Investigation of the Interaction between Berberine and Human Serum

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Investigation of the Interaction between Berberine and Human Serum Albumin Yan-Jun Hu,†,‡ Yi Liu,*,† and Xiao-He Xiao§ State Key Laboratory of Virology & Department of Chemistry, Wuhan University, Wuhan 430072, People’s Republic of China, 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 October 7, 2008; Revised Manuscript Received November 12, 2008

Berberine is an important traditional medicinal herb, which has been effectively used in the treatment of dysentery, diarrhea, stomatitis, throat infections, and hepatitis in folk medicine. In this study, the interaction between Berberine and human serum albumin (HSA) was investigated by fluorescence spectroscopy and UV-vis absorbance spectroscopy. In the mechanism discussion, it was proved that the fluorescence quenching of HSA by berberine is a result of the formation of berberine-HSA complex. Fluorescence quenching constants were determined using the Stern-Volmer equation and Scatchard equation to provide a measure of the binding affinity between berberine and HSA. The results of thermodynamic parameters ∆G, ∆H, and ∆S at different temperatures indicate that the electrostatic interactions play a major role for berberine-HSA association. Site marker competitive experiments indicated that the binding of berberine to HSA primarily took place in subdomain IIA. Furthermore, the distance r between donor (Trp-214) and acceptor (berberine) was obtained according to fluorescence resonance energy transfer (FRET).

1. Introduction Plasma protein binding is an important factor to understand the pharmacokinetics and pharmacodynamic properties of drug candidates, as it strongly influences drug distribution and determines the free fraction, which is available to the target.1 Human serum albumin (HSA), the most prominent protein in plasma, has the capability to bind a wide range of endogenous and exogenous compounds such as nonesterified fatty acids, heme, bilirubin, thyroxine, and bile acids, as well as an extraordinarily broad range of drugs.2-6 The crystallographic analyses of HSA revealed that the protein, a 585 amino acid residue monomer, contains three homologous R-helical domains (I-III), each of which is composed by two subdomains A and B. The protein is stabilized by 17 disulfide bridges.7 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. These binding sites are known as Sudlow I and Sudlow II,8,9 respectively, and the sole tryptophan residue in HSA is located in Sudlow I (Trp-214). The hydrophobic cavities in HSA often increase the apparent solubility of hydrophobic drugs in plasma and modulates their delivery to cells in vivo and in vitro; they can play a dominant role in drug disposition and efficacy.10 This paper investigates the association of HSA with berberine. Berberine (molecular structure: Figure 1; CAS Registry Number: 633-65-8), an alkaloid isolated from Chinese herbs, is an important traditional medicinal herb, mainly grows in Asia and Europe, which has been found to have analgesic,11 antibacterial,12 antimalarial,13 antipyretic,14 antitubercular,15 antiphoto* To whom correspondence should be addressed. Tel.: +86 27 87218284. Fax: +86-27-68754067. E-mail: [email protected]. † Wuhan University. ‡ Hubei Normal University. § Institute of Chinese Materia Medica.

Figure 1. Molecular structures of berberine.

oxidative,16 antileishmanial,17 antisecretory,18 and antitumor19 activities in vitro and in vivo. In addition to its medicinal uses, berberine is also used as a fluorescent probe of cells, DNA, and energized mitochondria20 in biochemical researches. Berberine hydrochloride (berberine) was selected to investigate because of its physiological and pharmacological effects mentioned above. 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. In addition, many promising new drugs are rendered ineffective because of their unusually high affinity for this abundant protein.21 Obviously, an understanding of the chemistry of the various classes of pharmaceutical interactions with albumin can suggest new approaches to drug therapy and design. Despite much information on the antimicrobial potential of Berberine, the interaction of HSA with berberine has not yet been detailed investigated. Spectral methods are powerful tool for the study of the reactivity of chemical and biological systems since it allows nonintrusive measurements of substances in low concentration under physiological conditions, and there are several studies of albumin induced by drugs or other bioactive small molecules using spectral methods.22-25 In this work, we employ spectral methods to obtain information related to the binding mechanisms of berberine to HSA such as quenching rate constants, binding modes, binding

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constants, binding sites, and intermolecular distances. We show that berberine binds specifically in Sudlow site I.

