Site-Selective Binding of Human Serum Albumin by Palmatine

106

Biomacromolecules 2010, 11, 106–112

Site-Selective Binding of Human Serum Albumin by Palmatine: Spectroscopic Approach Yan-Jun Hu,†,‡ Yu Ou-Yang,‡ Chun-Mei Dai,§ Yi Liu,*,† and Xiao-He Xiao§ State Key Laboratory of Virology and Department of Chemistry, Wuhan University, Wuhan 430072, People’s Republic of China, Hubei Key Laboratory of Pollutant Analysis and Reuse Technology, Department of Chemistry, Hubei Normal University, Huangshi 435002, People’s Republic of China, and Institute of Chinese Materia Medica, 302 Hospital of PLA, Beijing 100039, People’s Republic of China Received August 24, 2009; Revised Manuscript Received October 22, 2009

In this study, fluorescence spectroscopy in combination with UV-vis absorption spectroscopy and circular dichroism (CD) spectroscopy was employed to investigate the high affinity binding of palmatine to human serum albumin (HSA) under the physiological conditions. In the mechanism discussion it was proved that the fluorescence quenching of HSA by palmatine is a result of the formation of palmatine/HSA complex. Binding parameters calculating from Stern-Volmer method and Scatchard method showed that palmatine bind to HSA with the binding affinities of the order 104 L · mol-1. The thermodynamic parameters studies revealed that the binding was characterized by negative enthalpy and positive entropy changes and the electrostatic interactions play a major role for palmatine-HSA association. Site marker competitive displacement experiments demonstrating that palmatine bind with high affinity to site I (subdomain IIA) of HSA. The specific binding distance r (2.91 nm) between donor (Trp-214) and acceptor (palmatine) was obtained according to fluorescence resonance energy transfer (FRET). Furthermore, the CD spectral result indicates that the secondary structure of HSA was changed in the presence of palmatine.

1. Introduction Palmatine (molecular structure: inset of Figure 1; CAS Registry Number: 3486-67-7), an isoquinoline alkaloid originally isolated from Rhizoma coptidis, Cortex phellodendri, Radix tinosporae, and Enantia chlorantha, is an important traditional medicinal herb, which has been reported to be effective against experimental tumors by inhibiting the activity of reverse transcriptase.1 In another study, palmatine was shown to possess antipyretic, antibacterial, antifungal, antivirus, antiphotooxidative, dispelling dampness, antidote, antinociceptive, and anti-inflammatory activities in vitro and in vivo.2-10 In addition to its medicinal uses, palmatine is also used as a fluorescent probe of DNA, RNA, and enzyme11-14 in biochemical researches. In spite of these broad uses of palmatine mentioned above, its effects on plasma protein remain unclear. It is widely accepted in the pharmaceutical industry that the overall distribution, metabolism, and efficacy of many drugs can be altered based on their affinity to serum albumin.15-18 In addition, many promising new drugs are rendered ineffective because of their unusually high affinity for this abundant protein.19 Obviously, an understanding of the chemistry of the various classes of pharmaceutical interactions with albumin can suggest new approaches to drug therapy and design. Human serum albumin (HSA) is the most prominent protein in plasma, which carries several endogenous compounds including fatty acids.20,21 HSA has long been the center of attention of the pharmaceutical industry due to its ability to bind various drug molecules and alter their pharmacokinetic properties.22-24 The crystallographic * To whom correspondence should be addressed. Tel.: +86-27-68756667. Fax: +86-27-68754067. E-mail: [email protected]. † Wuhan University. ‡ Hubei Normal University. § Institute of Chinese Materia Medica.

