Phenotypic Characterization of the Binding of Tetracycline to Human

Biomacromolecules 2011, 12, 203–209

203

Phenotypic Characterization of the Binding of Tetracycline to Human Serum Albumin Zhenxing Chi and Rutao Liu* Shandong Provincial Key Laboratory of Water Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Shandong University, China-America CRC for Environment and Health, Shandong Province, 27 Shanda South Road, Jinan 250100, People’s Republic of China Received September 28, 2010; Revised Manuscript Received October 29, 2010

Because of the widely usage of the veterinary drug tetracycline (TC), its residue exist extensively in the environment (e.g., animal food, soils, surface water, and groundwater) and can enter human body, being potential harmful. Human serum albumin (HSA) is a major transporter for endogenous and exogenous compounds in vivo. The aim of this study was to examine the interaction of HSA with TC through spectroscopic and molecular modeling methods. The inner filter effect was eliminated to get accurate binding parameters. The site marker competition experiments revealed that TC binds to site II (subdomain IIIA) of HSA mainly through electrostatic interaction, illustrated by the calculated negative ∆H° and ∆S°. Furthermore, molecular docking was applied to define the specific binding sites, the results of which show that TC mainly interacts with the positively charged amino acid residues Arg 410 and Lys 414 predominately through electrostatic force, in accordance with the conclusion of thermodynamic analysis. The binding of TC can cause conformational and some microenvironmental changes of HSA, revealed by UV-visible absorption, synchronous fluorescence, and circular dichroism (CD) results. The accurate and full basic data in the work is beneficial to clarifying the binding mechanism of TC with HSA in vivo and understanding its effect on protein function during the blood transportation process.

1. Introduction Tetracycline (TC) (structure with atom numbers shown in the inset of Figure 1) is a veterinary drug that is widely used for the therapy of infectious diseases of animals in intensive farming systems and aquaculture because of its broad-spectrum antibiotic activity.1-3 Because of its low bioavailability,4 only a fraction of the ingested TC is metabolized in the animals and the residual TC is excreted and released into soils, surface water, and groundwater, being a potential risk to human health.5,6 The toxicity of TC residues in the environment including animal food,7 soils,8 and surface and groundwater5 has attracted widespread attention.9 Tetracycline can significantly disturbed the structure of microbial communities and the enzymatic activities (urease, acid phosphatase, and dehydrogenase) of soil,8 induce reproductive toxicity in male rats,10 cause tissue damage to both liver and pancreas of adult male albino rats,11 and interfere with processes of secretion as well as synthesis of pancreatic protein in pigeons.12 Human serum albumin (HSA), the most abundant protein in blood plasma,13 is involved in the transport of a variety of endogenous and exogenous ligands such as drugs and chemical contaminants.14 Transportation, distribution, and physiological and toxicological actions of chemical contaminants in vivo are closely related to their binding with proteins, and the binding of the contaminants with active sites of the proteins can change their structure and function and cause toxic effects.15 So it is very significant to investigate the interaction between the ligands and the major carrier protein like HSA. Although the interaction between TC and HSA at the molecular level by using spectroscopic methods has been studied,16 the inner filter effect was not considered, which affects * To whom correspondence should be addressed. Phone/Fax: 86-53188364868. E-mail: [email protected].

Figure 1. Effect of TC on HSA fluorescence (corrected). Conditions: HSA, 3.0 × 10-6 mol L-1; TC/ (10-5 mol L-1), (a) 0, (b) 0.5, (c) 1, (d) 1.5, (e) 2, (f) 2.5, (g) 3, (h) 3.5, (i) 4, (j) 4.5, (k) 5; pH 7.4; T ) 288 K.

the binding parameters calculated from the fluorescence data. In addition, the binding sites of TC to HSA have not previously been identified. The two aspects hinder our proper and comprehensive understanding of the interaction between the veterinary drug TC and HSA. In the present work, we investigated the interaction between TC and HSA in vitro under physiological conditions by spectroscopic and molecular modeling methods. The inner filter effect was corrected before we estimated the association constants, thermodynamic parameters, the number of binding sites, the binding forces, and the energy transfer distance of the interaction between TC and HSA. The specific binding site of TC on HSA was investigated. The effect of TC on the

10.1021/bm1011568  2011 American Chemical Society Published on Web 12/13/2010

204 Biomacromolecules, Vol. 12, No. 1, 2011

Chi and Liu

microenvironment and conformation of HSA was also discussed. The study provides an accurate and full basic data for clarifying the binding mechanisms of TC with HSA and is helpful for understanding its effect on protein function during the blood transportation process and its toxicity in vivo.