2. Materials and Methods 2.1. Materials. HSA and Warfarin were obtained from SigmaAldrich (St. Louis, MO, U.S.A.); berberine was obtained from Northeast General Pharmaceutical Factory (Liaoning, China); ibuprofen was obtained from Hubei Biocause Pharmaceutical Co., Ltd. (Hubei, China; the purity no less than 99.7%); and 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 weighted on a microbalance (Sartorius, ME215S) with a resolution of 0.1 mg. 2.2. Equipments and Spectral Measurements. 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. 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 since it provides no excitation of tyrosine residues and therefore neither emission nor energy transfer to the lone indole side chain would be nonnegligible. Appropriate blanks corresponding to the buffer were subtracted to correct the fluorescence background. 2.3. Principles of Fluorescence Quenching. Fluorescence quenching is described by the well-known Stern-Volmer equation:26

F0 ) 1 + KSV[Q] F

Figure 2. Emission spectra of HSA in the presence of various concentrations of berberine. The inset corresponds to the Stern-Volmer plot. c (HSA) ) 1.0 × 10-5 mol · L-1; c (berberine)/(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 berberine only (T ) 298 K, λex ) 295 nm). Table 1. Stern-Volmer Quenching Constants for the Interaction of Berberine with HSA at Various Temperatures pH

T (K)

10-4 KSV (L · mol-1)

Ra

S.D.b

7.4

298 304 310

4.883 4.674 4.548

0.9986 0.9996 0.9995

0.0135 0.0070 0.0074

a

R is the correlation coefficient.

b

S.D. is standard deviation.

(1)

where F0 and F denotes the steady-state fluorescence intensities in the absence and in the presence of quencher (berberine), 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].

3. Results and Discussions 3.1. Effect of Berberine on HSA Spectra. A variety of molecular interactions can result in quenching, including excitedstate reactions, molecular rearrangements, ground-state complex formation, and collisional quenching, and so on. The different mechanisms of quenching 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.26 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. In the experiment, the concentrations of HSA solution were stabilized at 1.0 × 10-5 mol · L-1, and the concentrations of berberine varied from 0 to 2.4 × 10-5 mol · L-1 at increments of 0.2 × 10-5 mol · L-1. The effect of berberine on HSA fluorescence intensity at 298 K is shown in Figure 2. It was observed from Figure 2 that a progressive decrease in the fluorescence intensity was caused by quenching, accompanied by a decrease of wavelength emission maximum λmax (a blue shift) in the albumin spectrum. This suggests an increased hydrophobicity of the region surrounding the tryptophan site (Trp-214).27 The inset in Figure 2 shows that, within the investigated concentrations range, the results agree with the Stern-Volmer equation. The calculation of KSV from Stern-Volmer plots (Table 1) demonstrated the effect on fluorescence quenching by berberine

at each temperature studied, the result shows that the Stern-Volmer quenching constant KSV is inversely correlated with temperature, which indicate that the probable quenching mechanism of berberine-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 wiIl frequently result in perturbation of the absorption spectrum of the fluorophore.26 For reconfirming the probable quenching mechanism of fluorescence of HSA by berberine is initiated by ground-state complex formation, we used the difference absorption spectroscopy to obtain spectra, the UV-vis absorption spectra of HSA and the difference absorption spectra between HSA-berberine and berberine at the same concentration could not be superposed within experimental error (Figure 3), this result reconfirm that the probable quenching mechanism of fluorescence of HSA by berberine is a static quenching procedure. Therefore, the quenching data were analyzed according to the modified Stern-Volmer equation:28

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

Interaction between Berberine and Human Serum Albumin

Figure 3. UV-visible spectra of HSA in the presence of berberine. A (solid): the absorption spectrum of HSA only; B (dash dot): the difference absorption spectrum between berberine-HSA and berberine at the same concentration; c (HSA) ) c (berberine) ) 1.0 × 10-5 mol · L-1. The absorbance spectra for the wavelength range from 250 to 300 nm are depicted in the inset.

