Keywords daidzein; genistein; serum albumin; interaction studies; binding pocket Correspondence Dr A.G.. The bind-ing of genistein to human serum albumin HSA has been investigated by equ
Trang 1with human serum albumin
H G Mahesha1, Sridevi A Singh1, N Srinivasan2and A G Appu Rao1
1 Department of Protein Chemistry & Technology, Central Food Technological Research Institute, Mysore, India
2 Molecular Biophysics unit, Indian Institute of Science, Bangalore, India
Isoflavones) naturally occurring oestrogen-like
mole-cules) play a beneficial role in the prevention of
osteoporosis Light is yet to be thrown on the cellular
mechanisms through which dietary isoflavones enhance
the retention of calcium in the bone [1] They offer
alternative therapies for a range of hormone dependent
conditions such as cancer, menopausal symptoms,
car-diovascular disease and osteoporosis [2] Isoflavones
have also been demonstrated to act as oestrogen
mim-ics via classical mediated signalling, apart from
func-tioning as tyrosine kinase inhibitors [3,4] and can
interact with oestrogen receptors It is believed that
their structural similarity to 17b-oestradiol molecule bears explanation for this mimicry [5] These molecules share several features in common with the oestradiol structure (Fig 1), including a pair of hydroxyl groups separated by a similar distance One of the hydroxyl groups is a substituent of the aromatic A ring, while the second lies at the opposite end of the molecule [6] However, the interaction with the receptors is not equivalent, since both the occupancy time and affinity are significantly less for isoflavones In addition, small differences in the structures of individual isoflavones drastically alter their oestrogenicity
Keywords
daidzein; genistein; serum albumin;
interaction studies; binding pocket
Correspondence
Dr A.G Appu Rao, Department of Protein
Chemistry & Technology, Central Food
Technological Research Institute,
Mysore 570 020, India
Fax: +91 821 2517233
Tel: +91 821 2515331
E-mail: appurao@cftri.res.in
(Received 5 October 2005, accepted
22 November 2005)
doi:10.1111/j.1742-4658.2005.05071.x
Genistein and daidzein, the major isoflavones present in soybeans, possess
a wide spectrum of physiological and pharmacological functions The bind-ing of genistein to human serum albumin (HSA) has been investigated by equilibrium dialysis, fluorescence measurements, CD and molecular visuali-zation One mole of genistein is bound per mole of HSA with a binding constant of 1.5 ± 0.2· 105m)1 Binding of genistein to HSA precludes the attachment of daidzein The ability of HSA to bind genistein is found
to be lost when the tryptophan residue of albumin is modified with N-bromosuccinimide At 27C (pH 7.4), van’t Hoff’s enthalpy, entropy and free energy changes that accompany the binding are found to be )13.16 kcalÆmol)1, )21 calÆmol)1K)1 and )6.86 kcalÆmol)1, respectively Temperature and ionic strength dependence and competitive binding meas-urements of genistein with HSA in the presence of fatty acids and 8-ani-lino-1-naphthalene sulfonic acid have suggested the involvement of both hydrophobic and ionic interactions in the genistein–HSA binding Binding measurements of genistein with BSA and HSA, and those in the presence
of warfarin and 2,3,5-tri-iodobenzoic acid and Fo¨rster energy transfer measurements have been used for deducing the binding pocket on HSA Fluorescence anisotropy measurements of daidzein bound and then dis-placed with warfarin, 2,3,5-tri-iodobenzoic acid or diazepam confirm the binding of daidzein and genistein to subdomain IIA of HSA The ability of HSA to form ternery complexes with other neutral molecules such as war-farin, which also binds within the subdomain IIA pocket, increases our understanding of the binding dynamics of exogenous drugs to HSA
Abbreviations
ANS, 8-anilino-1-naphthalene sulfonic acid; HSA, human serum albumin; TIB, 2,3,5-tri-iodo benzoic acid.
