Flavonoid binding to human serum albumin

Biochemical and Biophysical Research Communications 398 (2010) 444–449

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Flavonoid binding to human serum albumin Alessandro Bolli a, Maria Marino a, Gerald Rimbach b, Gabriella Fanali c, Mauro Fasano c, Paolo Ascenzi a,d,* a

Department of Biology, University Roma Tre, Viale Guglielmo Marconi 446, I-00146 Roma, Italy Institute of Human Nutrition and Food Science, Christian Albrechts University, Hermann-Rodewald-Strasse 6, D-24098 Kiel, Germany c Department of Structural and Functional Biology, and Center of Neuroscience, University of Insubria, Via Alberto da Giussano 12, I-21052 Busto Arsizio (VA), Italy d Interdepartmental Laboratory of Electron Microscopy, University Roma Tre, Via della Vasca Navale 79, I-00146 Roma, Italy b

a r t i c l e

i n f o

Article history: Received 21 June 2010 Available online 27 June 2010 Keywords: Human serum albumin Flavonoid binding Daidzein Daidzein metabolites Genistein Naringenin Quercetin Oleate Thermodynamics Allostery

a b s t r a c t Dietary flavonoid may have beneficial effects in the prevention of chronic diseases. However, flavonoid bioavailability is often poor probably due to their interaction with plasma proteins. Here, the affinity of daidzein and daidzein metabolites as well as of genistein, naringenin, and quercetin for human serum albumin (HSA) has been assessed in the absence and presence of oleate. Values of the dissociation equilibrium constant (K) for binding of flavonoids and related metabolites to Sudlow’s site I range between 3.3  106 and 3.9  105 M, at pH 7.0 and 20.0 °C, indicating that these flavonoids are mainly bound to HSA in vivo. Values of K increase (i.e., the flavonoid affinity decreases) in the presence of saturating amounts of oleate by about two folds. Present data indicate a novel role of fatty acids as allosteric inhibitors of flavonoid bioavailability, and appear to be relevant in rationalizing the interference between dietary compounds, food supplements, and drugs. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Human serum albumin (HSA), the most abundant protein in plasma (reaching a blood concentration of about 7.0  104 M), provides a depot and carrier for many endogenous and exogenous compounds, affects pharmacokinetics of many drugs, and holds some ligands in a strained orientation which results in their metabolic modification. Furthermore HSA renders potential toxins harmless transporting them to disposal sites, accounts for most of the antioxidant capacity of human serum, and displays (pseudo-)enzymatic properties [1–8]. HSA is a single non-glycosylated all-a chain protein, constituted by 585 amino acids, containing three homologous domains (labeled I, II, and III). Each domain is made by two separate sub-domains (named A and B) connected by random coils. Inter-domain helical regions link sub-domains IB–IIA and IIB–IIIA (Fig. 1) (see [2,6,9,10]). The structural organization of HSA provides several ligand binding sites (Fig. 1). HSA display seven binding clefts hosting chemically-diverse ligands including fatty acids (FAs), and therefore Abbreviations: FA, Fatty acid; HSA, Human serum albumin. * Corresponding author at: Department of Biology, University Roma Tre, Viale Guglielmo Marconi 446, I-00146 Roma, Italy, fax: +39 06 57336321. E-mail address: [email protected] (P. Ascenzi). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.06.096

labeled FA1–FA7 (Fig. 1). In particular, FA3 and FA4 compose the so-called Sudlow’s site II (located in sub-domain IIIA) that recognizes preferentially aromatic carboxylates with an extended conformation, and FA7 represents the so-called Sudlow’s site I that binds especially bulky heterocyclic anions. Remarkably, warfarin and ibuprofen (commonly used as anti-coagulant and anti-inflammatory drugs, respectively) are considered to be stereotypical ligands for Sudlow’s site I and II, respectively [4,8,11–17]. Flavonoids are plant phenolic secondary metabolites widely distributed in the human diet (see [18,19]). Although flavonoid consumption has been associated with the prevention of several degenerative diseases [20], their bioavailability is often poor probably due to their interaction with plasma proteins [21]. Since scarce information is available on HSA recognition by flavonoids and their metabolites [21], the binding mode of isoflavones (daidzein, daidzein metabolites, and genistein), flavanones (naringenin), and flavanols (quercetin) to HSA has been investigated by automated docking simulation. Moreover, values of the equilibrium constant for flavonoid binding to HSA Sudlow’s site I (i.e., FA7) have been determined in the absence and presence of oleate. Oleate inhibits allosterically flavonoid binding to HSA, highlighting the role of FAs in modulating ligand binding to HSA. This appears to be relevant in rationalizing the interference between dietary compounds, food supplements, and drugs.