Figure 1. Emission spectra of HSA in the presence of various concentrations of palmatine. c(HSA) ) 1.0 × 10-5 mol · L-1; c(palmatine)/(10-5 mol · L-1), a-m: from 0.0 to 2.4 at increments of 0.20; curve n (dashed line) shows the emission spectrum of palmatine only. (T ) 298 K, λex ) 295 nm). The inset corresponds to the molecular structure of palmatine.

analyses of HSA revealed that the protein, a 585 amino acid residue monomer, contains three structurally similar domains (I, II, and III), each containing two subdomains (A and B) and stabilized by 17 disulfide bridges.25 The crystal structure analyses also indicate that the principal regions of ligand binding sites in albumin are located in hydrophobic cavities in subdomains IIA and IIIA. According to Sudlow’s nomenclature, two primary sites (I and II) have been identified for ligand binding to HSA. Warfarin, an anticoagulant drug, and ibuprofen, a nonsteroidal antiinflammatory agent, have been considered as stereotypical ligands for Sudlow’s site I and II, respectively. Warfarin, as other bulky heterocyclic anions, binds to Sudlow’s

10.1021/bm900961e CCC: $40.75  2010 American Chemical Society Published on Web 11/09/2009

Binding of Human Serum Albumin by Palmatine

site I located in subdomain IIA, whereas ibuprofen, as other aromatic carboxylates with an extended conformation, prefers Sudlow’s site II, located in subdomain IIIA.26-28 Even though the binding sites of fatty acids on HSA have been located, the interaction of palmatine with HSA has not been fully investigated. Therefore, it was of interest to study the binding of palmatine with HSA. In this article, we present a spectroscopic analysis of the interaction of HSA with palmatine at physiological conditions, using constant protein concentration and various palmatine compositions. The interaction information regarding quenching mechanisms, binding parameters, thermodynamic parameters, binding modes, high-affinity binding site, intermolecular distances, and conformation investigation is reported here.

2. Materials and Methods 2.1. Materials. HSA and warfarin were obtained from SigmaAldrich (St. Louis, MO); palmatine was obtained from national institute for control of pharmaceutical and biological products (Beijing, China); ibuprofen was obtained from Hubei biocause pharmaceutical Co., Ltd. (Hubei, China; the purity no less than 99.7%); the buffer Tris had a purity of no less than 99.5% and NaCl, HCl, and so on, were all of analytical purity. All samples were dissolved in Tris-HCl buffer solution (0.05 mol · L-1 Tris, 0.15 mol · L-1 NaCl, pH 7.4), Appropriate blanks, run under the same conditions, were subtracted from the sample spectra. Sample masses were accurately weighed on a microbalance (Sartorius, ME215S) with a resolution of 0.1 mg. 2.2. Equipments and Spectral Measurements. All fluorescence spectra were recorded on F-2500 Spectrofluorimeter (Hitachi, Japan) equipped with 1.0 cm quartz cells and a thermostat bath. The widths of both the excitation slit and the emission slit were set to 2.5 nm. An excitation wavelength of 295 nm was chosen because it is exclusively due to the intrinsic tryptophan (Trp) fluorophore. Appropriate blanks corresponding to the buffer were subtracted to correct the fluorescence background. The UV spectrum was recorded at room temperature on a TU-1901 spectrophotometer (Puxi Analytic Instrument Ltd. of Beijing, China) equipped with 1.0 cm quartz cells. Circular dichroism (CD) spectra were measured with a Jasco J-810 Spectropolarimeter (Jasco, Tokyo, Japan) at room temperature over a wavelength range of 260-200 nm and under constant nitrogen flush. Quartz cells having path lengths of 1.0 cm were used at a scanning speed of 200 nm/min.

3. Results and Discussion 3.1. HSA Fluorescence Characteristics and Quenching Mechanism. Fluorescence is the process of photon emission as a result of the return of an electron in a higher energy orbital back to a lower orbital, a variety of molecular interactions can result in quenching, including excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching. In the experiment, the concentrations of HSA solution were stabilized at 1.0 × 10-5 mol · L-1, and the concentrations of palmatine varied from 0 to 2.4 × 10-5 mol · L-1 at increments of 0.2 × 10-5 mol · L-1. The effect of palmatine on HSA fluorescence intensity at 298 K is shown in Figure 1. It was observed from Figure 1 that a progressive decrease in the fluorescence intensity was caused by quenching, accompanied by a decrease of wavelength emission maximum λmax (a blue shift, from 340 to 327 nm) in the albumin spectrum. As a result, we predict that Trp-214 is relatively buried inside the HSA. This inference is based on the fact that the emission λmax of Trp-214 was 327 nm, while fluorescence emission of the