Trang 2Genistein, daidzein and glycitein are the major
iso-flavones of raw soybeans Both ingestion and injection
of genistein can affect the development of the
repro-ductive system, decrease thymic weight and delayed
type hypersensitivity response, modulate immune
response or reduce thyroid peroxidase [7] Soybeans
are the only natural dietary source of these diphenolic
compounds These molecules function as antioxidants
in plants and act as partial agonists of oestrogens in
mammalian tissues [8] Genistein exerts its influence on
oesteoblast-like cells, at dietarily achievable
concentra-tions The beneficial effects of genistein may be
partic-ularly related to the inhibition of oesteoclastogenesis
(mediated by cytokine production in oesteoblasts) [9]
Daidzein and genistein share similarity in structure
except for an additional hydroxyl group on the A ring
of genistein However, genistein may have up to
five-to sixfold greater oestrogenic activity in some assays [10] Genistein, in micromolar concentrations, alters the function of numerous ion channels and other mem-brane proteins [11]
Binding of isoflavones to serum albumin can be an important determinant of pharmaco-kinetics that restricts the unbound concentration and affects dis-tribution and elimination Human serum albumin (HSA)) a 585-residue monomeric protein ) is the major component of blood plasma and other intersti-tial fluid of body tissues [12] The binding sites for both endogenous and exogenous ligands on HSA are limited Binding of drug to the protein may be affected
by a variety of factors and genetic polymorphism could be one of them
Structural studies have helped map the locations of fatty acids and primary drug binding sites on the pro-tein [12,13] Fatty acid binding sites are distributed throughout the protein and involve all six subdomains while many drugs bind to one of the two primary binding sites on the protein known as drug sites I and
II [14] These investigations have used competitive binding methods to arrive at the selectivity of the pri-mary drug-binding site Drug site I, where warfarin binds, has been characterized to be conformationally adaptable with up to three subcompartments [15] Fur-ther work on site I and site II drugs is needed to build
a more comprehensive picture of drug interactions with HSA, which may provide a structural basis for a rational approach for drug design to exploit or exclude the impact of HSA on drug delivery [16] Most ligands are bound reversibly and the typical binding constants (Kb) range from 104to 106 m)1
Proteins⁄ enzymes are often the target molecules for all the isoflavones’ interactions We have explored the interaction of isoflavones with HSA at the molecular level using direct ligand binding measurements) equi-librium dialysis and intrinsic protein⁄ isoflavone fluo-rescence) as a probe, for both quantitative and qualitative perspectives, in detail The energetics of interactions has been followed by varying binding con-stant with temperature The nature of the interaction was identified by temperature and ionic strength dependence of binding constant, competitive ligand binding measurements with fatty acids and 8-anilino-1-naphthalene sulfonic acid (ANS) The binding pocket for isoflavones on HSA has been identified based on binding measurements of warfarin or 2,3,5-tri-iodo benzoic acid (TIB), in the presence of genistein, Fo¨rster energy transfer measurements and binding of genistein with HSA and BSA Based on the experimental work the possibility of simultaneous binding of warfarin and
OH
17 bb-oestradiol
Warfarin
Genistein
Daidzein
OH
OH
O
A
1 2 2’
3
4 5
6
7
8
3’
4’
5’
6’
1’
B
C
O
O
HO
HO
HO
O
H 3 C
Fig 1 Structures of 17b- oestradiol, warfarin, genistein and
daidz-ein Daidzein does not have a hydroxyl group at position 5 of the A
ring compared to genistein The positions of the A, B and C rings
and the functional groups are indicated for genistein The A and C
rings of the isoflavones are similar to the A and B rings of
oestra-diol The actual distance between the two hydroxyl groups on both
the molecules is nearly identical; these hydroxyl groups are critically
located to enable binding to the estrogen receptor protein.