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. The corresponding results at different temperatures are shown in Table 2. The decreasing trend of Ka with increasing temperature was in accordance with KSV’s dependence on temperature, as mentioned above. It shows that the binding constant between berberine and HSA is moderate and the effect of temperature is not significant. Thus, berberine can be stored and carried by this protein in the body. 3.2. Determination of Binding Mode between Berberine and HSA. 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. To elucidate the interaction between berberine and HSA, the thermodynamic parameters were calculated from the van’t Hoff plots. If the enthalpy change (∆H) does not vary significantly in the temperature range studied, both the enthalpy change (∆H) and entropy change (∆S) can be evaluated from the van’t Hoff equation:

ln Ka ) -

∆H ∆S + RT R

(3)

where Ka is analogous to the associative binding constants at the corresponding temperature and R is the gas constant. The temperatures used were 298, 304, and 310 K. The enthalpy change (∆H) is calculated from the slope of the van’t Hoff relationship (Figure 4). The free energy change (∆G) is then estimated from the following relationship:

∆G ) ∆H - T∆S

(4)

Table 2 summarizes the values of ∆H and ∆S obtained for the binding site from the slopes and ordinates at the origin of

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Figure 4. Van’t Hoff plots of berberine-HSA system, pH ) 7.4, c (HSA) ) 1.0 × 10-5 mol · L-1.

the fitted lines. The negative values of free energy (∆G), seen in Table 2, supports the assertion that the binding process is spontaneous. The research6 revealed that albumin itself carries a net negative charge at physiological pH; berberine carries a positive charge in aqueous solution (Figure 1). Hence, the binding of berberine to HSA might involve electrostatic interactions. The negative enthalpy (∆H) and positive entropy (∆S) values of the interaction of berberine and HSA is also indicating that the electrostatic interactions played a major role in the binding reaction.29,30 3.3. Identification of the Binding Site of Berberine on HSA. When small molecules bind to a set of equivalent sites on a macromolecule, the equilibrium binding constant (Kb) and the numbers of binding sites (n) can be analyzed according to the Scatchard equation:31 r

⁄Df ) nKb - rKb

(5)

where r is the moles of ligand bound per mole of protein, Df is the molar concentration of free ligand, n is the binding site multiplicity per class of binding sites, and K is the equilibrium binding constant. For the system of berberine and HSA, the values of Kb and n at different temperatures were shown in Table 2, respectively. The value of n was approximately equal to 1, which indicated that there was one class of binding sites of berberine on HSA. The crystal structure analyses revealed that HSA contains three homologous R-helical domains (I-III), each of which is composed of two subdomains A and B. According to Sudlow’s nomenclature, two primary sites (1 and 2) have been identified for ligand binding to HSA. Warfarin, an anticoagulant drug, and ibuprofen, a nonsteroidal anti-inflammatory agent, have been considered as stereotypical ligands for Sudlow’s sites 1 and 2, respectively. Warfarin, as other bulky heterocyclic anions, binds to Sudlow’s site 1, located in subdomain IIA, whereas ibuprofen, as other aromatic carboxylates with an extended conformation, prefers Sudlow’s site 2, located in subdomain IIIA. In addition,

Table 2. Binding Constants and Relative Thermodynamic Parameters of Berberine-HSA Interaction at pH 7.4 T (K)

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

10-4 Kb (L · mol-1) scatchard method

n

∆H (kJ · mol-1)

∆G (kJ · mol-1)

∆S (J · mol-1 · K-1)

298 304 310

6.643 5.906 5.371

5.415 5.152 4.894

0.968 0.929 0.963

-13.61

-27.50 -27.78 -28.06

46.62

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Figure 5. Effect of site marker to the berberine-HSA system (T ) 310 K, λex ) 295 nm). c (warfarin) ) c (ibuprofen) ) c (HSA) ) 1.0 × 10-5 mol · L-1; c (berberine)/(10-5 mol · L-1), A-I: 0; 0.4; 0.8; 1.2; 1.6; 2.0; 2.4; 2.8; 3.2. The insets correspond to the molecular structures of the site marker.