2. Experimental Section 2.1. Reagents. HSA (Sigma) was dissolved in ultrapure water to form a 3.0 × 10-5 mol L-1 solution, then preserved at 0-4 °C and diluted as required. We prepared a stock solution of TC (1.0 × 10-3 mol L-1) by dissolving 0.04809 g tetracycline hydrochloride (Sigma) in 100 mL of water. Phosphate buffer (0.2 mol L-1, mixture of NaH2PO4 · 2H2O and Na2HPO4 · 12H2O, pH 7.6) was used to control pH. NaH2PO4 · 2H2O and Na2HPO4 · 12H2O were of analytical reagent grade, obtained from Tianjin Damao Chemical Reagent Factory. 2.2. Apparatus and Methods. All fluorescence spectra were recorded on an F-4600 spectrofluorimeter (Hitachi, Japan). The excitation and emission slit widths were set at 5.0 nm. The scan speed was 1200 nm/min. PMT (photo multiplier tube) voltage was 700 V. UV-visible absorption spectra were measured on a UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan). CD spectra were recorded on a J-810 CD spectrometer (JASCO). The pH measurements were made with a pHs-3C acidity meter (Pengshun, Shanghai, China). 2.2.1. CD Measurements. CD spectra were collected from 190 to 260 at 0.2 nm intervals on a JASCO J-810 CD spectrometer. Three scans were made and averaged for each CD spectrum. The CD spectra were analyzed using SELCON3 software to calculate the secondary structural elements. 2.2.2. Fluorescence Measurements. The fluorescence measurements were carried out as follows: to each of a series of 10 mL test tubes, 1.0 mL of 0.2 mol L-1 phosphate buffer (pH 7.4) and 1.0 mL of 3 × 10-5 mol L-1 HSA were added, and then different amounts of 1.00 × 10-3 mol/L stock solution of TC were added. The fluorescence spectra were then measured (excitation at 278 nm and emission wavelengths of 285-450 nm). The synchronous fluorescence spectra were measured at λex ) 250 nm, ∆λ ) 15 nm, and ∆λ ) 60 nm. To eliminate the inner filter effects of protein and ligand, absorbance measurements were performed at excitation and emission wavelengths of the fluorescence measurements. The fluorescence intensity was corrected using the equation17

Fcor ) Fobsd10(A1+A2)/2

log

(F0 - F) ) log Ka + n log[Q] F

where F0, F, and [Q] are the same as in eq 2, Ka is the binding constant, and n is the number of binding sites per HSA molecule. 2.2.4. Thermodynamic Parameters. If the enthalpy change (∆H°) does not vary significantly over the temperature range studied, the enthalpy change (∆H°), free-energy change (∆G°), and the entropy change (∆S°) can be calculated based on the van’t Hoff equation (eq 4) and thermodynamic equation (eq 5):

( ) (

ln

)( )

(Ka)2 1 1 ∆H° ) (Ka)1 T1 T2 R

∆G° ) ∆H° - T∆S° ) -RT ln Ka

E)

(5)

R06 R06 + r6

(6)

where r is the distance between the acceptor and the donor, R0 is the critical distance when the transfer efficiency is 50%, which can be calculated by

R06 ) 8.79 × 10-25K2n-4ΦJ

(7)

where K2 is the spatial orientation factor of the dipole, n is the refractive index of the medium, Φ 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

J)

∫

∞

0

F(λ)ε(λ)λ4dλ

∫

∞

0

(8) F(λ)dλ

where F(λ) is the fluorescence intensity of the fluorescence donor at wavelength λ and ε(λ) is the molar absorption coefficient of the acceptor at wavelength λ. The energy transfer efficiency is given by