Biomacromolecules, Vol. 11, No. 1, 2010

107

Table 1. Stern-Volmer Quenching Constants for the Interaction of Palmatine with HSA at Various Temperatures pH

T (K)

10-4KSV (L · mol-1)

7.4

298 304 310

6.198 6.040 5.828

a

R is the correlation coefficient.

b

R

a

0.9994 0.9993 0.9978

S.D.b 0.0116 0.0137 0.0234

S.D. is standard deviation.

exposed tryptophan molecule is around 340 nm due to solvent relaxation.20 This result indicates the HSA conformation bury Trp-214 in a hydrophobic environment.29 In Figure 1, curve n (dashed line) shows the emission spectrum of palmatine only, the data show that the palmatine absorbs at the excitation wavelength of tryptophan (295 nm) produce a very weak fluorescence at around 370 nm, but the emission peak interference at around 340 nm could be negligible; the fluorescence spectrum of HSA splits from a single peak to dual peaks is caused by palmatine-HSA binding. For fluorescence quenching, the decrease in intensity is usually described by the well-known Stern-Volmer equation:30

F0 ) 1 + KSV[Q] F

(1)

where F0 and F denotes the steady-state fluorescence intensities in the absence and in the presence of quencher (palmatine), respectively, KSV is the Stern-Volmer quenching constant, and [Q] is the concentration of the quencher. Hence, eq 1 was applied to determine KSV by linear regression of a plot of F0/F against [Q]. The quenching mechanisms are usually classified as either dynamic quenching or static quenching. Dynamic and static quenching can be distinguished by their differing dependence on temperature and viscosity.30 Higher temperatures result in faster diffusion and hence larger amounts of dynamic quenching. Higher temperatures will typically result in the dissociation of weakly bound complexes and, hence, smaller amounts of static quenching. The calculation of KSV from Stern-Volmer plots (Table 1) demonstrated the effect on fluorescence quenching by palmatine at each temperature (298, 304, and 310 K) studied; the result shows that the Stern-Volmer quenching constant KSV is inversely correlated with temperature, which indicates that the probable quenching mechanism of the palmatine-HSA binding reaction is initiated by compound formation rather than by dynamic collision. One additional method to distinguish static and dynamic quenching is by careful examination of the absorption spectra of the fluorophore. Collisional quenching only affects the excited states of the fluorophores, and thus, no changes in the absorption spectra are expected. In contrast, ground-state complex formation will frequently result in perturbation of the absorption spectrum of the fluorophore.30 For reconfirming the probable quenching mechanism of fluorescence of HSA by palmatine is initiated by ground-state complex formation, we used the difference absorption spectroscopy to obtain spectra. Figure 2 shows the absorption spectra of HSA in the presence of palmatine. Two absorption peaks were observed, at 276 and 342 nm, and palmatine has absorption in the same place as HSA 276 nm. The changes in absorbance at 276 nm indicate that palmatine affects HSA molar absorbance rather than a simple absorption spectra overlapping, as predicted by the temperature results. The absorbance of a series of HSA-palmatine com-

108


Hu et al.

