Journal of Molecular Structure 798 (2006) 6974 www.elsevier.

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Antioxidant avonoids bind human serum albumin

C.D. Kanakis a, P.A. Tarantilis a, M.G. Polissiou H.A. Tajmir-Riahi b,*
a

a,*

, S. Diamantoglou b,

Laboratory of Chemistry, Department of Science, Agricultural University of Athens, 75 Iera Odos, 118 55 Athens, Greece b bec at Trois-Rivie res, TR, Que., Canada G9A 5H7 Department of ChemistryBiology, University of Que Received 21 December 2005; received in revised form 28 February 2006; accepted 1 March 2006 Available online 27 April 2006

Abstract Human serum albumin (HSA) is a principal extracellular protein with a high concentration in blood plasma and carrier for many drugs to dierent molecular targets. Flavonoids are powerful antioxidants and prevent DNA damage. The antioxidative protections are related to their binding modes to DNA duplex and complexation with free radicals in vivo. However, avonoids are known to inhibit the activities of several enzymes such as calcium phospholipid-dependent protein kinase, tyrosine protein kinase from rat lung, phosphorylase kinase, phosphatidylinositol 3-kinase and DNA topoisomerases that exhibit the importance of avonoid-protein interaction. This study was designed to examine the interaction of human serum albumin (HSA) with quercetin (que), kaempferol (kae) and delphinidin (del) in aqueous solution at physiological conditions, using constant protein concentration of 0.25 mM (nal) and various drug contents of 1 lM1 mM. FTIR and UVvis spectroscopic methods were used to determine the polyphenolic binding mode, the binding constant and the eects of avonoid complexation on protein secondary structure. The spectroscopic results showed that avonoids are located along the polypeptide chains through H-bonding interactions with overall anity constant of Kque = 1.4 104 M1, Kkae = 2.6 105 M1 and Kdel = 4.71 105 M1. The protein secondary structure showed no alterations at low pigment concentration (1 lM), whereas at high avonoid content (1 mM), major reduction of a-helix from 55% (free HSA) to 4246% and increase of b-sheet from 15% (free HSA) to 1719% and b-anti from 7% (free HSA) to 1020% occurred in the avonoidHSA adducts. The major reduction of HSA a-helix is indicative of a partial protein unfolding upon avonoid interaction. 2006 Elsevier B.V. All rights reserved.
Keywords: Flavonoid; Crocus sativus L.; Protein; Binding mode; Binding constant; Secondary structure; FT-IR; UVvis spectroscopy

1. Introduction Human serum albumin (HSA) is a principal extracellular protein with a high concentration in blood plasma (40 mg/ml) [13]. HSA is a globular protein composed

Abbreviations: Fla, avonoid; quer, querectin; kae, kaempferol; del, delphinidin; HSA, human serum albumin; FTIR, Fourier transform infrared. * Corresponding authors. Tel.: +30 210 529 4241; fax: +30 210 529 4265 (M.G. Polissiou); tel.: +1 819 376 5011x3310; fax: +1 819 376 5084 (H.A. Tajmir-Riahi). E-mail addresses: mopol@aua.gr (M.G. Polissiou), tajmirri@uqtr.ca (H.A. Tajmir-Riahi). 0022-2860/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2006.03.051

of three structurally similar domains (I, II and III), each containing two subdomains (A and B) and stabilized by 17 disulphide bridges [47]. Aromatic and heterocyclic ligands were found to bind within two hydrophobic pockets in subdomains IIA and IIIA, namely site I and site II [47]. Seven binding sites are localized for fatty acids in subdomains IB, IIIA, IIIB and on the subdomain interfaces [2]. HSA has also high anity metal binding site at the N-terminus [1]. The multiple binding sites underlie the exceptional ability of HSA to interact with many organic and inorganic molecules and make this protein an important regulator of intercellular uxes, as well as the pharmacokinetic behavior of many drugs [18].