E)1F0 ) 1 + KSV[Q] ) 1 + kqτ0[Q] F

(4)

where (Ka)1 and (Ka)2 are the binding constants at T1 and T2 and R is the universal gas constant. 2.2.5. Energy Transfer Calculation. According to the Fo¨rster’s nonradiation energy transfer theory, the parameters related to energy transfer can be calculated based on the equations as follows. The energy transfer effect is related not only to the distance between the acceptor and the donor but also to the critical energy transfer distance, that is

(1)

where Fcor and Fobsd are the corrected and observed fluorescence intensities, respectively, whereas A1 and A2 are the sum of the absorbance of protein and ligand at the excitation and emission wavelengths, respectively. To confirm the quenching mechanism, the fluorescence quenching data were analyzed according to the Stern-Volmer equation18

(3)

F F0

(9)

(2)

where F0 and F are the fluorescence intensities in the absence and presence of the quencher, respectively, KSV is the Stern-Volmer quenching constant, [Q] is the concentration of the quencher, kq is the quenching rate constant of the biological macromolecule, and τ0 is the fluorescence lifetime in the absence of quencher. 2.2.3. Binding Parameters. For the static quenching interaction, when small molecules bind independently to a set of equivalent sites on a macromolecule, the binding constant (Ka) and the number of binding sites (n) can be determined by the following equation:

where F0 and F are the same in eq 2. 2.2.6. Molecular Modeling Study. Docking calculations were carried out using DockingServer.19 The MMFF94 force field20 was used for energy minimization of the ligand molecule (TC) using DockingServer. Gasteiger partial charges were added to the ligand atoms. Nonpolar hydrogen atoms were merged, and rotatable bonds were defined. Docking calculations were carried out on a HSA protein model (PDB code 1BJ5). Essential hydrogen atoms, Kollman united atom type charges, and solvation parameters were added with the aid of AutoDock tools.21 Affinity (grid) maps of 50 × 50 × 50 Å grid points and 0.375 Å spacing were generated using the Autogrid program.21 The AutoDock

Binding of Tetracycline to Human Serum Albumin

Biomacromolecules, Vol. 12, No. 1, 2011

205

parameter set and distance-dependent dielectric functions were used in the calculation of the van der Waals and the electrostatic terms, respectively. Docking simulations were performed using the Lamarckian genetic algorithm (LGA) and the Solis and Wets local search method.22 Initial positions, orientations, and torsions of the ligand molecules were set randomly. All rotatable torsions were released during docking. Each run of the docking experiment was set to terminate after a maximum of 250000 energy evaluations. The population size was set to 150. During the search, a translational step of 0.2 Å and quaternion and torsion steps of 5 were applied.

3. Results and Discussion 3.1. Characterization of the Binding Interaction of TC with HSA by Fluorescence Measurements Based on the Elimination of the Inner Filter Effects. Although the interaction between TC and HSA has been studied by fluorescence technique,16 the inner filter effect was not considered. In this study, we eliminated the inner filter effect for all of the fluorescence and synchronous fluorescence results to obtain accurate data. The fluorescence intensity was corrected using the eq 1. 3.1.1. Fluorescence Spectra. The fluorescence intensity (F) of HSA decreased regularly with increasing TC concentration (Figure 1). Furthermore, a small blue shift was observed with increasing TC concentration, which suggests that the fluorescence chromophore of HSA was placed in a more hydrophobic environment after the addition of TC.23 The quenching mechanism was determined to investigate whether TC interacts with HSA to form a complex. Quenching mechanisms include static and dynamic quenching. To confirm the quenching mechanism, the fluorescence quenching data were analyzed according to the Stern-Volmer eq 2. The Stern-Volmer plots before and after correction are shown in Figure 2A. It can be seen that the plot before correction was not linear, so the Stern-Volmer equation cannot be used to calculate KSV and kq. After correction with eq 1 to remove the inner filter effect, the plot shows results that agree with the Stern-Volmer eq 2. The Stern-Volmer plots for the quenching of HSA by TC at two different temperatures are shown in Figure 2B. The quenching type should be single static or dynamic quenching. Because higher temperature results in larger diffusion coefficients, the dynamic quenching constants will increase with increasing temperature. In contrast, increased temperature is likely to result in decreased stability of complexes, and thus the static quenching constants are expected to decrease with increasing temperature.24 The maximum scatter collision quenching constant of various quenchers with the biopolymer was 2.0 × 1010 L mol-1 s-1.25 In this paper, KSV and kq at two different temperatures are listed in Table 1. The KSV values decreased with increasing temperature and kq was greater than 2.0 × 1010 L mol-1 s-1. These results indicate that the quenching was not initiated from dynamic collision but from the formation of a complex. 3.1.2. Binding Parameters. The quenching mechanism was determined to be static quenching in 3.1.1, so the binding constant (Ka) and the number of binding sites (n) can be calculated according to eq 3. A plot of log[(F0 - F)/F] versus log[TC] yields log Ka as the intercept on the y axis and n as the slope (shown in SI Figure 1, Supporting Information). Table 2 shows the calculated Ka and n. The number of binding sites, n, is approximately 1, indicating that there is one binding site in HSA for TC during their interaction. 3.1.3. Thermodynamic Parameters and Binding Forces. The acting forces between small organic molecules and biomolecules