Figure 2. UV-visible spectra of HSA in the presence of palmatine: A, absorption spectrum of palmatine/HSA 1:1 complex; B, absorption spectrum of palmatine only; C, absorption spectrum of HSA only; D, difference between absorption spectrum of palmatine/HSA 1:1 complex and palmatine. c(HSA) ) c(palmatine) ) 1.0 × 10-5 mol · L-1. The inset corresponds to the absorbance at 276 and 342 nm, the data obtained from a titration experiment in Tris-HCl buffer pH 7.4, 298 K, 1.0 × 10-5 mol · L-1 HSA and palmatine in the range 0.0 to 4.0 × 10-5 mol · L-1.

plexes at 276 and 342 nm in the inset of Figure 2, a good linear relationship shows that a small experimental error. The UV-vis absorption spectra of HSA (Figure 2, curve C) and the difference absorption spectra between HSA/palmatine 1:1 complex and palmatine (Figure 2, curve D) could not be superposed within experimental error; this result reconfirms that the probable fluorescence quenching mechanism of HSA by palmatine is a static quenching procedure. 3.2. Binding Parameters. For a static quenching procedure, the data were analyzed according to the modified Stern-Volmer equation:31

F0 1 1 1 ) + ∆F faKa [Q] fa

(2)

In the present case, ∆F is the difference in fluorescence in the absence and presence of the quencher at concentration [Q], fa is the fraction of accessible fluorescence, and Ka is the effective quenching constant for the accessible fluorophores, which are analogous to associative binding constants for the quencher-acceptor system. The dependence of F0/∆F on the reciprocal value of the quencher concentration [Q]-1 is linear with the slope equaling to the value of (faKa)-1. The value fa-1 is fixed on the ordinate. The constant Ka is a quotient of the ordinate fa-1 and the slope (faKa)-1. When small molecules bind to a set of equivalent sites on a macromolecule, the equilibrium binding constant and the numbers of binding sites can be also analyzed according to the Scatchard equation:32 r

/Df ) nKb - rKb

(3)

where r is the moles of ligand bound per mole of protein, Df is the molar concentration of free ligand, n is binding site

Figure 3. Modified Stern-Volmer plots (A) and Scatchard plots (B) of the palmatine-HSA system at different temperatures.

multiplicity per class of binding sites, and Kb is the equilibrium binding constant. Figure 3A and B show the modified Stern-Volmer plots and Scatchard plots for the palmatine-HSA system at different temperatures, respectively. The corresponding results at different temperatures are shown in Table 2. The decreasing trend of Ka and Kb with increasing temperature were in accordance with KSV’s dependence on temperature, as mentioned above. The linearity of Scatchard plots indicate that palmatine binds to a single class of binding site on HSA. The binding site n (approximately equal to 1) increased slightly with the temperatures rising, which shows the interaction of palmatine to HSA seems to be the presence of one high affinity binding site; the interaction between HSA and palmatine accelerated with the temperature increases, hence, the high affinity binding sites of the interaction is slightly strengthened. The results in Table 2 also show that the binding constant between palmatine and HSA is moderate and the effect of temperature is not significant. Thus, palmatine can be stored and carried by this protein in the body. 3.3. Thermodynamic Parameters and Binding Mode. The interaction forces between drugs and biomolecules may include electrostatic interactions, multiple hydrogen bonds, van der Waals interactions, hydrophobic and steric contacts within the antibody-binding site, and so on. If the enthalpy change (∆H) does not vary significantly in the temperature range studied, both the enthalpy change (∆H)



109

Table 2. Binding Constants and Relative Thermodynamic Parameters of Palmatine-HSA Interaction at pH ) 7.4

T(K)

10-4Ka (L · mol-1) modified Stern-Volmer method

10-4Kb (L · mol-1) Scatchard method

n

298 304 310

5.447 4.995 4.462

5.569 4.948 4.223

1.07 1.13 1.22

∆H (kJ · mol-1)

∆G (kJ · mol-1)

∆S (J · mol-1 · K-1)

-12.76

-27.03 -27.32 -27.61

47.90

and entropy change (∆S) can be evaluated from the van’t Hoff equation:

ln Ka ) -

∆H ∆S + RT R

(4)

where Ka is analogous to the associative binding constants at the corresponding temperature and R is the gas constant. To elucidate the interaction between palmatine and HSA, the thermodynamic parameters were calculated from the van’t Hoff plots. The enthalpy change (∆H) is calculated from the slope of the van’t Hoff relationship. The free energy change (∆G) is then estimated from the following relationship:

∆G ) ∆H - T∆S

(5) Figure 4. Van’t Hoff plots of the palmatine-HSA system.