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Diets rich in antioxidants contribute to a lower incidence of several major chronic diseases [911]. In particular, cancer development or growth is inhibited by antioxidants. Antioxidants delay or prevent the oxidation of a given substrate by free radicals [12,13]. Crocus sativus L. ower is known and the widely used spice saron comes from its dried red stigmas. Its petals though are a by-product rich in avonoids [14] and can be used as a potential source of dietary avonoids. Flavonoids (Scheme 1) are naturally occurring polyphenolic compounds used as food supplements. Dietary avonoids are receiving increasing attention as potential protectors against a variety of human diseases, in particular cardiovascular disease [911] and cancer [12,13,15]. A large number of mechanisms of action have been attributed to avonoids, including antioxidant properties [1619] and eects on enzymes and signal transduction pathways. However, avonoids are known to inhibit the activities of several enzymes such as calcium phospholipid-dependent protein kinase, tyrosine protein kinase from rat lung, phosphorylase kinase, phosphatidylinositol 3-kinase and DNA topoisomerases that exhibit the importance of avonoidprotein interaction [2023]. Recent reports have shown a high anity for quercetin towards HSA complexation [2429]. Therefore, the interaction of antioxidant polyphenols with HSA has a major biological importance and can be used as a model for avonoidprotein interaction. Thus, it was of interest to study the interaction of human serum albumin with avonoids in aqueous solution and to
OH OH

examine the eects of polyphenol complexation on protein secondary structure. We now report the FT-IR and UV vis spectroscopic results on the interaction of human serum albumin with quercetin, kaempferol and delphinidin in aqueous solution at physiological conditions with constant HSA concentration (0.25 mM) and various avonoid contents (1 lM1 mM). Structural information regarding the avonoid binding mode, binding constant and the eects of drug complexation on the protein secondary structure are provided here. 2. Experimental 2.1. Materials and methods Human serum albumin fraction V was purchased from Sigma Chemical Co., and used as supplied. Quercetin, delphinidin and kaempferol were purchased from Extrasynthese (France). Other chemicals were of reagent grade and used without further purication. 2.2. Preparation of stock solutions Human serum albumin was dissolved in aqueous solution (20 mg/ml or 0.5 mM) containing potassium dihydrogen phosphate buer 0.1 M and 0.1 M NaOH. The protein concentration was determined spectrophotometrically using the extinction coecient of 36,500 M1 cm1 at 280 nm [8]. In this study, HSA did not have its fatty acids removed, thus being in a situation closer to that which occurs in vivo. Indeed, under normal physiological conditions, between 0.1 and 2 fatty acid molecules are bound to the albumin [2,7]. The avonoid solutions (0.0020 2 mM) were prepared in ethanol/water mixture (25/75%). It should be noted that 25% ethanol does not aect protein structure, while higher ethanol content (>50%) can alter protein structure. In the nal step, avonoid solution was added dropwise to the protein solution with constant stirring to ensure the formation of homogeneous solution and to attain the nal avonoid concentrations of 0.001, 0.01, 0.1 and 1 mM with a nal protein concentration of 0.25 mM (or with drug/protein molar ratios of 0.004, 0.04, 0.4 and 4) for infrared measurements. The pH of the solution was adjusted to 6.97.2 by pH meter ORION Model 210A. 2.3. Absorption spectroscopy

HO

OH OH O

Quercetin
OH

HO

OH OH O

Kaempferol
OH OH + O OH

HO

OH OH

Delphinidin

Scheme 1.