Figure 2. (A) Stern-Volmer plots for the quenching of HSA by TC at 288 K before and after correction; (B) Stern-Volmer plots for the quenching of HSA by TC at different temperatures (corrected). Conditions: HSA: 3.0 × 10-6 mol L-1; pH 7.4.

include hydrogen bonds, van der Waals interactions, electrostatic forces and hydrophobic interaction forces. Because the temperature effect is very small, the interaction enthalpy change (∆H°) can be regarded as a constant if the temperature range is not too wide. The enthalpy change (∆H°), free-energy change (∆G°), and the entropy change (∆S°) for the interaction between TC and HSA were calculated according to the van’t Hoff equation (eq 4) and thermodynamic equation (eq 5; Table 2). The negative ∆H° and positive ∆S° indicated that electrostatic forces play the major role during the interaction. Because under the experimental conditions used, the pH (7.4) is much greater than the isoelectric point of HSA (4.7-4.9),26 HSA is negatively charged. TC is also negatively charged at pH 7.4.27 However, TC can interact with the positively charged amino acid residues of HSA through electrostatic forces.28 In addition, the negative sign of ∆G° indicates the binding of TC with HSA is spontaneous.24 3.1.4. Energy Transfer between TC and HSA. The overlap of the absorption spectrum of TC and the fluorescence emission spectrum of HSA is shown in SI Figure 2, Supporting Information. According to Fo¨rster’s nonradiative energy transfer theory,29 the energy transfer will happen under the conditions: (i) the donor can produce fluorescence light; (ii) the absorption spectrum of the receptor overlaps enough with the donor’s fluorescence emission spectrum; (iii) the distance between the donor and the acceptor is less than 8 nm.

206

Biomacromolecules, Vol. 12, No. 1, 2011

Chi and Liu

Table 1. Stern-Volmer Quenching Constants for the Interaction of TC with HSA at Different Temperatures

a

pH

T (K)

KSV (×105 L mol-1)

kq (×1012 L mol-1 s-1)

Ra

S.D.b

7.4

288 308

0.05543 0.04249

0.5543 0.4249

0.99753 0.99852

0.00682 0.00404

R is the correlation coefficient.

b

SD is the standard deviation for the KSV values.

Table 2. Binding Constants and Relative Thermodynamic Parameters of the TC-HSA System T (K)

Ka (×103 L mol-1)

n

Ra

∆H° (kJ mol-1)

∆S° (J mol-1 K-1)

288 308

4.87181 3.10735

0.99101 0.96796

0.99545 0.99748

-16.582

13.02

a

∆G° (kJ mol-1) -20.3317 -20.5920

R is the correlation coefficient for the Ka values.

Table 3. Effects of Site Probe on the Binding Constant of TC to HSA

Figure 3. Domain structure of human serum albumin (HSA).