Trang 3genistein has been raised It is important to check if the
binding site of HSA has space and appropriate shape
and residues to accommodate both warfarin and
geni-stein A crystal structure of HSA bound to warfarin is
available (PDB no 1h9z and 1 ha2) [16] We used this
complex structure to explore the accommodation of
genistein and to generate a 3D model of the ternary
complex of HSA–warfarin–genistein
Results
Equilibrium dialysis
To determine the classes and number of genistein
bind-ing sites, saturation of these sites on HSA is required
The binding data are given in Fig 2 The number of
genistein molecules bound by a mole of protein (m) is plotted against free genistein concentration [L] Human serum albumin was saturated at 50 lm genistein (Fig 2A) Scatchard plot [17] of the above data shows only one high affinity binding site for genistein with a binding constant of 1.0 ± 0.2· 105m)1 (Fig 2B) Non-linear fitting algorithms for the data given in Fig 2A (m versus [L]) were given similar results for the maximum number of binding sites and binding con-stant for single occupancy
Fluorescence measurements Human serum albumin, when excited at 295 nm, has
an emission maximum at 333 nm (Fig 3) The absorp-tion spectra of isoflavones overlap in the emission region of HSA Genistein and daidzein have absorp-tion peaks at 325 and 340 nm, respectively (Fig 3, inset) With the addition of genistein, there is a quenching of fluorescence intensity, indicating efficient Fo¨rster type energy transfer The overlap integral J has been calculated by integrating the spectra in the wavelength range 310–400 nm to be 8.5· 10)15 and 9.28· 10)15 cm3Æmol)1 for genistein and daidzein, respectively The energy transfer efficiency E (k2¼
2⁄ 3, N ¼ 1.45 [18], F ¼ 0.118 [19]) for genistein and daidzein was 0.05 and 0.022, respectively The Fo¨rster distance R0, was 2.26 and 2.29 nm for genistein and daidzein, respectively The distance between the
Fig 2 Human serum albumin interaction with genistein:
equilib-rium dialysis One mililitre of HSA (63.64 lm) was dialysed against
3 mL of genistein (10–100 l M ) in 50 m M Tris ⁄ HCl pH 7.4 for 24 h
at 27 C Corresponding blanks containing 1 mL of the above buffer
were dialysed against 3 mL of 10–100 l M genistein The tubes
were kept in a water bath at 27 C with shaking at 100 r.p.m.
for the entire period The concentrations of free genistein in
equilibrium were determined by molar absorption coefficient
37.3 · 10 3
M )1Æcm)1 (A) A plot of m (moles of ligand bound to
pro-tein) vs free ligand concentration (L) (B) Scatchard plot depicting
the plot of m ⁄ (L) versus m.
Fig 3 Resonance energy transfer from HSA to genistein and daidz-ein Emission spectra of HSA in 50 m M Tris ⁄ HCl pH 7.4 Excitation wavelength was 295 nm Emission range was 300–400 nm with slit widths of 5 nm for excitation and 10 nm for emission Protein concentration was 1 l M Temperature was maintained at 27 C
using a water bath Inset, absorption spectra of genistein (n) and
daidzein (s) showing peak at 325 and 340 nm for genistein and daidzein, overlapping the emission maxima of 333 nm for HSA.
Trang 4compounds studied and the tryptophan residue was
obtained and the r0, distance between acceptor and
donor was 3.6 and 4.35 nm for these compounds,
respectively The maximal critical distance for R0 is
from 5 to 10 nm [20] and the maximum distance
between donor and acceptor for r0 is in the range
7–10 nm [21] The values of R0 and r0 for genistein
and daidzein suggested that nonradiation transfer
occurred between these isoflavones and HSA A
com-parison of the J, Ro and r-values of different ligands
bound to HSA is given in Table 1
Fluorescence quenching studies with genistein Interaction of genistein with HSA has been monitored following the quenching of relative fluorescence inten-sity of HSA Quenching of fluorescence by genistein does not lead to detectable changes in wavelength of maximum emission or the band shape Quantitation
of genistein–HSA interaction is shown in Fig 4A A maximum quench of 17% has been observed at 12 lm
of genistein, representing 59% completion of the reac-tion as deduced from the linear double reciprocal plot
of Q versus genistein concentration to be 28 ± 3 (Fig 4B) The stoichiometry of the genistein–HSA complex has been estimated from the Job’s plot [22] (Fig 4C) to be 1 : 1 ± 0.2 The mass action plot, pre-sented in Fig 4D has been constructed (using the value of n¼ 1 and the extent of reaction calculated from Fig 4B) The binding constant given by the slope
of this plot is 1.5 ± 0.2· 105 m)1 However, trypto-phan-modified HSA did not interact with genistein in the concentration range studied Genistin and daid-zin) the glycosylated forms of genistein and daidz-ein) did not interact with HSA as shown by the fluorescence quenching measurements
Binding energetics The effect of temperature on the interaction of geni-stein with HSA has been followed in the range 17–47 C The binding constant, K, exhibits a
recipro-Table 1 Comparison of the genistein (ligand) distance to
trypto-phan (HSA) measured by Forster nonradiative energy transfer with
other ligands bound to HSA.
o (nm) r (nm)
Bendroflumethiazide [52] 5.86 · 10 –16 1.55 1.47
3-hydroxy flavone [53] 1.64 · 10 –14
Genistein (present study) 8.35 · 10)15 2.25 3.68
Daidzein (present study) 9.28 · 10)15 2.29 4.35
a From [54] b From [55].