The UVvis spectra are recorded on Jasco UVvis, V-550 spectrophotometer with nal pigment concentrations of 0.0010.1 mM and constant protein concentration of 0.025 mM. The values of the binding constants K were obtained according to the method described by Zhong et al. [31] and Stephanos [32]. By assuming that there is only one type of interaction between avonoids and protein in aqueous solution, the Eqs. (1) and (2) can be established:

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HSA flavonoid HSA : flavonoid K HSA : flavonoid HSAflavonoid

1 2

where K is the binding constant and avonoids = quercetin, kaempferol and delphinidin Assuming [HSA : avonoid] = CB K CB ; C HSA C B C flavonoid C B 3

where CHSA and Cavonoid are the analytical concentrations of HSA and avonoid in solution, respectively. According to the BeerLambert law: C HSA A0 eHSA A A0 CB eB 4 5

ver. 3.1). Thus the root-mean square (rms) noise of every spectrum was calculated. By means of the second derivative in the spectral region 16001700 cm1 eight major peaks for HSA and the complexes were resolved. The above spectral region was deconvoluted by the curve-tting method with the LevenbergMarquadt algorithm and the peaks corresponding to a-helix (16561658 cm1), b-sheet (16141640 cm1), turn (16601677 cm1), random coil (16421648 cm1) and b-antiparallel (16801692 cm1) were adjusted and the area was measured with the Gaussian function. The area of all the component bands assigned to a given conformation was then summed up and divided by the total area [36]. The curve-tting method analysis was performed using the PEAKSOLVE software (ver. 1.05) of the Galactic Industries Corporation (Peaksolve Guide, 1991). 3. Results and discussion 3.1. FT-IR spectra of avonoidHSA adducts Evidence regarding the avonoidHSA complexation comes from infrared spectroscopic results. Since there was no major spectral shifting for the protein amide I band at 1656 cm1 (mainly C@O stretch) and amide II band at 1545 cm1 (CN stretching coupled with NH bending modes) [33] upon pigment interaction, the dierence spectra [(protein solution + avonoid solution) (protein solution)] were obtained, in order to monitor the intensity variations of these vibrations and the results are shown in Fig. 1. Similarly, the infrared self-deconvolution with second derivative resolution enhancement and curve-tting procedures [35] were used to determine the protein secondary structures in the presence of avonoid and the results are presented in Fig. 2.

where A0 and A are the absorbance of HSA at 280 nm in the absence and presence of avonoid, respectively. eHSA and eB are the molar extinction coecients of HSA and the bound avonoid, respectively. is the light path of the cuvette (1 cm). By displacing eHSA and eB in Eq. (3) by Eqs. (4) and (5), Eq. 6 can be deduced: A0 eHSA eHSA 1 . A A0 eB eB K C flavonoid 6

Thus, the double reciprocal plot of 1/(A A0) vs. 1/Cavonoid is linear and the binding constant (K) can be estimated from the ratio of the intercept to the slope [31,32]. 2.4. FTIR spectroscopic measurements Infrared spectra were recorded on a Nicolet Magna 750 FT-IR spectrophotometer (DTGS detector, Ni-chrome source and KBr beamsplitter) with a total of 100 scans and resolution of 4 cm1. Spectra were collected and manipulated using the OMNIC (ver. 3.1) software supplied by the manufacturer of the spectrophotometer. Spectra (dried lm) were recorded after 1 h of incubation, using AgBr windows. The dierence spectra [(protein solution + avonoid solution) (protein solution)] were generated using the polypeptide antisymmetric and symmetric CH stretching bands [33], located at 29002800 cm1 as internal standard. Details regarding infrared spectral treatment are given in our recent publication [34]. 2.5. Determination of protein secondary structure Analysis of the secondary structure of HSA and its avonoid complexes was carried out on the basis of the procedure reported [35]. The protein secondary structure is determined from the shape of the amide I band, located at 16501660 cm1. The FT-IR spectra were smoothed and their baselines were corrected automatically using the built-in software of the spectrophotometer (OMNIC

Fig. 1. FTIR spectra in the region of 18001500 cm1 of hydrated lm (physiological pH), for free HSA (top rst curve) and dierence spectra for avonoidHSA adducts (bottom six curves) obtained at dierent pigment concentrations.