It has been reported for HSA that K2 ) 2/3, n ) 1.36, and Φ ) 0.074.13 J in eq 8 was evaluated by integrating the UV absorption and fluorescence emission spectra (SI Figure 2, Supporting Information). Based on these data and eqs 6-9, we found that J ) 1.552 × 10-14 cm3 L mol-1, R0 ) 2.41 nm, E ) 0.0127, and r ) 4.98 nm. So the distance between TC and the tryptophan residue in HSA after interaction is 4.98 nm, which is lower than 8 nm. These accords with conditions of Fo¨rster’s nonradiative energy transfer theory, indicating again the static quenching interaction between TC and HSA. In summary, after elimination of the inner filter effect, we obtain accurate data of the interaction of TC with HSA including the quenching rate constant, the association constant, the number of binding sites, the thermodynamic parameters and the average distance between the bound TC and the tryptophan residue 214 in HSA, which is different from previous study (SI Table 1, Supporting Information).16 3.2. Identification of the Specific Binding Sites on HSA. HSA is a globular protein composed of three homologous R-helical domains (I-III). Each domain contains two subdomains (A and B; Figure 3).30 The principal regions of ligand binding sites of albumin are located in hydrophobic cavities in subdomains IIA and IIIA.30 Many ligands bind specifically to serum albumin, for example warfarin and phenylbutazone for site I (subdomain IIA), flufenamic acid (FA) and ibuprofen for site II (subdomain IIIA), and digitoxin for site III.31 Table 3 shows the changes in fluorescence of TC bound to HSA on the addition of other drugs. TC was not significantly displaced by phenylbutazone or by digitoxin. However, flufenamic acid

K (without the site probe; 103 L mol-1)

K (with PB; 103 L mol-1)

K (with FA; 103 L mol-1)

K (with Dig; 103 L mol-1)

3.11

3.07

1.29

3.78

(subdomain IIIA) gave a significant displacement of TC, suggesting that TC’s binding site on HSA is site II (subdomain IIIA). The environment in site II is composed mainly of Leu 387, Cys 392, Phe 395, Phe 403, Leu 407, Arg 410, Tyr 411, Leu 430, Cys 438, Arg 445, and Leu 453.32 To further define the binding site, molecule docking was employed by setting the simulation box to site II. The crystal structure of HSA was taken from the Protein Data Bank (entry PDB code 1BJ5). The best energy ranked result is shown in Figure 4 and Figure 5. The docking result (Figure 5) shows that TC mainly interacts with the positively charged amino acid residues Arg 410 and Lys 414 predominately through electrostatic force (Table 4), in agreement with our conclusion of thermodynamic analysis. In addition, the modeled TC-HSA structure showed that hydrogen bonds (TC with Lys 414), hydrophobic interactions (TC with Leu 387 and Leu 394), and other forces (TC with GLN 390, Arg 410, Tyr 411, and Lys 414) are also present, but the electrostatic forces play a major role in the binding of TC to HSA. 3.3. Investigation of HSA Conformation Changes. 3.3.1. UV-Vis Absorption Spectra Studies. UV-vis absorption spectroscopy technique can be used to explore the structural

Figure 4. Binding site of TC on HSA. HSA is shown in cartoon. TC is represented using spheres. The atoms of TC are color-coded as follows: O, red; N, blue; C, green; H, white.

Binding of Tetracycline to Human Serum Albumin

Biomacromolecules, Vol. 12, No. 1, 2011

207

Figure 6. UV-vis spectra of HSA in presence of different concentrations of TC (vs the same concentration of TC solution). Conditions: HSA, 1.0 × 10-6 mol L-1; TC (10-5 mol L-1), (a) 0, (b) 2, (c) 4, (d) 6, (e) 8; pH 7.4; T ) 288 K.