Fig 4 Quantitation of the interaction of HSA with genistein by fluorescence quench-ing HSA (1 l M ) in 50 m M Tris ⁄ HCl pH 7.4 was titrated with increasing aliquots of stock genistein solution (2 lL equivalent to
1 l M genistein per aliquot) in 80% methanol and the percentage quench was recorded Blank titrations with N-acetyl tryptophana-mide of equivalent absorbance at 280 nm as HSA in presence of varying concentration of genistein were carried out (A) Percentage quench of fluorescence intensity, as a func-tion of constituent genistein concentrafunc-tion (B) Double-reciprocal plot of data in A;
Qmax¼ 28 ± 3 (± indicates probable error in all cases) (C) Job’s plot, C HSA + C genistein ¼
10 l M showing the stoichiometry of 1 : 1 (D) Mass action plot of data (in A) in accord-ance with [47].
Trang 5cal relationship with temperature (Fig 5A) Thus,
van’t Hoff enthalpy, DH, is determined to be
)13.16 kcalÆmol)1 The binding reaction is entropy
driven DS has been determined as )21.0 calÆmol)1Æ
K)1and DG is found to be)6.86 kcalÆmol)1at 27C
Effect of ionic strength on binding of genistein– HSA interaction
To determine whether ionic interactions play a role in the genistein–HSA interaction, the ionic strength of the buffer was increased by the addition of potassium chloride (0–200 mm) It was observed that Qmax remained unaltered on increasing the ionic strength of the buffer implying no change in the binding geometry The binding constant decreased with increasing ionic strength (Fig 5B), establishing the role of ionic inter-action in the binding
The Stokes radius of HSA in the presence of increasing concentrations of potassium chloride in buf-fer was measured by size exclusion chromatography The elution volume of the protein increased with ionic strength indicating a decrease in Stokes radius (Fig 5B, inset) The decreased Stokes radius of the molecule could also contribute to the observed decrease in affinity
Fluorescence of albumin bound daidzein Daidzein is the only intrinsically fluorescent isoflavone among those studied This property has been exploited
to study the nature of binding to HSA There is a shift
of the emission maxima of the daidzein bound albumin towards shorter wavelengths (from 465 to 457 nm) compared to unbound daidzein (Fig 6) This indicates that daidzein is binding on the hydrophobic pocket in HSA
Fluorescence quenching studies with defatted HSA and BSA
HSA and BSA have similar folding with a well-known primary structure The important difference is that BSA has two tryptophan residues (W134 and W212) located in domain I and domain II, respectively, while HSA has only one tryptophan at position 214 in domain II This property is used to identify the bind-ing pocket for isoflavones in HSA Primary quenchbind-ing curves of both HSA and BSA and the defatted HSA are plotted (Fig 7A) The different intercepts of the double reciprocal plots (data not shown) correspond
to different Qmax values The overlap of the mass action plots (Fig 7B), indicates that the binding con-stant for genistein is the same for both HSA and BSA, both of which are known to contain bound fatty acid The quenching curve for genistein with fatty acid-free
Fig 5 (A) Effect of temperature on the binding constant of
geni-stein to HSA: van’t Hoff’s plot HSA (1 l M ) in 50 m M Tris ⁄ HCl
pH 7.4 was titrated with increasing aliquots of stock genistein
solu-tion (2 lL equivalent to 1 l M genistein per aliquot) in 80%
meth-anol at different temperatures (17, 27, 37 and 47 C and the
percentage quench was recorded Blank titrations were carried out
as described for Fig 4 van’t Hoff’s plot was constructed to obtain
the thermodynamic parameters (B) Effect of ionic strength on the
binding constant of genistein to HSA A plot of the binding constant
as a function of ionic strength to show the effect of ionic strength
on the binding constant of genistein Human serum albumin (1 l M )
in 50 m M Tris ⁄ HCl pH 7.4 was titrated at different ionic strengths
adjusted by using potassium chloride (0, 50, 100 and 200 m M ) with
increasing aliquots of stock genistein solution (2 lL equivalent to
1 l M genistein per aliquot) in 80% methanol The percentage
quench of the intrinsic fluorescence of HSA was recorded Blank
titrations were carried out as described for Fig 4 Inset, Stokes
radius of HSA at different molarities of KCl (0–200 m M ) was
deter-mined by size exclusion chromatography on HPLC using a TSK SW
2000 column (300 · 4.6 mm, 4 l) The column was pre-equilibrated
at the required ionic strength attained using KCl of buffer 50 m M
Tris ⁄ HCl pH 7.4 Equilibrated samples (20 lL) of the protein
(1 mgÆmL)1) were injected at 27 C at a flow rate of 0.2 mLÆmin)1.