Table 2 summarizes the values of ∆H and ∆S obtained for the binding site from the slopes and ordinates at the origin of the fitted lines (Figure 4). The negative values of free energy (∆G), seen in Table 2, support the assertion that the binding process is spontaneous. The positive entropy (∆S) and negative enthalpy (∆H) values generally represent hydrophobic interactions,33,34 which is consistent with the observed spectral shifting form Figure 1. The research24 revealed that albumin itself carries a net negative charge at physiological pH, whereas palmatine carries a positive charge in aqueous solution (inset of Figure 1). Hence, the binding of palmatine to HSA might involve electrostatic interactions. 3.4. Site-Selective Binding of Palmatine on HSA. Warfarin and ibuprofen were used as site marker fluorescence probes for monitoring sites I and II of HSA, respectively.26-28 To identify the palmatine binding site on HSA, site marker competitive experiments are carried out using drugs (warfarin and ibuprofen) that specifically bind to a known site or region on HSA. Then information about the palmatine binding site can be gained by monitoring the changes in the fluorescence of palmatine-bound HSA that was brought about by site I and site II markers. In the site marker competitive experiment, palmatine was gradually added to the solution of HSA and site markers held in equimolar concentrations (1.0 × 10-5 mol · L-1). As shown in Figure 5A, with the addition of warfarin into HSA, the maximum emission wavelength of HSA had an obvious red shift (from 340 to 372 nm), and the fluorescence intensity was significantly higher than that without warfarin. Then, with the addition of palmatine, the fluorescence intensity of the HSA decreased gradually, accompanied by an increase of wavelength emission maximum λmax (a red shift, from 372 to 376 nm) in the albumin spectrum. This suggests an increased polar of the region surrounding the tryptophan site (Trp-214)29 and indicating that the bound palmatine to HSA was obviously affected by adding warfarin. Figure 5B shows the comparison of the fluorescence spectra of palmatine-HSA system in the absence and presence of ibuprofen. By contrast, with the presence of ibuprofen, the fluorescence property of the palmatine-HSA system was almost the same as in the absence of ibuprofen,

which indicated that ibuprofen did not prevent the binding of palmatine in its usual binding location. To facilitate the comparison of the influence of warfarin and ibuprofen on the binding of palmatine to HSA, the binding constants in the presence of site markers were analyzed using the modified Stern-Volmer method (Figure 6A) and the Scatchard method (Figure 6B), respectively. The corresponding results are shown in Table 3. The results show that the binding constant was surprisingly variable in the presence of warfarin, while a small influence in the presence of ibuprofen in the binding parameters (somewhat lower than with isolated HSA). These results and plots demonstrating that the decrease in probe fluorescence may result from competitive displacement of the probe, and palmatine bind with high affinity to site I (subdomain IIA) of HSA. 3.5. Binding Distance. FRET has been used as a “spectroscopic ruler” for measuring molecular distances in biological and macromolecular systems;35 it takes place when the fluorescence emission band of one molecule (donor) overlaps with an excitation band of a second (acceptor) that is within 2-8 nm.36,37 Using FRET, the distance r between palmatine and HSA (Trp-214) could be calculated by the equation:30 6

E)1-

R0 F ) 6 F0 R0 + r6

(6)

where E denotes the efficiency of transfer between the donor and the acceptor, r is the average distances between donor and acceptor, and R0 is the critical distance when the efficiency of transfer is 50%.

R60 ) 8.79 × 10-25K2n-4φJ

(7)

In eq 7, K2 is the orientation factor related to the geometry of the donor and acceptor of dipoles and K2 ) 2/3 for random orientation as in fluid solution; n is the average refracted index

110


Hu et al.