A. Bolli et al. / Biochemical and Biophysical Research Communications 398 (2010) 444–449

445

Fig. 1. HSA structure. FA binding sites are indicated by arrows and labelled. Oleate molecules bound to FA sites (represented as space filled) are shown in blue. The Trp214 residue is rendered with red sticks. Atomic coordinates have been taken from PDB entry 1GNI [32]. This picture was drawn with the UCSF Chimera software version 1.4.1 [47]. For further details, see text. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

2. Materials and methods

free flavonoid concentration (i.e., [flavonoid]), according to Eq. (1) [26]:

FA-free HSA (P96%), daidzein, genistein, naringenin, quercetin, and oleate were purchased from Sigma–Aldrich (St. Louis, MO, USA). 6,30 -Dihydroxydaidzein, 8-hydroxydaidzein, 8,30 -dihydroxydaidzein, 40 -daidzeinsulfate, 7-daidzeinsulfate, and 7,40 -daidzeindisulfate were purchased as previously reported [22,23]. All products were of analytical or reagent grade and were used without further purification. The stock HSA solution (=5.0  104 M) was prepared by dissolving the protein in 1.0  101 M phosphate buffer (pH 7.0) at 20.0 °C. The flavonoid stock solution (=1.0  103 M) was prepared by dissolving daidzein, 6,30 -dihydroxydaidzein, 8-hydroxydaidzein, 8,30 -dihydroxydaidzein, 40 -daidzeinsulfate, 7-daidzeinsulfate, 7,40 -daidzeindisulfate, genistein, naringenin, and quercetin in dimethylsulfoxide. The oleate stock solution (=1.0  102 M) was prepared by dissolving the FA in 1.0  102 M NaOH. Values of the dissociation equilibrium constant for flavonoid binding to HSA (i.e., K) were obtained spectrofluorimetrically, in the absence and presence of oleate, at pH 7.0 (1.0  101 M phosphate buffer) and 20.0 °C. Briefly, small aliquots of the flavonoid stock solutions were added to the buffered HSA solution; the final volume was 600 lL. The final HSA concentration was 5.0  106 M. The final flavonoid concentration ranged between 1.0  106 and 7.5  105 M. The final oleate concentration ranged between 1.0  106 and 1.0  103 M. The flavonoid-dependent changes of the intrinsic tryptophan fluorescence of HSA were recorded after incubation for 20 min, after each addition. Flavonoid-dependent spectrofluorimetric changes were recorded between 300 nm and 500 nm (the excitation wavelength was 280 nm) [24,25]. Test measurements performed after 2 h excluded slow kinetic events. The intrinsic fluorescence of ligands and dimethylsulfoxide (<15%) was subtracted from the flavonoid-induced quenching of HSA intrinsic fluorescence to determine values of K. Flavonoid binding to HSA was analyzed by plotting the molar fraction of HSA-flavonoid complexes (i.e., a) as a function of the

a ¼ ½flavonoid=ðK þ ½flavonoidÞ

ð1Þ

Data concerning the effect of the oleate concentration (i.e., 1.0  106 M 6 [oleate] 6 1.0  103 M) on the flavonoid affinity for HSA have been analyzed according to Eq. (2) [26]:

log K  ¼ log KfðH þ 10½oleate Þ=ðH þ 10½oleate Þg þ log ðH =HÞ

ð2Þ

where K and K* are the dissociation equilibrium constants for flavonoid binding to oleate-free and oleate-bound HSA, respectively, and H and H* are the dissociation equilibrium constants for oleate binding to flavonoid-free and flavonoid-bound HSA, respectively. Values of the molar fraction of the flavonoid-bound HSA (W), of the molar fraction of the flavonoid-free HSA (X), of the molar fraction of the HSA-bound flavonoid (Y), and of the molar fraction of the HSA-free flavonoid (Z) have been calculated according to Eqs. (3)–(6) [27,28]:

p W ¼ f½flavonoid=½HSA þ K=½HSA þ 1  ðð½flavonoid=½HSA þ K=½HSA þ 1Þ2  4  ½flavonoid=½HSAÞg=2

ð3Þ

X ¼1W

ð4Þ

Y ¼ W  ½HSA=½flavonoid

ð5Þ

Z ¼1Y

ð6Þ

Data were analyzed using the MatLab program (The Math Works Inc., Natick, MA, USA). The results are given as mean values of at least four experiments plus or minus the corresponding standard deviation. Automatic flexible ligand docking simulation for flavonid binding to HSA was performed by using Autodock 4.0 and the graphical user interface AutoDockTools [29–31]. The structure of HSA was downloaded from the Protein Data Bank (PDB entry: 1GNI) [32].