The protein was eluted isocratically using the same buffer and
detected at 280 nm.
Trang 6HSA (Fig 7A) shows that fatty acid-free HSA binds
genistein with a lower affinity (1.25· 105m)1) than
the control Bound fatty acid may enhance the affinity
of genistein to HSA
Studies with fatty acid
Among the various ligands, fatty acids alone can
attach to the primary binding site of HSA
Experi-ments have been conducted using palmitic acid and
defatted HSA to understand the affinity
characteris-tics of genistein bound HSA The increase in the
fluorescence of genistein bound protein with the
increase in fatty acid concentration evidences the
dis-placement of genistein by palmitic acid (data not
shown) It has been suggested that hydrophobic
inter-actions are the dominant contributing factors to the
affinity of fatty acid to HSA apart from electrostatic
interactions [13]
ANS binding studies
ANS, known to bind to hydrophobic pockets of
pro-teins, is a much-utilized fluorescent ‘hydrophobic
probe’ for examining the nonpolar character of
pro-teins and membranes [23] To examine systematically
the role of hydrophobic interactions in the binding of
genistein to HSA, ANS-bound HSA was titrated with
genistein The replacement of ANS by genistein in
the protein indicates that ANS and genistein bind to
the same site This is corroborated by the decrease in ANS-bound HSA fluorescence with increasing concentrations of genistein The binding constant, estimated by the competitive ligand binding measure-ments is (1.27 ± 0.2· 105 m)1), very similar to that
of the genistein–HSA interaction The hydrophobic amino acid residues in HSA that form hydrophobic cavities in each domain interact with the alkyl chain of fatty acids whereas two to three basic amino acid residues at the entrance of the hydrophobic
Fig 6 Emission spectra of daidzein showing blue shift on binding
to HSA Daidzein (2.75 l M ) in 50 m M Tris ⁄ HCl pH 7.4 was titrated
against increasing concentrations of HSA in the same buffer The
final concentration of HSA was 14.75 l M Stock HSA (835 l M ) was
added in 5 lL aliquots and the spectra recorded between 400 and
550 nm after excitation at 340 nm, the excitation maxima for
daidz-ein Excitation slit width was 5 nm and emission slit width was
10 nm Dotted line, free daidzein; dashed line, daidzein bound to
HSA Concentration of HSA is 14.75 l M
Fig 7 (A) Interaction of genistein with HSA, defatted HSA and BSA HSA (1 l M ) was titrated with increasing aliquots of genistein and the percentage quench was recorded Human serum albumin was defatted by the procedure described previously [41] and the effect of fatty acid removal on genistein binding was followed
by fluorescence quenching measurements Human serum albumin (– O-), defatted HSA (– x-), BSA ( )m-) The excitation and emission slit widths were at 5 and 10 nm, respectively Conditions were same as described for Fig 4 (B) Mass action plot of HSA and BSA HSA (1 l M ) or BSA in 50 m M Tris ⁄ HCl pH 7.4 was titrated with increasing aliquots of genistein and the percentage quench in fluorescence was recorded as described for Fig 4 The mass action plot was constructed from the double reciprocal data to obtain the binding constant d, HSA; h, BSA.