Figure 5. Effect of site marker to palmatine-HSA system. (T ) 310 K, λex ) 295 nm) a-g: c(site marker) ) c(HSA) ) 1.0 × 10-5 mol · L-1, c(palmatine)/(10-5 mol · L-1), from 0.0 to 2.4 at increments of 0.4; curve h shows the emission spectrum of site marker only. The insets correspond to the molecular structures of site marker warfarin (A) and ibuprofen (B).

of medium in the wavelength range where spectral overlap is significant; φ is the fluorescence quantum yield of the donor; J is the effect of the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor (Figure 7), which could be calculated by the equation:

J)

∫0∞ F(λ)ε(λ)λ4dλ ∫0∞ F(λ)dλ

Figure 6. Modified Stern-Volmer plots (A) and Scatchard plots (B) of site marker competitive experiments of the palmatine-HSA system. Table 3. Binding Constants of Competitive Experiments of Palmatine-HSA System (T ) 310 K) modified Stern-Volmer method 10-4Ka site marker (L · mol-1) / (blank) warfarin ibuprofen

(8)

where F(λ) is the corrected fluorescence intensity of the donor in the wavelength range, from λ to λ + ∆λ; ε(λ) is the extinction coefficient of the acceptor at λ. In the present case, n ) 1.36, φ ) 0.074,38 according to the eqs 6-8, we could calculate that J ) 2.92 × 10-14 cm3 · L · mol-1; E ) 0.379; R0 ) 2.68 nm; and the binding distance r ) 2.91 nm. The values for R0 and r are on the 2-8 nm scale,39 and 0.5R0 < r < 1.5R0,40 indicating an existence of an interaction between palmatine and HSA (Trp-214). 3.6. Conformation Investigation. The far-UV CD spectra of HSA exhibit a typical shape of an R-helix rich secondary structure (two minima at approximately 208 and 222 nm).20,41,42 To ascertain the possible influence of palmatine binding on the secondary structure of HSA, we measured the far-UV CD spectra in the range of 200-260 nm. Figure 8 shows the CD spectra of

a

4.462 1.585 3.634

Ra

S.D.b

0.9999 0.0489 0.9999 0.0381 0.9999 0.0096

R is the correlation coefficient.

b

Scatchard method 10-4Kb (L · mol-1) 4.223 1.605 3.582

Ra

S.D.b

0.9929 0.0072 0.9976 0.0010 0.9995 0.0019

S.D. is standard deviation.

the free HSA and its palmatine-HSA complexes obtained at pH ) 7.4 and room temperature. As expected for a protein that is predominately R-helical, the CD spectrum of free HSA shows a strong negative ellipticity at 208 and 222 nm (Figure 8, curve a). The addition of palmatine causes a decrease in band intensity at all wavelengths of the far-UV CD (Figure 8, curve b-f), comparing curve b-f with a, the difference indicates that the interaction of palmatine with HSA changes the conformation of HSA. The decrease in negative ellipticity indicates a decrease in the R-helical content, which means the peptide strand unfolding even more. The CD results were expressed in terms of mean residue ellipticity (MRE) in deg · cm2 · dmol-1 according to the following equation:

MRE )

observed CD(mdeg) cpnl × 10

(9)



111

spectroscopy, UV-visible absorption spectroscopy, and circular dichroism spectroscopy. The experimental results indicate that the quenching mechanism of fluorescence of HSA by palmatine is a static quenching procedure, the binding reaction is spontaneous, and electrostatic interactions played a major role in the reaction. Site marker competitive experiments demonstrating that the binding site is located in the hydrophobic pocket of site I (subdomain IIA). Furthermore, the binding parameters, binding site, binding distance, and conformation changes were also obtained.

Figure 7. Spectral overlap of palmatine absorption (curve A) with HSA fluorescence (curve F; T ) 298 K). c(HSA) ) c(palmatine) ) 1.0 × 10-5 mol · L-1.

