1981) 17. [9] J. Tang, W. Wang, F. Luan, X. Chen, Int. J. Biol. Macromol. 37 (2005) 85. [10] H. M. Zhang, Y. Q. Wang, M. L. Jiang, Dyes Pigm. 82 (2009) 156. [11] W. He, Y. Li, J. Tian, H. Liu, Z. Hu, X. Chen, J. Photochem. Photobiol. A: Chem. 174 (2005) 53. [12] U. Kragh-Hansen, V. T. G. Chuang, M. Otagiri, Biol. Pharma. Bull. 25 (2002) 695. [13] J. H. Lin, D. M. Cocchetto, D. E. Duggan, Clin. Pharmacokinet. 12 (1987) 402. [14] Y. Ni, X. Zhang, S.Kokot, Spectrochim. Acta Part A 71 (2009) 1865. [15] X. Chen, J. C. Fan, Y. Wang, C. P. Fan, Z. C. Shang, Anal. Sci. 22 (2006) 427. [16] R. G. Berger, J. Am. Acad. Orthop. Surg. 2 (1994) 255. [17] J. Li, C. Ren, Y. Zhang, X. Liu, X. Yao, Z. Hu, J. Mol. Struct. 881 (2008) 90. [18] A. Sulkowska, M. Maciazek-Jurczyk, B. Bojko, J. Rownicka, I. Zubic-Skupien, E. Temba, D. Pentak, W. W. Sulkowski, J. Mol. Struct. 881 (2008) 97. [19] Y. Zhang, Y. Li, L. Dong, J. Li, W. He, X. Chen, Z. Hu, J. Mol. Struct. 875 (2008) 1. [20] Y. Q. Wang, H. M. Zhang, G. C. Zhang, S. X. Liu, Int. J. Biol.Macromol. 41 (2007) 243. [21] C. Z. Huang, Y. F. Li, X. D. Liu, Anal. Chim. Acta 375 (1998) 89. [22] A. M. Khan, S. Muzammil, J. Musarrat, Int. J. Biol. Macromol. 30 (2002) 243. [23] S. Era, K. B. Itoh, M. Sogami, K. Kuwata, T. Iwama, H. Yamada, H. Watari, Int. J. Pept. Protein Res. 35 (1990) 1. [24] SYBYL Software, Version 6. 9, Tripos Associates Inc. , St. Louis, 2002. [25] A. Pedretti, L. Villa, G. Vistoli, J. Mol. Graph. Model 21 (2002) 47. [26] B. Valeur, in: Molecular Fluorescence: Principles and Applications, Wiley, VCH, Weinheim, 2002. [27] S. Rubino, A. Gomez-Hens, M.Ualcarcel, Talanta 33 (1986) 633. [28] J. Jin, X. Zhang, J. Lumin. 128 (2008) 81. [29] J. Tang, F. Luan, X. Chen, Bioorg. Med. Chem. 14 (2006) 3210. [30] J. Zhu, X. Zhang, D. Li, J. Jin, J. Mol. Struct. 843 (2007) 38. Colchicine in ? exible active site Lome? oxacin in ? exible active site Colchicine into ? exible active site with Lome? oxacin already docked Lome? oxacin into ? exible active site with Colchicine already docked 325. 75 fM 809. 14 aM 803. 23 zM 149. 79 mM A 1. 12 7. 89 0. 3 It probably inhibits the access of LMF to the HSA due to hydrogen bonding which may occur between COL and HSA.On the contrary, LMF dose not affect to the formation of the HSA-COL complex. The simultaneous presence of these drugs in binding to HSA indicates that precautions are required in the treatment when the combinations of these drugs used at the same time. This is due to the fact that each of them interferes with the binding of the other one to HSA. Acknowledgements The ? nancial support of the Research Council of the Islamic Azad University-Mashhad Branch is gratefully acknowledged. The authors thank Dr. Ljungberg for English editing. 2486 J. Chamani et al. / Journal of Luminescence 130 (2010) 2476–2486 31] [32] [33] [34] B. Klajnert, M. Bryszewska, Bioelectrochemistry 55 (2002) 33. S. S. Lehrer, Biochemistry 10 (1971) 3254. M. R. Eftink, C. A. Ghiron, Biochemistry 15 (1976) 672. B. Bojko, A. Sulkowska, M. Maciazek, J. Rownicka, F. Njau, W. W. Sulkowska, Int. J. Biol. Macromol. 42 (2008) 314. [35] J. Kang, Y. Liu, M. X. Xie, S. Li, M. Jiang, Y. D. Wang, Biochim. Biophys. Acta 1674 (2004) 205. [36] A. Sulkowska, M. Maciazek, J. Rownicka, B. Bojko, D. Pentak, W. W. Sulkowski, J. Mol. Struct. 834–836 (2007) 162. [37] F. T. Cui, J. Fan, Z. D. Hu, Bioorg. Med. Chem. 12 (2004) 151.