Trang 7pocket interact with the carboxy group of fatty acids
[24]
Effect of genistein on tertiary and secondary
structure of HSA
The effect of increasing genistein concentration on the
tertiary and secondary structure of HSA has been
studied by measuring CD spectra in near and far UV
region, respectively The characteristic patterns in the
near UV region, caused by the asymmetric
environ-ment of tryptophan, tyrosine and phenyl alanine
resi-dues in the native structure, are not affected in
presence of genistein, upto a concentration of 50 lm
This indicates that genistein has no effect on the
ter-tiary structure of HSA There are no changes in the
far UV CD bands up to a concentration of 50 lm
genistein, indicating that genistein had no effect on the
secondary structure of HSA These results helped to
establish that genistein does not affect the
conforma-tion of HSA
Warfarin binding using induced CD
measurements
CD spectra in the near UV region (250–350 nm) were
recorded for genistein (0–50 lm), HSA in presence of
varying concentrations of genistein (0–50 lm), HSA
(15 lm) in the presence or absence of warfarin
(50 lm), with the concentration of genistein varying
from 0 to 50 lm Genistein does not exhibit any CD
bands in the above wavelength region Human serum
albumin does not induce any CD band for genistein (0
to 50 lm) However, the addition of warfarin to HSA
induced a CD band at 310 nm and 255 nm (Fig 8A)
There was no decrease in the CD signal when genistein
was added to the warfarin bound HSA; there was an
additional CD band at 270 nm (Fig 8B), which is not
observed in the absence of warfarin Warfarin,
report-edly, binds to subdomain IIA [16] It is evident that
genistein does not replace warfarin but binds alongside
warfarin to HSA
Binding of genistein in the presence of daidzein
The fluorescence of daidzein was found to increase on
binding to HSA as mentioned earlier The saturation
was reached at 14.75 lm HSA (Fig 9A) Quenching of
fluorescence was observed on adding genistein to the
daidzein bound HSA (Fig 9B) indicating the
replace-ment of daidzein by genistein The quench was
maxi-mum at 27 lm of genistein The binding constant of
the competing ligand (Fig 9C) was evaluated from a
plot of Fmax⁄ F vs molarity of genistein [25]; the binding constant of genistein was calculated to be 5.63· 105m)1
Fluorescence anisotropy measurements Fluorescence anisotropy measurements were made for the daidzein–HSA system by exciting at 340 nm (max-ima for daidzein) and emission at 465 nm There was
an increase in fluorescence anisotropy of daidzein on binding to HSA Anisotropy of daidzein increased from 0.01 to 0.25 on binding (Fig 10) The increase in anisotropy could be due to the restriction imposed by
Fig 8 Competitive ligand interactions of HSA: warfarin and geni-stein CD measurements were carried out in the near UV region of 250–350 nm in 50 m M Tris ⁄ HCl pH 7.4 The cell path length was
1 cm and spectra were recorded at a speed of 10 nmÆmin)1 All scans are an average of three runs A mean residue weight of 115 was used for calculating the molar ellipticity values (A) Effect of warfarin on the near UV CD of HSA The concentration of HSA was
15 l M and those of warfarin 0–50 l M Dashed line, HSA in buffer; solid line, HSA with 10 l M warfarin; dotted line, HSA with 50 l M
warfarin (B) Effect of genistein on near UV CD of warfarin-bound HSA Spectra were recorded after genistein (50 l M ) was added to HSA with 50 l M warfarin Dashed line, HSA in the presence of warfarin (50 l M ); solid line, 50 l M genistein in the presence of war-farin (50 l M )-bound HSA.
Trang 8the binding on the rotation around the daidzein mole-cule
The anisotropy of daidzein bound to HSA remained constant in the presence of diazepam Diazepam is known to bind to the domain IIIA of HSA, which is the primary binding site for fatty acids Warfarin also did not affect the anisotropy of daidzein bound to HSA TIB decreased the anisotropy of daidzein from 0.16 to 0.08 The anisotropy of free daidzein was 0.02 Hence, TIB partially displaced the daidzein in HSA (Table 2)
The anisotropy of warfarin bound to HSA was measured in the presence of genistein The anisotropy
of warfarin bound to HSA (5 lm bound to 10 lm HSA) was found to be 0.5 This was unaltered with the addition of genistein even up to 100 lm revealing that warfarin was not displaced by genistein (Table 3)
Fig 10 Variation in fluorescence anisotropy of daidzein as a func-tion of HSA concentrafunc-tion Daidzein (2.75 l M ) was titrated against increasing concentrations of HSA The excitation and emission wavelengths were 340 and 465 nm, respectively Slit widths were
at 5 and 10 nm for excitation and emission, respectively.
Fig 9 Competitive ligand binding interactions of HSA, genistein and daidzein (fluorescence measurements) Daidzein (2.75 l M ) was titrated against increasing concentrations of HSA to a final concen-tration of 14.75 l M ) in 50 m M Tris ⁄ HCl buffer pH 7.4 The excitation wavelength was 340 nm and emission range was 400–550 nm Excitation slit width was 5 nm and emission slit width was 10 nm.