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Fig. 2. Curve-tted amide I region (17001600 cm1) with secondary structure determination of the free HSA and its avonoid complexes in aqueous solution with 1 mM pigment concentration. The percentages of turns, random, a-helix, parallel b-sheet and antiparallel (b-anti) structures were calculated as the ratio of the corresponding peak areas to the total of all seven amide I peaks.

At low avonoid concentration (1 lM), negative features centered at 16551661 cm1 (amide I) and 15391543 cm1 (amide II) were observed in the dierence spectra of the quercetin, kaempferol and delphinidinHSA complexes (Fig. 1, 1 lM). The observed spectral changes are due to the pigment interaction with protein C@O and CN groups. The interaction of avonoid with protein CN group is also evident from the shift of the amide A band at 3303 cm1 (peptide NH stretching mode) [33] towards a lower frequency at 3290 cm1 (spectra not shown). As the avonoid concentration was increased to 1 mM, the negative peaks exhibited increase in intensity and shifting towards higher frequencies, in the spectra of the avonoidHSA complexes (Fig. 1, 1 mM). The reduction in intensity and shifting of these derivative bands are related to continued drug-HSA complexation and major alterations of protein secondary structure at high avonoid concentrations. A quantitative analysis of the protein secondary structure for the free HSA and its avonoid complexes in H2O is given in Fig. 2. The free HSA contained major a-helix (1658 cm1) 55%, b-sheet (1614, 1622 and 1633 cm1) 15%, turn (1677 cm1) 13%, random (1645 cm1) 10% and b-antiparallel (1688 cm1) 7%. The b-sheet structure is composed of three components at 1614 cm1 (inter b-strand), 1622 cm1 (intra b-strand), and 1633 cm1 (hydrated) (Fig. 2), that are consistent with the recent

spectroscopic studies of the human serum albumin [37 39]. It should be noted that the C@O stretching and OH bending modes of avonoids occur in the region of 1620 1660 cm1, where the protein amide I band located. In order to avoid overlapping and interfering with our protein amide I band curve-tting, the contributions of the avonoid bands in this region are subtracted. The amount of protein a-helix structure calculated here (55%) is consistent with the values obtained by other spectroscopic methods [3739]. However, X-ray structural analysis showed higher a-helix content for HSA (60%) than those obtained by spectroscopic methods [2,5]. The reason can be due to the sample preparation and the protein structural arrangements in the solution (spectroscopy) and solid state (X-ray). Upon avonoid complexation, the a-helix structure was reduced from 55% to 4246%, while b-sheet increased from 15% to 1719% and the b-anti also increased from 7% to 1020% at high quercetin, kaempferol and delphinidin concentration (Fig. 2, 1 mM, Cav./CHSA = 4). It is important to note that at low avonoid content (1 lM, Cav./CHSA = 0.004), no major alterations of protein secondary structure occurred upon pigment interaction (spectra not shown). These results are in agreement with those obtained by Xie et al. [30] when they studied the interaction of HSA with the avonoid hesperitin where in high avonoid content they observed a similar reduction in a-helix and an increase in b-sheet while no obvious changes were observed in low drug

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concentration (Cav./CHSA < 0.1). The major alterations of HSA secondary structures are due to a partial protein unfolding at high avonoid concentrations. Similar protein conformational changes were observed upon protonation and heat denaturation [4042]. 3.2. Stability of avonoidHSA adducts Major reduction of intensity and shifting was observed for characteristic UV absorption bands of quercetin at 374 nm, kaempferol at 368 nm and delphinidin at 572 nm, upon HSA interaction (Fig. 3). This is indicative of a major pigment structural changes in these avonoid protein complexes. Zsila et al. [26] reported that as a result of specic binding of quercetin to HSA, its absorption spectrum exhibits red shift. The same observation was made here for quercetin, kaempferol and delphinidin. In addition to the observed red shift, the decrease in absorption intensity suggests that quercetin binds HSA in ionic form in non-planar conformation [26]. This decrease in absorption intensity is observed here also for kaempferol