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A. Bolli et al. / Biochemical and Biophysical Research Communications 398 (2010) 444–449

Flavonoid structures were calculated using the Dundee PRODRG server [33]. Single bonds were allowed to rotate freely during the Monte Carlo simulated annealing procedure. The analysis of the conformational space was restricted to a cubic box of 70 Å edge centered on the coordinates of Sudlow’s site I. Monte Carlo simulated annealing was performed by starting from a temperature of 900 K with a relative cooling factor of 0.95/cycle, in order to reach the temperature of 5 K in 100 cycles [29–31].

A

3. Results and discussion

1.00 0.75

α 0.50 0.25

B 0.00

0

10

20

30

40

50

60

[Genistein] × 10 (M) 6

-4.7 -4.8

Log K*

Flavonoid binding to HSA was investigated by analyzing the perturbation of the intrinsic HSA fluorescence, mainly due to the unique Trp residue present at position 214 (Fig. 1) close to FA7 cleft (i.e., Sudlow’s site I). Flavonoid binding induces a decrease in the intrinsic HSA fluorescence (Fig. 2A), which appears flavonoid-independent (data not shown). The effect of flavonoids on the intrinsic HSA fluorescence is super-imposable to that previously reported for the binding of several endogenous and exogenous compounds (e.g., drugs), including warfarin, to Sudlow’s site I [2,6,8,10,24,34–39]. Therefore, the perturbation of the tryptophan fluorescence of HSA appears to be essentially independent of the ligands that bind to the FA7 site [2,6,8,10,24,34– 37,39]. Figure 2B shows the binding isotherms for genistein association with HSA, at pH 7.0 and 20.0 °C in the absence and presence of 4.0  105 M oleate. Flavonoid binding to HSA follows a simple equilibrium (Fig. 2B), values of the Hill coefficient n ranging between 0.95 and 1.05. Data analysis, according to Eq. (1), allowed the determination of K values for daidzein, 6,30 -dihydroxydaidzein, 8-hydroxydaidzein, 8,30 -dihydroxydaidzein, 40 -daidzeinsulfate, 7daidzeinsulfate, 7,40 -daidzeindisulfate, genistein, naringenin, and quercetin binding to HSA (Fig. 2B and Table 1). Values of K for flavonoid binding to HSA range between 3.3  106 and 3.9  105 M, at pH 7.0 and 20.0 °C, quercetin and 40 -daidzeinsulfate displaying the lowest and highest K values, respectively (Table 1). Values of K for quercetin and genistein binding to HSA determined here (Table 1) are in agreement with those previously reported [25,40]. Accounting for the physiological concentration of HSA (=7.0  104 M) [2], the average flavonoid plasma concentration (=1.0  106 M) and K values given in Table 1, data analysis according to Eqs. (3)–(6) indicates that the molar fraction of the flavonoid bound to HSA (i.e., Y) ranges between 0.95 and 0.99, and the molar fraction of the HSA-free flavonoid (i.e., Z) ranges between 0.01 and 0.05. An automated docking analysis of all flavonoids into Sudlow’s site I has been carried out in order to confirm flavonoid binding to FA7 (i.e., Sudlow’s site I) and to estimate values of intermolecular energies for ligand interaction with HSA. All flavonoids tested are able to enter the FA7 cleft with favourable values of intermolecular energy. It is worth mentioning that intermolecular energy values calculated from experimentally determined K values for flavonoid binding to HSA are in good agreement with those obtained from flavonoid docking calculations (Table 1). As shown in Fig. 2C and Table 1, oleate inhibits flavonoid binding to HSA dose-dependently over the 1.0  106 M–1.0  103 M concentration range, values of K increasing by about two folds, e.g. the affinity of genistein decreases from K = 7.8  106 M, in the absence of oleate, to K* = 1.5  105 M, in the presence of saturating levels of the FA. The analysis of data shown in Fig. 2C according to Eq. (2) allowed to determine values of the dissociation equilibrium constants for oleate binding to flavonoid-free and flavonoid-bound HSA (i.e., H = 1.5  105 M, and H* = 3.0  105 M, respectively).