To the above solution, 5 lL of stock genistein in 80% methanol (1.4 m M ) was added in aliquots and the spectra recorded at 27 C The final concentration of genistein was 27 l M (A) Emission spec-tra of daidzein with increasing micromolar concenspec-tration of HSA (solid line 0; dashed line, 1.66; dotted line, 4.98; dashed ⁄ dotted line, 8.26; + + + +, 11.52; short dashed ⁄ dotted line, 14.75) (B) Emission spectra of daidzein–HSA complex with increasing micro-molar concentration of genistein (solid line, 0; dashed line, 5.48; dotted line, 10.92; dashed ⁄ dotted line, 16.29; )±)±), 21.64; ++ 26.94) (C) Fmax ⁄ F vs genistein concentration to obtain the binding constant of the competing ligand—genistein.
Trang 9The characteristic of albumin to allow a variety of
lig-ands to bind to it is amazing Albumin is the principal
carrier of fatty acids that are otherwise insoluble in the
circulating plasma Human serum albumin is
com-posed of three homologous domains (I, II and III)
Each domain, in turn, is the product of two
subdo-mains, which are predominantly helical and extensively
cross-linked by several disulfide bridges [26] The
typical binding constants for various ligands range from 104 to 106m)1 The vast majority of ligands bind reversibly on one or both sites within specialized cavit-ies of subdomains IIA and IIIA of albumin The bind-ing property of the subdomain IIIA of albumin is general, whereas that of subdomain IIA is more
speci-fic The amino acid residues that line the cavities are quite similar in charge distribution for both the sub-domains IIA and IIIA Yet, they impart desired selec-tivity In each of the two subdomains, there is an asymmetric charge distribution, leading to a hydropho-bic surface on one side and a basic or positively charged surface on the other This explains the dis-criminatory affinity of albumin for small anionic com-pounds The van der Waals’ surface of the binding pocket in IIA appears like an elongated sock wherein the foot region is primarily hydrophobic and the leg is primarily hydrophilic The opening to the pocket is clearly accessible to the solvent The affinity of flavo-noids for HSA is in line with its general ability to bind small negatively charged ligands [12,26,27]
Results of the present study indicate that the binding
of genistein to HSA by equilibrium dialysis is charac-terized by the equilibrium constant 1.0 ± 0.2· 105 (Fig 2B) The binding constants obtained by fluo-rescence quenching measurements for genistein and daidzein to HSA are 1.5 ± 0.2· 105m)1 and 1.4 ± 0.2· 105m)1, respectively Thus, there is good agreement in the binding constants obtained for geni-stein–HSA interaction by both direct and indirect methods The binding of the isoflavones to HSA is similar and the R2 group at position 5 of the aromatic A-ring does not play a significant role in the binding
of either genistein or daidzein (Fig 1) The B-ring of the flavonoids is electron richer than the A-ring, ren-dering it more susceptible to ionization at physiologi-cal pH [28] The reported plasma concentrations of daidzein and genistein are in the range of 50–800 lgÆL)1 [2] Thus, the concentrations used to determine the equilibrium constant are physiologically relevant The interaction of genistein and daidzein with HSA could not be followed by isothermal calorimetry due to the limited solubility of the above in aqueous buffers used in the study
The decrease in the binding constant with increase
in temperature (Fig 5A), suggests the involvement of noncovalent interactions and a major role for ionic interactions in the binding of genistein to HSA, which
is further corroborated by the observed decrease in the binding constant on the addition of potassium chlor-ide The negative free energy values indicate that the binding is spontaneous and that it is energetically more favorable for genistein or daidzein to link to HSA
Table 2 Corrected fluorescence anisotropy values of the daidzein
HSA complex, when different aliquots of warfarin, diazepam and
triiodobenzoic acid were added.
Warfarin
Daizepam
Triiodobenzoic acid
Table 3 Corrected fluorescence anisotropy values of the warfarin–
HSA complex, when different aliquots of genistein were added.