Fig. 4. The plot of 1/(A A0) vs. 1/avonoid concentration for HSA and its avonoid adducts, where A0 is the initial absorption band of free HSA (280 nm) and A is the recorded absorption at dierent pigment concentrations (0.0010.1 mM) and nal HSA content of 0.025 mM.

and delphinidin. The binding constants of avonoid HSA complexes were calculated as described in Section 2.1. The double reciprocal plot of pigment concentrations versus protein absorption is shown in Fig. 4. The overall binding constants for pigmentprotein adducts were estimated to be Kque = 1.4 104 M1, Kkae = 2.6 105 M1 and Kdel = 4.71 105 M1 (Fig. 4). The binding constant of quercetin is in good agreement with those reported in the literature [26,27]. The larger stability of delphinidin HSA adduct over quercetin and kaempferol complexes can be attributed to the presence of a positive charge on delphinidin (Scheme 1), which can provide additional binding site with protein and stabilize delHSA complexes. This could be the reason for the decrease in the absorption intensity of delphinidin. The overall association constants calculated for the avonoidHSA adducts show a weak pigmentprotein interaction (H-bonding) [26] with respect to the other strong ligandprotein complexes with binding constants ranging from 106 to 108 M1 [43]. Similar binding constants were observed for several pigments, polymers and biogenic polyamineHSA complexes [34,44,45]. 4. Conclusions
Fig. 3. UVvis spectra characteristics of avonoids and their HSA adducts for quercetin at 374 nm (A), kaempferol at 368 nm (B) and delphinidin at 572 nm (C) with nal HSA concentration of 0.025 mM and avonoid contents of 0.05 mM.

Recent structural modeling and spectroscopic results showed moderate binding for avonoidHSA complexes [2628]. Flavonoids are mainly located in the vicinity of