-4.9 -5.0 -5.1 -5.2

C -6

-5

-4

-3

-2

Log [Oleate] Fig. 2. Flavonoid binding to HSA. (Panel A) Effect of genistein concentration on the fluorescence spectrum of HSA. Genistein concentration ranged between 0.0 M (spectrum a), 1.7  106 (spectrum b), 3.3  106 (spectrum c), 5.0  106 (spectrum d), 1.0  105 (spectrum e), 2.0  105 (spectrum f), 3.5  105 (spectrum g), and 5.0  105 M (spectrum h). (Panel B) Binding isotherms for genistein association with HSA, in the absence (circles) and presence (squares) of 4.0  105 M oleate. The continuous lines were calculated according to Eq. (1) by non-linear regression curve fitting with values of K and K* reported in Table 1. (Panel C) Effect of oleate concentration on values of K* for genistein binding to HSA. The filled square on ordinate indicates the dissociation equilibrium constant for genistein binding to oleate-free HSA. The continuous line was calculated according to Eq. (2) by non-linear regression curve fitting with K = 7.8  106 M, K* = 1.5  105 M, H = 1.5  105 M, and H* = 3.0  105 M. The flavonoid and oleate concentration corresponds to that of the free ligand. Where not shown, the standard deviation is smaller than the symbol. All data were obtained at pH 7.0 and 20.0 °C. For details, see text.

Since oleate binding to HSA does not perturb significantly the intrinsic emission of the Trp214 residue, the inhibitory effect of oleate on flavonoid binding to HSA should not be due to direct competition in the FA7 binding site, rather oleate exerts allosterically a structural perturbation of the FA7 cavity. Note that FA7 is a low affinity FA binding site [41]. On structural bases [32], we postulate that the interaction of oleate within the FA2 cleft induce a conformational rearrangement that involves amino acid side chains in contact with the flavonoid. Indeed, oleate makes a salt

447

A. Bolli et al. / Biochemical and Biophysical Research Communications 398 (2010) 444–449 Table 1 Values of thermodynamic parameters for flavonoid binding to HSA in the absence (K, DGexp, and DGcalc) and presence (K*) of oleate, at pH 7.0 and 20.0 °Ca. Flavonoid

a

K (M)

DGexp (kJ/mol)

DGcalc (kJ/mol)

K* (M)

Daidzein

2.6  10

5

26.0

23.5

3.5  105

6,30 -Dihydroxydaidzein

2.3  105

26.4

22.3

3.9  105

8-Hydroxydaidzein

2.7  105

26.0

28.2

4.3  105

8,30 -Dihydroxydaidzein

1.5  105

27.4

24.1

2.5  105

40 -Daidzeinsulfate

3.9  105

25.1

22.3

6.1  105

40 ,7-Daidzeindisulfate

4.5  106

30.4

30.0

2.0  105

7-Daidzeinsulfate

2.7  105

26.0

28.0

5.1  105

Genistein

7.8  106

29.1

22.3

1.5  105

Naringenin

5.3  106

30.1

22.5

1.5  105

Quercetin

3.3  106

31.2

33.2

7.2  106

Values of the intermolecular energies (DGcalc) were obtained from the docking simulation of flavonoids in Sudlow’s site I (FA7).

bridge with Arg257 [32], a residue involved together with Tyr150, His242, and His288 in the formation of an extended network of hydrogen bonds which contributes to the stability of bound flavonoids (e.g., by establishing polar contacts with phenolic OH groups) (Fig. 3). 4. Conclusion and perspectives The interest in flavonoids increased in the last decade principally due to epidemiological evidence indicating that diets rich in fruits and vegetables may reduce the incidence of chronic diseases such as cardiovascular diseases, diabetes, cancer, neuronal diseases, and stroke (see [18–20]). The mode of action by which flavonoids elicit potential protective effects against degenerative pathologies may principally involve the sex steroid hormone receptor activities [18–20,42]. However, flavonoids are subjected to various biotransformation reactions in different tissues [18]. In particular, during passage through the wall of the small intestine

