CHEM. RES. CHINESE UNIVERSITIES 2012, 28(2), 287—290
Characterization of Gallic Acid Interaction with
Human Serum Albumin by Spectral and
Molecular Modeling Methods
LIU Zuo-jia, LI Dan and NIU Feng-lan*
School of Public Health, Jilin University, Changchun 130021, P. R. China
Abstract The binding of drugs with human serum albumin(HSA) is a crucial factor influencing the distribution and
bioactivity of drugs in the body. To understand the action mechanisms between gallic acid(GA, 3,4,5-trihydroxybenzoic acid) and HSA, the binding of GA with HSA was investigated by a combined experimental and computational
approach. The fluorescence properties of HSA and the binding parameters of GA collectively indicate that the binding
is characterized by static quenching mechanism at one high affinity binding site. According to the estimated molecular distance between the donor(HSA) and the acceptor(GA), the binding is related to the fluorescence resonance
energy transfer. As indicated by the thermodynamic parameters, hydrophobic interaction plays a major role in the
GA-HSA complex. Further, the experimental results reveal that GA is bound in the large hydrophobic cavity of subdomain IIA in the site I of HSA, which is well approved by molecular docking.
Keywords Gallic acid; Human serum albumin; Fluorescence quenching; Molecular modeling
Article ID 1005-9040(2012)-02-287-04
1
Introduction
Distribution and transport of drugs in the body are correlated with their affinities for human serum albumin(HSA)[1]. In
most cases, drug-HSA interactions will significantly affect the
distribution volume and the elimination rate of drugs[2]. In previous studies[3], many drugs binding reversibly to albumin have
been probed with a combined spectroscopic and crystallographic approach. Gallic acid(GA, 3,4,5-trihydroxybenzoic
acid) as a natural plant polyphenol is a kind of important active
component in Chinese traditional medicines and is of multiple
biological and pharmacological properties[4―6]. The binding
parameters in polyphenolic compound-HSA or -membrane
mimetic environments have been determined by means of different experimental approaches[7]. However, few efforts have
been focused on the molecular binding between GA and HSA
in aqueous solution under physiological conditions in detail.
We examined the occurrence and nature of GA binding HSA
using a combined experimental and computational approach.
The quenching mechanism, binding constants, and the number
of binding sites were evaluated by the fluorescence quenching
method. The identification of binding sites, the effect of GA on
the conformation of HSA, and the nature of forces involved in
the interactions were further discussed based on the synchronous fluorescence spectra and thermodynamic parameters.
Furthermore, the experimental observations were also interpreted by molecular modeling. The study presented herein clarified the details of GA-HSA interactions and provided a structural basis for the possible design of GA derivatives.
2
2.1
Experimental
Materials
HSA(Sigma) was freshly dissolved in 0.1 mol/L phosphate
buffered saline(PBS) of pH 7.4 to a concentration of 20 µmol/L
based on its molecular weight of 66500. Stock solution of GA
of 1.0 mmol/L(Sigma Co., Ltd.) was prepared in MilliQ pure
water, stored and protected from light at 4 °C and diluted to
various concentrations. All the chemicals were of analytical
reagent grade and MilliQ water was used throughout.
2.2
Apparatuses and Methods
Fluorescence measurement was carried out on a LS-55
fluorescence spectrofluorimeter equipped with a thermostat
bath(Perkin Elmer). The fluorescence quenching of HSA was
recorded in a wavelength range of 300―500 nm at the excitation wavelength of 290 nm. Synchronous fluorescence spectra
of HSA were recorded in the wavelength range of 250―450
nm at 296 K. The titration solution was prepared in a 2 mL
quartz cuvette and incubated in dark for 1.0 min. The bandwidth for measuring emission was 5 nm. The temperature of
sample was kept by recycle water throughout experiment.
Docking calculations were carried out on the system of
GA-HSA with the Autodock4.0 package[8]. The structure of
HSA was obtained from the protein data bank(PDB 1h9z[9]),
which was further refined and optimized by means of the
pdb2pqr1.3 software package[10] to add the missing side chains
of some residues and to remove clashes. The 3D structure of
———————————
*Corresponding author. E-mail: [email protected]
Received May 9, 2011; accepted July 29, 2011.
Supported by the Project of Department of Science and Technology of Jilin Province, China(No.20070424).