Trang 10Negative entropy indicates a loss in the degree of
free-dom of genistein when embedded in the HSA cavity
The effect of KCl and temperature point to the
pres-ence of electrostatic interactions apart from the
hydro-phobic interactions
The blue shift of daidzein bound protein
fluores-cence (Fig 6) is indicative of the role of hydrophobic
interactions in the binding of this aglycone to HSA
with the emission maxima shifting from 465 to
457 nm The binding of daidzein to a hydrophobic
pocket in HSA may be a cause for this phenomenon
Further, fluorescence of the albumin bound ANS is
found to be quenched by the addition of either
geni-stein or daidzein The observed concentration
depend-ence of quenching of fluorescdepend-ence indicates that the
binding sites of ANS and genistein are the same
apparently leading to possible replacement of ANS
by the isoflavones These experiments suggest the
involvement of hydrophobic interactions in the
bind-ing of genistein or daidzein to HSA Isoflavones,
genistein and daidzein (Fig 1), have a flavone nucleus
made up of two benzene rings (A and B) linked
through a heterocyclic pyrane C ring These aromatic
rings may be involved in hydrophobic interactions
with hydrophobic pockets of domain IIA of HSA
The complete three-dimensional structure of HSA has
recently been determined by X-ray crystallography,
and the binding sites for several drugs have been
identified ANS reportedly binds to two sites on
HSA, IIA and IIIA, with a binding constant of
7.9· 104m)1 and 8.7· 105m)1, respectively
Subdo-main IIIA is the site where ANS binds to HSA with
a higher affinity [29]
The intrinsic fluorescence of albumin is due to the
tryptophan residue (W214) [26], conserved in all
mam-malian albumins and located strategically in the
domain IIA for developing van der Waals’ interactions
with ligands bound at that site [30] Domain IIA has
five lysine residues (positions 203, 210, 220, 231 and
241) and one arginine residue at position 218 These
residues are positively charged at the pH used in the
present study and could contribute to ionic
inter-actions with genistein or daidzein Genistein and
daidzein have a phenolic structure with conjugated
double bonds Albumin is known to reversibly
com-plex with phenols via hydrogen bonding and
hydro-phobic interactions [31]
The increase in anisotropy of daidzein bound HSA
with increase in protein concentration (Fig 10),
indi-cates the reduction of freedom of rotation of daidzein
bound HSA Increase in anisotropy could be due to
decreased Brownian motion or energy transfer between
identical chromophores The high value of anisotropy
(0.25) indicates that daidzein is binding at a motionally restricted site on HSA
Identification of the binding pocket for isoflavones on HSA
The binding pocket on HSA for isoflavones was identi-fied through: (a) Fo¨rster energy transfer measurements; (b) binding of genistein with HSA and BSA; and (c) competitive ligand binding measurements using war-farin
Fo¨rster distance (R0) and the distance between acceptor and donor (r0) for the genistein and daidzein were in the range known to prove that nonradiation transfer occurred between these isoflavones and HSA The quenching of intrinsic fluorescence measure-ments of HSA and BSA by genistein (Figs 7A,B) assist
in identification of the binding site on the albumin molecule The Qmax for HSA is 28% compared to 53% with BSA The difference between HSA and BSA
is the presence of an additional tryptophan in BSA at position 134 This is at site II, the interface of domain
IA and IIA of HSA [27] The conserved tryptophan is
at position 214 The binding constants for genistein with BSA and HSA are same, the stoichiometry for binding being 1 : 1 The isoflavone has an identical binding site on both the molecules Hence, the binding site on both the albumins for genistein is the same Our extrinsic CD measurements of genistein binding
in presence of warfarin suggest that the binding is inclusive There is enough conformational flexibility in domain IIA of HSA to accommodate both warfarin and genistein The binding of warfarin and its crystal structure with HSA–myristic acid is reported [16] Warfarin has only one binding site in domain IIA hav-ing tryptophan at 214 The structures of genistein and warfarin are similar (Fig 1) Tryptophan residue (W214) is in domain IIA, which explains the quenching
of protein fluorescence due to genistein binding In the case of BSA, the additional tryptophan W134, is very near to W214[27] The accommodation of genistein at site I may therefore quench the fluorescence due to both tryptophans in BSA, corroborating the higher quenching observed in case of BSA The modification
of tryptophan residues on HSA has resulted in the loss
of interaction of genistein with albumin Quercetin (3,5,7,3,‘4’-pentahydroxy flavone, a plant derived flavo-noid compound) binds to HSA with an association constant of 1.46 · 104m)1at 37C in the large hydro-phobic cavity of subdomain IIA and the protein microenvironment of this site is rich in polar (basic) amino acid residues which are able to help to stabilize the negatively charged ligand bound in nonplanar