a secondary binding cleft has been found for ibuprofen located at the interface between subdomains IIA and IIB.8,9,32,33 To identify the berberine 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 berberine binding site can be gained by monitoring the changes in the fluorescence of berberine bound HSA that are brought about by site I (warfarin) and site II (ibuprofen) markers (Figure 5). In the site marker competitive experiment, berberine 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, and the fluorescence intensity was significantly higher than that of without warfarin. Then, with the addition of

berberine, the fluorescence intensity of the HSA decreased gradually, accompanied by an increase of wavelength emission maximum λmax (a red shift) in the albumin spectrum. This suggests an increased polar of the region surrounding the tryptophan site (Trp-214),27 and indicating that the bound berberine to HSA was affected by adding warfarin. Figure 5b shows the comparison of the fluorescence spectra of the berberine-HSA system in the absence and presence of ibuprofen. By contrast, with the presence of ibuprofen, the fluorescence property of the berberine-HSA system was almost the same as in the absence of ibuprofen, which indicated that ibuprofen did not prevent the binding of berberine in its usual binding location. To facilitate the comparison of the influence of warfarin and ibuprofen on the binding of berberine to HSA, the binding constants in the presence of site markers were analyzed using the modified Stern-Volmer equation and Scatchard equation (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 analysis indicated that the binding site of berberine was mainly located within site I (subdomain IIA) of HSA. 3.4. Energy Transfer from HSA to Berberine. FRET is a nondestructive spectroscopic method that can monitor the proximity and relative angular orientation of fluorophores, the donor and acceptor fluorophores can be entirely separate or attached to the same macromolecule. A transfer of energy could take place through direct electrodynamic interaction between the primarily excited molecule and its neighbors,34 which will happen under the following conditions: (i) the donor can produce fuorescence light; (ii) fuorescence emission spectrum of the donor and UV-vis absorbance spectrum of the acceptor have more overlap; and (iii) the distance between the donor and the acceptor approach and is lower than 8 nm.35 Using FRET, the distance r between berberine and HSA (Trp-214) could be calculated by the following equation26

E)1-

R60 F ) 6 F0 R + r6

(6)

0

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)

2

In eq 7, K 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 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

Table 3. Binding Constants of Competitive Experiments of the Berberine-HSA System (T ) 310 K) modified Stern-Volmer method

a

Scatchard method

site marker

10-4 Ka (L · mol-1)

Ra

S.D.b

10-4 Kb (L · mol-1)

Ra

S.D.b

/ (blank) warfarin ibuprofen

5.371 1.034 4.654

0.9995 0.9997 0.9943

0.1104 0.1582 0.1665

4.894 2.484 4.709

0.9852 0.9787 0.9909

0.0103 0.0034 0.0069

R is the correlation coefficient.

b

S.D. is standard deviation.

Interaction between Berberine and Human Serum Albumin

Figure 6. Spectral overlap of berberine absorption (curve A, dashed line) with HSA fluorescence (curve F, solid line) (T ) 298 K). c (HSA) ) c (berberine) ) 1.0 × 10-5 mol · L-1.

spectrum of the donor and the absorption spectrum of the acceptor (Figure 6), which could be calculated by the following equation

∫0∞ F(λ)ε(λ)λ4dλ J) ∫0∞ F(λ)dλ

(8)

where F(λ) is the corrected fluorescence intensity of the donor in the wavelength range from λ to λ + ∆λ and ε(λ) is the extinction coefficient of the acceptor at λ. In the present case, n ) 1.36 and φ ) 0.074;36 according to eqs 6-8, we could calculate that J ) 3.13 × 10-14 cm3 · L · mol-1, E ) 0.316, R0 ) 2.71 nm, and r ) 3.10 nm. The average distances between a donor fluorophore and acceptor fluorophore are on the 2-to-8 nm scale,37 and 0.5R0 < r < 1.5R0,38 which indicate that the energy transfer from HSA to berberine occurs with high probability.

4. Conclusions In this paper, the interaction of berberine with HSA was studied by spectroscopic methods including fluorescence spectroscopy and UV-visible absorption spectroscopy. The experimental results indicate that the probable quenching mechanism of fluorescence of HSA by berberine is a static quenching procedure; the binding reaction is spontaneous, and electrostatic interactions played a major role in the reaction. In addition, the binding site is located in the hydrophobic pocket of subdomain IIA according to the site competitive study. Acknowledgment. The authors gratefully acknowledge financial support of National Natural Science Foundation of China (Grant Nos. 20803019, 20621502, and 20873096), the Research Foundation of Education Bureau of Hubei Province, China (Grant No. Q20082205), and Hubei Normal University Foundation, China (Grant No. 2007F10).

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