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CHEM. RES. CHINESE UNIVERSITIES
[11]
GA was built via the web-tools corina3D . An Autogrid of
60×60×60 in grid size with a spacing of 0.0375 nm centered on
the special position in the potential binding site was prepared
with autodock tools. Docking was performed via the empirical
free energy function and the Lamarckian genetic algorithm.
The molecular modeling was performed based on the following
parameters: the energy evaluations of 100000, the maximum
number of 27000 iterations for an initial population of 100
randomly placed individuals with a mutation rate of 0.02, a
crossover rate of 0.80, and an elitism value of 1. The other
parameters were defaults. The number of docking runs was
10000. Evaluation of the results was performed by sorting the
binding energy predicted by docking conformations. A cluster
of analysis based on the root mean square deviation value that
is lower than 0.2 nm was performed subsequently.
3
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whereas a slight blue shift of tryptophan residue fluorescence
occurred[Fig.2(B)]. The blue shift of the maximum emission
wavelength indicates the chromophore of protein, Trp214, was
placed in a more hydrophobic environment after the addition of
GA[14].
Results and Discussion
3.1 Characterization of Interaction Between GA
and HSA
To confirm the binding of GA to HSA, the fluorescence
quenching spectra of HSA at various concentrations of GA
were determined(Fig.1). Obviously, with the increasing of the
concentration of GA, the fluorescence intensity of HSA decreased tardily. A blue shift was observed for the maximum
emission wavelength of HSA with progressively titrating GA to
HSA solution. This observation indicates that the fluorescence
of HSA was quenched by GA, suggesting that the complex of
GA -HSA was formed.
Fig.2
Synchronous fluorescence spectra of HSA
in the presence of GA
(A) Δλ=15 nm; (B) Δλ=60 nm. T=296 K, pH=7.40. c(HSA)=2.0×10–5
mol/L; 106 c(GA)/(mol·L–1): a. 0; b. 4; c. 12; d. 20; e. 24; f. 32; g. 40;
h. 50; i. 60; j.70; k. 80; l. 90; m. 100; n. 110.
3.3 Quenching Mechanism and Binding Constants
Fig.1
Fluorescence quenching spectra of HSA
in the presence of GA
c(HSA)=2.0×10–5 mol/L; 106 c(GA)/(mol·L–1): a. 0; b. 4; c. 12;
d. 20; e. 24; f. 32; g. 40; h. 50; i. 60; j.70; k. 80; l. 90; m. 100.
T=296 K; λex=290 nm, λem=340 nm.
3.2 Analysis of HSA Conformation After GA
Binding
The synchronous fluorescence spectra can provide the information about molecular environment in the vicinity of the
chromosphere molecules with the help of the fluorescence
quenching and the possible shift of the maximum emission
wavelength. Δλ was stabilized at 15 or 60 nm, which gives the
characteristic information of tyrosine(Tyr) or tryptophan(Trp)
residues, respectively[12,13]. It is clearly seen that the addition of
GA led to a dramatic decrease in the fluorescence intensity but
the emission maximum of tyrosine kept the position[Fig.2(A)],
The possible quenching mechanism between GA and HSA
can be analyzed by the Stern-Volmer equation[15]:
F0/F=1+Kqτ0[Q]
(1)
where F and F0 are the steady state fluorescence intensities in
the presence and absence of quencher, respectively; Kq, τ0 and
[Q] are the quenching rate constant, the average lifetime of the
molecule without quencher and the concentration of quencher,
respectively. With the fluorescence lifetime of Trp in HSA
taken at around 10−8 s[16], an approximate quenching rate
constant(Kq, L·mol−1·s−1) can be obtained via the Stern-Volmer
curve. As shown in Fig.3, the values of Kq are 1.12×1012
L·mol−1·s−1(r=0.998) at 296 K and 1.58×1012 L·mol−1·s−1
(r=0.996) at 303 K, respectively. The maximum dynamic collision quenching constant Kq of various kinds of quenchers for
biopolymers fluorescence is around 2.0×1010 L·mol−1·s−1[16].
From the results, each rate constant is greater than that of the
scatter procedure, which means that the quenching is not
initiated by dynamic collision but static one, i.e., the formation
of GA-HSA complex.
For the static quenching interaction, the relationship between the fluorescence intensity and the quencher can be expressed as the Lineweaver-Burk equation[15]:
F0/(F0–F)=1+KA–1[Q]–1
(2)
No.2
LIU Zuo-jia et al.