Figure 5. (A) Enlarged binding mode between TC and HSA. HSA is shown in cartoon. The interacting side chains of HSA are displayed in surface mode. TC is represented using balls and sticks. The atoms of TC are color-coded as follows: O, red; N, blue; C, green; H, white. (B) Molecular modeling of the interaction between TC and HSA. The atoms of TC are marked with blue and the atoms of amino acid residues of HSA are labeled with gray. Table 4. Atoms Involved in the Electrostatic Interaction of TC with HSA and the Distances between Them, Analyzed from the Molecular Docking Result protein atom (HSA)

ligand atom (TC)

distance (Å)

ARG 410 (CB) ARG 410 (CB, CD, CG) ARG 410 (CD, CG, CZ, NE) ARG 410 (CD, CG, CZ, NE) ARG 410 (CD, CG, NE) ARG 410 (CD) ARG 410 (CD) ARG 410 (CZ, NE, NH2) ARG 410 (NE) LYS 414 (NZ) LYS 414 (NZ) LYS 414 (NZ) LYS 414 (NZ) LYS 414 (NZ)

O19 H41 N30 H50 H51 O20 O22 O24 O29 O29 N30 H50 H51 H42

3.83 3.38 2.68 2.41 2.73 3.70 3.60 3.05 3.84 3.71 2.63 2.71 2.87 3.90

changes of protein and to investigate protein-ligand complex formation. The UV-vis absorption spectra of HSA in the presence and absence of TC are shown in Figure 6. HSA has two absorption peaks. The strong absorption peak at about 208 nm reflects the framework conformation of the protein.33 The weak absorption peak at about 279 nm appears to be due to the aromatic amino acids (Trp, Tyr, and Phe).34 With gradual addition of TC to HSA solution, the intensity of the peak at

208 nm decreases and red shifts and the intensity of the peak at 279 nm also decreases. The results indicate that the interaction between TC and HSA leads to the loosening and unfolding of the protein skeleton and increases the hydrophobicity of the microenvironment of the aromatic amino acid residues.35 3.3.2. Synchronous Fluorescence. Synchronous fluorescence spectroscopy can give information about the molecular environment in the vicinity of chromophores. The spectrum is obtained through the simultaneous scanning of the excitation and emission monochromators while maintaining a constant wavelength interval between them. When the wavelength intervals (∆λ) are stabilized at 15 or 60 nm, the synchronous fluorescence gives the characteristic information of tyrosine residues or tryptophan residues, respectively.36 The synchronous fluorescence spectra of HSA with various amounts of TC in Figure 7A show that the emission peaks do not shift over the investigated concentration range, which indicates that TC has little effect on the microenvironment of the tyrosine residues in HSA. In Figure 7B, the emission maximum of the tryptophan residue shows a slight blue shift (from 278.6 to 277.6 nm), which indicates that the conformation of HSA was changed such that the polarity around the tryptophan residue 214 decreased and the hydrophobicity was increased.13 3.3.3. Circular Dichroism. To ascertain the possible influence of TC binding on the secondary structure of HSA, CD measurements were performed in the presence of different TC concentrations (Figure 8). The estimates for the secondary structural elements are listed in Table 5. The CD spectra of HSA exhibited two negative bands in the ultraviolet region at about 208 and 218 nm, which is characteristic of the R-helix of proteins.37 The calculated secondary structure content of HSA was 59.3% R-helix, 6% β-pleated sheet, 11.1% β-turn, and 23.9% random coil. With the addition of TC at HSA/TC ratios of 1:4 and 1:16, the R-helix decreased by 4.2% and 10.2%, β-pleated sheet increased by 1.4% and 3.6%, β-turn increased by 0.9% and 3.2%, and random coil increased by 2% and 3.4%, respectively. These results suggest that the interaction of TC with HSA can affect the secondary structure of HSA, which may affect its physiological function.38 As mentioned above, the binding of TC can lead to the loosening and unfolding of the protein skeleton and increases

208

Biomacromolecules, Vol. 12, No. 1, 2011

Chi and Liu Table 5. Effects of TC on the Percentage of Secondary Structural Elements in HSA secondary structural elements in HSA molar ratio of HSA to TC

helix ((2%)

beta ((1%)

turn ((1%)

random ((1%)

1:0 1:4 1:16

59.3 55.1 49.1

6 7.4 9.6

11.1 12.0 14.3

23.9 25.9 27.3

molecular modeling techniques under physiological conditions. The inner filter effect of fluorescence was corrected. The experimental results indicate that TC can interact with HSA in site II (subdomain IIIA) mainly with one binding site through electrostatic forces. The secondary structure and the microenvironment of the tryptophan residue 214 of HSA were altered for bound TC. Molecular docking were employed to further define the specific binding sites, the results of which show that TC mainly interacts with the positively charged amino acid residues Arg 410 and Lys 414 predominately through electrostatic force, in accordance with the conclusion of thermodynamic analysis. The study provides accurate and comprehensive basic data for clarifying the binding mechanism of TC with human serum albumin and is helpful for understanding its effect on protein function during its transportation and distribution in blood. The established research route to investigate the specific binding sites of TC on HSA can also be applied to explore other organic molecules.