into the circulatory system and subsequent passage into the liver, flavonoids are subjected to deglycosylation, glucuronidation, sulfation, and methylation [18,43]. Some of these metabolites maintain their positive biological activity(ies), even if this issue has not been extensively studied [44]. The role of flavonoids in preventing chronic diseases has been questioned by the low bioavailability of these compounds and their metabolites [43]. Importantly, our data indicate that flavonoids and their metabolites circulate in the human blood stream mostly bound to HSA (0.95 6 Y 6 0.99). Note that HSA, but not low density lipoproteins, acts as a carrier and a plasma store of quercetin metabolites guaranteeing their translocation to vascular targets [45]. The protective role of flavonoids, reflecting their transport and storage by HSA, can be inhibited by the increase of plasma FA levels (see Table 1). Accordingly, patients affected by the metabolic syndrome, characterized by high FA plasma levels, [46] may not fully benefit from potential protective effects of dietary flavonoids.

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Fig. 3. Binding of oleate and genistein to the FA2 and FA7 sites of HSA, respectively. Tyr150, His242, Arg257, and His288 involved in the formation of hydrogen bonds with oleate and genistein are shown. HSA and oleate coordinates have been taken from PDB entry 1GNI [32]. Genistein coordinates have been calculated by using Autodock 4.0 and the graphical user interface AutoDockTools [29–31]. For further details, see text.

Acknowledgments Authors wish to thank Dr. N. Botting, Dr. J. Coley, and Dr. B. Farley for providing us with the sulfated isoflavone test components. This work was partially supported by grants from the Ministero dell’Istruzione, dell’Università e della Ricerca of Italy (CLAR 2009, to P.A.) and from the Ministero della Salute of Italy (project Gender Medicine, to M.M.). References [1] G. Sudlow, D.J. Birkett, D.N. Wade, The characterization of two specific drug binding sites on human serum albumin, Mol. Pharmacol. 11 (1975) 824–832. [2] T. Peters Jr. (Ed.), All about Albumin: Biochemistry, Genetics and Medical Applications, Academic Press, San Diego and London, 1996. [3] U. Kragh-Hansen, V.T. Chuang, M. Otagiri, Practical aspects of the ligandbinding and enzymatic properties of human serum albumin, Biol. Pharm. Bull. 25 (2002) 695–704. [4] J. Ghuman, P.A. Zunszain, I. Petitpas, A.A. Bhattacharya, M. Otagiri, S. Curry, Structural basis of the drug-binding specificity of human serum albumin, J. Mol. Biol. 353 (2005) 38–52. [5] M. Fasano, S. Curry, E. Terreno, M. Galliano, G. Fanali, P. Narciso, S. Notari, P. Ascenzi, The extraordinary ligand binding properties of human serum albumin, IUBMB Life 57 (2005) 787–796. [6] P. Ascenzi, A. Bocedi, S. Notari, G. Fanali, R. Fesce, M. Fasano, Allosteric modulation of drug binding to human serum albumin, Mini Rev. Med. Chem. 6 (2006) 483–489. [7] P.A. Zunszain, J. Ghuman, T. Komatsu, E. Tsuchida, S. Curry, Crystal structural analysis of human serum albumin complexed with hemin and fatty acid, BMC Struct. Biol. 3 (2003) 6. [8] P. Ascenzi, M. Fasano, Serum heme-albumin: an allosteric protein, IUBMB Life 61 (2009) 1118–1122. [9] S. Sugio, A. Kashima, S. Mochizuki, M. Noda, K. Kobayashi, Crystal structure of human serum albumin at 2.5 Å resolution, Protein Eng. 12 (1999) 439–446. [10] P. Ascenzi, M. Fasano, Allostery in a monomeric protein: the case of human serum heme-albumin, Biophys. Chem. 148 (2010) 16–22. [11] S. Curry, H. Mandelkov, P. Brick, N. Franks, Crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites, Nat. Struct. Biol. 5 (1998) 827–835. [12] A.A. Bhattacharya, S. Curry, N.P. Franks, Binding of the general anesthetics propofol and halothane to human serum albumin. High resolution crystal structures, J. Biol. Chem. 275 (2000) 38731–38738. [13] A.A. Bhattacharya, T. Grüne, S. Curry, Crystallographic analysis reveals common modes of binding of medium and long-chain fatty acids to human serum albumin, J. Mol. Biol. 303 (2000) 721–732.

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