289
fluorescence emission spectrum of HSA is shown in Fig.4. The
donor-to-acceptor distance(r=2.04 nm) is lower than 8 nm,
which accords with non-radiative energy transfer theory[19] well.
This result indicates that the energy transfer from HSA to GA
occured with a high possibility, which will quench the florescence of HSA.
Fig.3
Stern-Volmer plots for quenching various
concentrations of GA with HSA at 296(a)
and 303 K(b)
c(HSA)=2.0×10–5 mol/L; λex=290 nm.
With this equation, the binding constant(KA, L·mol−1) at
various temperatures can be obtained: KA=9.91×103 L·mol−1
(r=0.991) at 296 K and KA=1.19×104 L·mol−1(r=0.982) at
303 K. It is found that the binding constant increases with the
increasing of temperature, which shows that the temperature
has an effect on the binding between GA and HSA. The number
of binding sites, n, can be calculated according to the
equation[15]:
lg[(F0–F)/F]=lgKA+nlg[Q]
(3)
The values of n are noticed to be 1.07(r=0.997) at 296 K
and 1.15(r=0.998) at 303 K, respectively. It can be suggested
that there is one independent binding site on HSA for GA.
3.4
Fluorescence Resonance Energy Transfer
(FRET) Mechanism
FRET occurs as long as the emission spectrum of fluorophore(donor) overlaps with the absorption spectrum of small
molecules(acceptor). Generally, the extent of energy transfer
depends on the distance r between donor and acceptor and the
extent of spectral overlap. The distance between Trp214 and the
bound small molecule can be calculated according to the
Förster theory[17]. The efficiency of energy transfer, E, is calculated with the following equation:
E =1−
6
0
F
R
=
F0 R06 + r 6
(4)
where r is the distance between donor and acceptor; R0 is the
critical distance when the transfer efficiency is 50%, which can
be calculated by the following equation:
R06=8.8×10–25k2N4ФJ
(5)
2
where k is the spatial orientation factor of the dipole; N is the
refractive index of the medium; Ф is the fluorescence quantum
yield of the donor; and J is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor. In the present case, k2=2/3, N=1.36 and
Ф=0.15[18]. J is given by the following equation:
∑ F (λ )ε (λ )λ Δλ
∑ F ( λ ) Δλ
4
J=
(6)
where F(λ) is the fluorescence intensity of the fluorescent
donor of wavelength λ; and ε(λ) is the molar absorption coefficient of the acceptor at wavelength λ.
The overlap of UV absorption spectrum of GA with the
Fig.4
Overlapping of fluorescence emission
spectrum of HSA(λex=290 nm)(a) with
UV absorption spectrum of GA(b)
c(GA)=1.0×10–5 mol/L; c(HSA)=1.0×10–5 mol/L;
pH=7.4; T=296 K.
3.5
Interaction Forces Between GA and HSA
To estimate the binding mode, the thermodynamic parameters, enthalpy change(ΔH), entropy change(ΔS) and free
energy change(ΔG), were mainly considered. In the present
study, the thermodynamic parameters listed in Table 1 were
evaluated according to the Van’t Hoff equation:
lnK= –ΔH/RT+ΔS/R
(7)
where, K is the binding constant at corresponding temperature
(K1=9.91×103 L·mol−1, T1=296 K and K2=1.19×104 L·mol−1,
T2=303 K); R(8.314 J·mol–1·K–1) is the gas constant. Then,
ΔH(19.14 kJ/mol) and ΔS(141.15 J·mol–1·K–1) can be calculated
by means of equation (6) and the above values. ΔG can be estimated based on the following relationship:
ΔG=ΔH –TΔS
(8)
Table 1
Thermodynamic parameters of GA
binding to HSA
T/K
ΔG/(kJ·mol–1)
ΔH/(kJ·mol–1)
ΔS/(J·mol–1·K–1)
296
303
–22.64
–23.72
19.14
19.14
141.15
141.15
Here, both the positive values of ΔH(19.14 kJ/mol) and
ΔS(141.15 J·mol–1·K–1) show that the hydrophobic interaction
played an absolutely key role in the binding of GA to HSA[20].