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C.D. Kanakis et al. / Journal of Molecular Structure 798 (2006) 6974 [11] E. Middelton, C. Kandaswami, T.C. Theoharides, Pharmacol. Rev. 52 (2000) 673. [12] B. Halliwell, R. Aeschbach, J. Loliger, O.I. Arouma, Food Chem. Toxicol. 33 (1995) 617. [13] S. Fleschin, M. Fleschin, S. Nita, E. Pavel, V. Magearu, Roun. Biotechnol. Lett. 5 (2003) 479. [14] R. Nrbaek, K. Brandt, J.K. Nielsen, M. rgaard, N. Jacobsen, Biochem. Syst. Ecol. 30 (2002) 763. [15] L.R. Ferguson, Mutat. Res. 475 (2001) 89. [16] D. Amic, D. Davidovic, D. Beleso, N. Trinajstic, N. Croatica, Chem. Acta 76 (2003) 55. [17] S.R. Husain, J. Cillard, P. Cillard, Phytochemistry 26 (1987) 2489. [18] G.C. Jagetia, V.A. Venkatesha, T.K. Reddy, Mutagenesis 8 (2003) 337. [19] I.B. Afanasev, E.A. Osttrachovitch, N.E. Abromanova, L.G. Korkina, Biochem. Pharmacol. 50 (1995) 627. [20] A.K. Srivastava, Biochem. Biophys. Res. Commun. 131 (1985) 1. [21] W.F. Matter, R.F. Brown, C. Vlahos, Biochem. Biophys. Res. Commun. 186 (1992) 624. [22] R.S. Brinkworth, M.J. Stoermer, D.P. Fairlie, Biochem. Biophys. Res. Commun. 188 (1992) 631. [23] F. Boege, T. Straub, A. Kehr, C. Boesenberg, K. Christiansen, A. Andersen, F. Jakob, J. Kohrle, J. Biol. Chem. 271 (1996) 2262. [24] B. Sengupta, P.K. Sengupta, Biochem. Biophys. Res. Commun. 299 (2002) 400. [25] M.I. Kaldas, U.K. Walle, H. Vander Houde, J.M. McMillan, T. Walle, J. Agric. Food Chem. 53 (2005) 4194. [26] F. Zsila, Z. Bikadi, M. Simonyi, Biochem. Pharmacol. 65 (2003) 447. [27] C. Dufour, O. Dangles, Biochim. Biophys. Acta 1712 (2005) 164. [28] S. Bi, L. Ding, Y. Tian, D. Song, X. Zhou, X. Liu, H. Zhang, Mol. Struct. 703 (2004) 37. [29] B. Sengupta, P.K. Sengupta, Biopolymers 72 (2003) 427. [30] M.X. Xie, X.Y. Xu, Y.D. Wang, Biochim. Biophys. Acta 1724 (2005) 215. [31] W. Zhong, Y. Wang, J.-S. Yu, Y. Liang, K. Ni, S. Tu, J. Pharm. Sci. 93 (2004) 1039. [32] J.J. Stephanos, J. Inorg. Biochem. 62 (1996) 155. [33] S. Krimm, J. Bandekar, Adv. Protein Chem. 38 (1986) 181. [34] A. Ahmed Ouameur, E. Mangier, S. Diamantoglou, R. Rouillon, R. Carpentier, H.A. Tajmir-Riahi, Biopolymers 73 (2004) 503. [35] D.M. Byler, H. Susi, Biopolymers 25 (1986) 469. [36] A. Ahmed, H.A. Tajmir-Riahi, R. Carpentier, FEBS Lett. 363 (1995) 65. [37] L. Boulkanz, N. Balcar, M.H. Baron, Appl. Spectrosc. 49 (1995) 1737. [38] E. Bramanti, E. Benedetti, Biopolymers 38 (1996) 639. [39] J.T. Pelton, L.R. McLean, Anal. Biochem. 277 (2000) 167. [40] F.S Parker, Applications of Infrared, Raman and Resonance Spectroscopy in Biochemistry, Plenum Press, New York, 1983. [41] W.K. Surewicz, M.A. Moscarello, H.H. Mantsch, J. Biol. Chem. 262 (1987) 8598. [42] I.E. Holzbaur, A.M. English, A.A. Ismail, Biochemistry 35 (1996) 5488. [43] U. Kragh-Hansen, Dan. Med. Bull. 37 (1990) 57. [44] A. Ahmed Ouameur, R. Marty, H.A. Tajmir-Riahi, Biopolymers 77 (2005) 129. [45] C. Ragi, M.R. Sedaghat-Herati, A. Ahmed Ouameur, H.A. TajmirRiahi, Biopolymers 78 (2005) 231.

protein subdomain IIA with both H-bonding and ionic interactions [26,27]. Quercetin binding involves both H-bonding and ionic interactions via ring OH groups with a moderate binding constant of 1.46 104 M1 [26]. We have also calculated similar binding constant K = 1.40 104 M1 for quercetinHSA adducts. However, larger binding constants were obtained for kaempferol K = 2.6 105 M1 and delphinidin K = 4.71 105 M1. The extra stability associated with delphinidin can be attributed to the presence of a positive charge on polyphenolic ring, which interacts with protein negative charges and stabilizes delHSA complexes. Additional stability of kaempferol over quercetin complexes can be due to the possible ionization of the ring OH group, which can interact with protein amino groups more eectively. The antioxidant avonoids induce no protein conformational changes at low pigment concentration (1 lM, Cav./CHSA = 0.004), while major reduction of a-helix from 55% (free HSA) to 4246% and increase of b-sheet from 15% (free HSA) to 1719% and b-anti from 7% (free HSA) to 1020% occur at high avonoid content (1 mM, Cav./CHSA = 4). The reduction of protein a-helical structure upon avonoid interaction was also reported for hesperetinHSA adducts [30]. This indicates that avonoid complexation causes protein unfolding at high pigment concentrations. Acknowledgments This work was supported by grants from Natural Sciences and Engineering Research Council of Canada (NSERC), NATO (Grants LST.NR.CLG. 981092) and the Agricultural University of Athens, Greece. References
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