Acknowledgment. The authors gratefully acknowledge financial support of National Natural Science Foundation of China (Grant Nos. 20803019, 20873096, and 20621502), the Research Foundation of Education Bureau of Hubei Province, China (Grant No. Q20082205), and Hubei Normal University Foundation, China (Grant No. 2007F10).

References and Notes

Figure 8. Circular dichroism spectra of free HSA and its palmatine-HSA complexes. a-f: c(HSA) ) 1.0 × 10-6 mol · L-1, c(palmatine)/(10-6 mol · L-1), from 0 to 5 at increments of 1 (T ) 298 K).

where cp is the molar concentration of the protein, n is the number of amino acid residues (585), and l is the path length (1 cm). The R-helix contents of free and combined HSA were calculated from the MRE value at 208 nm using the following equation:

R-helix(%) )

[

]

-MRE208 - 4000 × 100 33000 - 4000

(10)

where MRE208 is the observed MRE value at 208 nm, 4000 is the MRE of the β-form and random coil conformation cross at 208 nm, and 33000 is the MRE value of a pure R-helix at 208 nm. From the above eqs 9 and 10, the quantitative analysis results of the R-helix content were obtained and shown in Figure 8. A major reduction of the R-helix from 58.7% (free HSA) to 33.1% (palmatine-HSA 5:1 complex) was observed, which was indicative of the loss of R-helix upon the interaction. Therefore, we inferred that the binding of palmatine to HSA induced conformation changes markedly in HSA.

4. Conclusions In this work, the specific interaction of palmatine with HSA was studied by spectroscopic methods including fluorescence

(1) Schmeller, T.; Latz-Bru¨ning, B.; Wink, M. Phytochemistry 1997, 44, 257–266. (2) Kong, W. J.; Zhao, Y. L.; Xiao, X. H.; Li, Z. L.; Ren, Y. S. J. Hazard. Mater. 2009, 168, 609–613. (3) Yan, D.; Jin, C.; Xiao, X. H.; Dong, X. P. J. Biochem. Biophys. Methods 2008, 70, 845–849. (4) Ku¨peli, E.; Kos¸ar, M.; Yes¸ilada, E.; Bas¸er, K. H. C.; Bas¸er, C. Life Sci. 2002, 72, 645–657. (5) Chao, J.; Lu, T. C.; Liao, J. W.; Huang, T. H.; Lee, M. S.; Cheng, H. Y.; Ho, L. K.; Kuo, C. L.; Peng, W. H. J. Ethnopharmacol. 2009, 125, 297–303. (6) Singh, J.; Kakkar, P. J. Ethnopharmacol. 2009, 123, 22–26. (7) Kim, Y. M.; Ha, Y. M.; Jin, Y. C.; Shi, L. Y.; Lee, Y. S.; Kim, H. J.; Seo, H. G.; Choi, J. S.; Kim, Y. S.; Kang, S. S.; Lee, J. H.; Chang, K. C. Food Chem. Toxicol. 2009, 47, 2097–2102. (8) Yu, Y.; Yi, Z. B.; Liang, Y. Z. FEBS Lett. 2007, 581, 4179–4183. (9) Chae, S. H.; Jeong, I. H.; Choi, D. H.; Oh, J. W.; Ahn, Y. J. J. Agric. Food Chem. 1999, 47, 934–938. (10) Kim, J. P.; Jung, M. Y.; Kim, J. P.; Kim, S. Y. J. Agric. Food Chem. 2000, 48, 1058–1063. (11) Li, J.; Shuang, S.; Dong, C. Talanta 2009, 77, 1043–1049. (12) Islam, Md. M.; Kumar, G. S. J. Mol. Struct. 2008, 875, 382–391. (13) Islam, Md. M.; Chowdhury, S. R.; Kumar, G. S. J. Phys. Chem. B 2009, 113, 1210–1224. (14) Hirakawa, K.; Kawanishi, S.; Hirano, T. Chem. Res. Toxicol. 2005, 18, 1545–1552. (15) Zhang, G.; Keita, B.; Craescu, C. T.; Miron, S.; Oliveira, P.; Nadjo, L. Biomacromolecules 2008, 9, 812–817. (16) Monti, S.; Manet, I.; Manoli, F.; Capobianco, M. L.; Marconi, G. J. Phys. Chem. B 2008, 112, 5742–5754. (17) Beauchemin, R.; N’soukpoe´-Kossi, C. N.; Thomas, T. J.; Thomas, T.; Carpentier, R.; Tajmir-Riah, H. A. Biomacromolecules 2007, 8, 3177–3183. (18) Ahmad, B.; Parveen, S.; Khan, R. H. Biomacromolecules 2006, 7, 1350–1356. (19) Carter, D. C.; Ho, J. X. AdV. Protein Chem. 1994, 45, 153–203. (20) Charbonneau, D.; Beauregard, M.; Tajmir-Riahi, H. A. J. Phys. Chem. B 2009, 113, 1777–1784. (21) La´zaro, E.; Lowe, P. J.; Briand, X.; Faller, B. J. Med. Chem. 2008, 51, 2009–2017. (22) Nicoletti, F. P.; Howes, B. D.; Fittipaldi, M.; Fanali, G.; Fasano, M.; Ascenzi, P.; Smulevich, G. J. Am. Chem. Soc. 2008, 130, 11677– 11688. (23) Olson, R. E.; Christ, D. D. Annu. Rep. Med. Chem. 1996, 31, 327– 336. (24) Kragh-Hansen, U. Pharmacol. ReV. 1981, 33, 17–53. (25) He, X. M.; Carter, D. C. Nature 1992, 358, 209–215. (26) Sudlow, G.; Birkett, D. J.; Wade, D. N. Mol. Pharmacol. 1975, 11, 824–832. (27) Sudlow, G.; Birkett, D. J.; Wade, D. N. Mol. Pharmacol. 1976, 12, 1052–1061. (28) Wanwimolruk, S.; Birkett, D. J.; Brooks, P. M. Mol. Pharmacol. 1983, 24, 458–463. (29) Il’ichev, Y. V.; Perry, J. L.; Simon, J. D. J. Phys. Chem. B 2002, 106, 452–459.