In addition, positive entropy may also be a sign of electrostatic
interaction[20], which means that electrostatic interaction also
existed in the interaction between GA and HSA. Nevertheless,
it is clear from the values presented in Table 1 that the binding
of GA to HSA was an exothermic process accompanied by a
positive value of ΔS and negative values of ΔG. Both the negative values of ΔG at the various temperatures imply the spontaneity of the binding of GA to HSA. Moreover, it is noteworthy to mention that the major contribution to ΔG arises more
from the TΔS rather than from ΔH. Thus the binding process
was entropy-driven.
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3.6
CHEM. RES. CHINESE UNIVERSITIES
Molecular Modeling
To improve the understanding of interaction between GA
and HSA, the best energy ranked result is displayed in Fig.5. It
can be seen that GA is deeply inserted in the hydrophobic
cavity of the site I. On account of the charge property of GA
under the physiological condition, it can be also concluded that
electrostatic force is one of the major forces of interaction
between GA and HSA. Accordingly, this finding provides an
optimal structural basis to explain the very efficient fluorescence quenching of HSA emission in the presence of GA.
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phobic cavity of subdomain IIA. The expected output should
ultimately help one design GA derivatives with altered
HSA-binding properties.
References
[1]
Carter D. C., Ho J. X., Adv. Protein Chem., 1994, 45, 153
[2]
Kragh-Hansen U., Pharm. Rev., 1981, 33(1), 17
[3]
Rawel H. M., Frey S. K., Meidtner K., Kroll J., Schweigert F. J.,
Mol. Nutr. Food Res., 2006, 50, 705
[4]
Rawel H. M., Kroll J., Rohn S., Food Chem., 2001, 72(1), 59
[5]
Kroll J., Rawel H. M., J. Food Sci., 2001, 66(1), 48
[6]
Zhao W. J., Niu F. L., Chem. Res. Chinese Universities, 2009, 25(1),
56
[7]
Zhang Y. H., Dong L. J., Li J. Z., Chen X. G., Talanta, 2008, 76,
246
[8]
Morris G. M., Goodsell D. S., Halliday R. S., Huey R., Hart W. E.,
[9]
Petitpas I., Bhattacharya A. A., Twine S., East M., Curry S., J. Biol.
Belew R. K., Olson A. J., J. Comput. Chem., 1998, 19(14), 1639
Chem., 2001, 276(25), 22804
[10]
Huey R., Morris G. M., Olson A. J., Goodsell D. S., J. Comput.
Chem., 2007, 28(6), 1145
[11]
Dolinsky T. J., Nielsen J. E., McCammon J. A., Baker N. A., Nucleic Acids Res., 2004, 32(7), 665
Fig.5
Binding mode between GA and HSA
The residues of HSA are represented by yellow and red; and GA structure is
represented by a gray one. The hydrogen bonds between GA and HSA are
represented by yellow dashed lines.
4
Conclusions
The interaction between GA and HSA was investigated by
a combined spectral and modeling approach. According to the
synchronous fluorescence spectra, the binding of GA to HSA
leads to the changes in the conformation of HSA. As thermodynamic parameters showed, hydrophobic interactions dominated in the association reaction and the binding process was
entropy driven. Computational mapping of the possible binding
sites of GA revealed the drug to be bound in the large hydro-
[12]
Wang Y. Q., Zhang H. M., Zhang G. C., Tao W. H., Tang S. H., J.
Mol. Struct., 2007, 830(1), 40
[13]
Congdon R. W., Muth G. W., Splittgerber A. G., Anal. Biochem.,
1993, 213(2), 407
[14]
Klotz I. M., Ann. NY Acad. Sci., 1973, 226(1), 18
[15]
Gelamo E. L., Silva C. H. T. P., Imasato H., Tabak M., Biochim.
Biophys. Acta, 2002, 1594(1), 84
[16]
Lakowicz J. R., Weber G., Biochemistry, 1973, 12(21), 4161
[17]
Stryer L., Annu. Rev. Biochem., 1978, 47, 819
[18]
Förster T., Sinanoglu O., Modern Quantum Chemistry, Academic
Press, New York, 1965, 93
[19]
Valeur B., Brochon J. C., New Trends in Fluorescence Spectroscopy,
6th Ed., Springer Press, Berlin, 1999, 25
[20]
Tian J. N., Liu J., Hu Z. D., Chen X. G., Bioorg. Med. Chem., 2005,
13(12), 4124