Figure 7. (A, B) Synchronous fluorescence spectra of HSA (corrected): (A) ∆λ ) 15 nm, (B) ∆λ ) 60 nm. Conditions: HSA, 3.0 × 10-6 mol L-1; TC (10-5 mol L-1), (a) 0, (b) 1, (c) 2, (d) 3, (e) 4, (f) 5; T ) 288 K.

Acknowledgment. The work is supported by NSFC (20875055), the Cultivation Fund of the Key Scientific and Technical Innovation Project, Graduate Independent Innovation Foundation of Shandong University (yzc10056), Ministry of Education of China (708058) and Excellent Young Scientists and Key Science-Technology Project in Shandong Province (2008GG10006012) are also acknowledged. Supporting Information Available. Plot of log[(F0 - F)/ F] versus log[TC], overlap of the absorption spectrum of TC with fluorescence emission spectrum of HSA, and comparison of our results after the elimination of the inner filter effect with previous studies. This material is available free of charge via the Internet at http://pubs.acs.org.

References and Notes

Figure 8. CD spectra of HSA and HSA-TC system. Conditions: HSA, 1.0 × 10-7 mol L-1; TC, (a) 0, (b) 4.0 × 10-7 mol L-1, (c) 1.6 × 10-6 mol L-1; pH 7.4; T ) 288 K.

the hydrophobicity of the microenvironment of the tryptophan residue 214 of HSA. The R-helix of the secondary structure of HSA decreased and the β-pleated sheet, β-turn, and random coil increased due to bound TC.

4. Conclusions In this paper, we simulated the interaction of the widely used veterinary drug TC with HSA in vitro by spectroscopic and

(1) Aleksandrov, A.; Simonson, T. Biochemistry 2008, 47, 13594–603. (2) Jiao, S.; Zheng, S.; Yin, D.; Wang, L.; Chen, L. Chemosphere 2008, 73, 377–82. (3) Kong, W. D.; Zhu, Y. G.; Liang, Y. C.; Zhang, J.; Smith, F. A.; Yang, M. EnViron. Pollut. 2007, 147, 187–93. (4) Rigos, G.; Nengas, I.; Alexis, M.; Troisi, G. M. Aquat. Toxicol. 2004, 69, 281–8. (5) Ye, Z.; Weinberg, H. S.; Meyer, M. T. Anal. Chem. 2007, 79, 1135– 44. (6) Hakkarainen, M.; Hoglund, A.; Odelius, K.; Albertsson, A. C. J. Am. Chem. Soc. 2007, 129, 6308–6312. (7) Yang, M.; Xu, Y.; Wang, J. H. Anal. Chem. 2006, 78, 5900–5. (8) Wei, X.; Wu, S. C.; Nie, X. P.; Yediler, A.; Wong, M. H. J. EnViron. Sci. Health, Part B 2009, 44, 461–71. (9) Wollenberger, L.; Halling-Sorensen, B.; Kusk, K. O. Chemosphere 2000, 40, 723–30. (10) Farombi, E. O.; Ugwuezunmba, M. C.; Ezenwadu, T. T.; Oyeyemi, M. O.; Ekor, M. Exp. Toxicol. Pathol. 2008, 60, 77–85. (11) Asha, K. K.; Sankar, T. V.; Viswanathan Nair, P. G. J. Pharm. Pharmacol. 2007, 59, 1241–8. (12) Tucker, P. C.; Webster, P. D. Am. J. Dig. Dis. 1972, 17, 675–82. (13) Hu, Y. J.; Liu, Y.; Xiao, X. H. Biomacromolecules 2009, 10, 517– 521.