112


(30) Lakowicz, J. R. Principles of fluorescence spectroscopy, 3rd ed.; Springer: New York, 2006. (31) Lehrer, S. S. Biochemistry 1971, 10, 3254–3263. (32) Scatchard, G. Ann. N. Y. Acad. Sci. 1949, 51, 660–672. (33) Ross, P. D.; Subramanian, S. Biochemistry 1981, 20, 3096–3102. (34) Timaseff, S. N. Thermodynamics of protein interactions. In Proteins of biological fluids; Peeters, H., Ed.; Pergamon Press: Oxford, 1972. (35) Kapanidis, A. N.; Laurence, T. A.; Lee, N. K.; Margeat, E.; Kong, X.; Weiss, S. Acc. Chem. Res. 2005, 38, 523–533. (36) Stryer, L.; Haugland, R. P. Proc. Natl. Acad. Sci. U.S.A. 1967, 58, 719–726.

Hu et al. (37) Valeur, B.; Brochon, J. C. New Trends in Fluorescence Spectroscopy; Springer: Berlin, 1999. (38) Cyril, L.; Earl, J. K.; Sperry, W. M. Biochemists’ Handbook; E. & F. N. Spon: London, 1961. (39) Yang, J. Y.; Yang, W. Y. J. Am. Chem. Soc. 2009, 131, 11644–11645. (40) Valeur, B. Molecular Fluorescence: Principles and Applications; Wiley Press: New York, 2001. (41) Lu, G.; Ai, S.; Li, J. Langmuir 2005, 21, 1679–1682. (42) Sen, P.; Fatima, S.; Ahmad, B.; Khan, R. H. Spectrochim. Acta, Part A 2009, 74, 94–99.

BM900961E

Site-Selective Binding of Human Serum Albumin by Palmatine

Recommend Documents