Binding of Tetracycline to Human Serum Albumin (14) D’Eon, J, C.; Simpson, A. J.; Kumar, R.; Baer, A. J.; Mabury, S. A. EnViron. Toxicol. Chem. 2010, 29, 1678–88. (15) Guo, Y. M.; Yue, Q. Y.; Gao, B. Y. EnViron. Toxicol. Pharmacol. 2010, 30, 5–51. (16) Bi, S.; Song, D.; Tian, Y.; Zhou, X.; Liu, Z.; Zhang, H. Spectrochim Acta, Part A 2005, 61, 629–36. (17) Shyamali, S. S.; Lillian, D. R.; Lawrence, L.; Esther, B. Biochemistry 1979, 18, 1026–1036. (18) Qin, C.; Xie, M. X.; Liu, Y. Biomacromolecules 2007, 8, 2182–9. (19) Bikadi, Z.; Hazai, E. J. Cheminform. 2009, 1, 15. (20) Halgren, T. A. J. Comput. Chem. 1998, 17, 490–519. (21) Morris, G. M.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.; Olson, A. J.; Goodsell, D. S. J. Comput. Chem. 1998, 19, 1639–1662. (22) Solis, F. J.; Wets, R. J. B. Math. Oper. Res. 1981, 6, 19–30. (23) Yuan, T.; Weljie, A. M.; Vogel, H. J. Biochemistry 1998, 37, 3187– 95. (24) Khan, S. N.; Islam, B.; Yennamalli, R.; Sultan, A.; Subbarao, N.; Khan, A. U. Eur. J. Pharm. Sci. 2008, 35, 371–82. (25) Zhang, Y. Z.; Zhou, B.; Zhang, X. P.; Huang, P.; Li, C. H.; Liu, Y. J. Hazard. Mater. 2009, 163, 1345–52. (26) Leis, D.; Barbosa, S.; Attwood, D.; Taboada, P.; Mosquera, V. Langmuir 2002, 18, 8178–8185. (27) Figueroa, R. A.; Leonard, A.; MacKay, A. A. EnViron. Sci. Technol. 2004, 38, 476–83.

Biomacromolecules, Vol. 12, No. 1, 2011

209

(28) Pan, X. R.; Liu, R. T.; Qin, P. F.; Wang, L.; Zhao, X. C. J. Lumin. 2010, 130, 611–617. (29) Fo¨rster, T. Modern Quantum Chemistry; Academic Press: New York, 1965. (30) Beauchemin, R.; N’Soukpoe-Kossi, C. N.; Thomas, T. J.; Thomas, T.; Carpentier, R.; Tajmir-Riahi, H. A. Biomacromolecules 2007, 8, 3177–83. (31) Bian, H. D.; Li, M.; Yu, Q.; Chen, Z. F.; Tian, J. N.; Liang, H. Int. J. Biol. Macromol. 2006, 39, 291–297. (32) Celiesˇiujte˙, R.; Zˇiemys, A.; Kulys, J. Biologija 2004, 3, 27–31. (33) Yang, Q.; Liang, J.; Han, H. J. Phys. Chem. B 2009, 113, 10454–8. (34) Zhao, X. C.; Liu, R. T.; Chi, Z. X.; Teng, Y.; Qin, P. F. J. Phys. Chem. B 2010, 114, 5625–5631. (35) Wu, T.; Wu, Q.; Guan, S.; Su, H.; Cai, Z. Biomacromolecules 2007, 8, 1899–906. (36) Wang, Y. Q.; Zhang, H. M.; Zhang, G. C.; Liu, S. X.; Zhou, Q. H.; Fei, Z. H.; Liu, Z. T. Int. J. Biol. Macromol. 2007, 41, 243–250. (37) Lu, J. Q.; Jin, F.; Sun, T. Q.; Zhou, X. W. Int. J. Biol. Macromol. 2007, 40, 299–304. (38) Huang, B. X.; Kim, H. Y.; Dass, C. J. Am. Soc. Mass Spectrom. 2004, 15, 1237–47.

BM1011568