Spectroscopic characterization of effective components anthraquinones in Chinese medicinal herbs binding with serum albumins

Spectrochimica Acta Part A 62 (2005) 203–212

Spectroscopic characterization of effective components anthraquinones in Chinese medicinal herbs binding with serum albumins Shuyun Bia,b , Daqian Songa , Yuhe Kanc , Dong Xuc , Yuan Tiana , Xin Zhoua , Hanqi Zhanga,∗ b

a College of Chemistry, Jilin University, Linyuan Road 1788, Changchun 130023, PR China Department of Chemistry, College of Science and Engineering, Yanbian University, Yanji 133002, PR China c Department of Chemistry, Northeast Normal University, Changchun, Jilin 130024, PR China

Received 27 November 2004; accepted 16 December 2004

Abstract The interactions of serum albumins such as human serum albumin (HSA) and bovine serum albumin (BSA) with emodin, rhein, aloeemodin and aloin were assessed employing fluorescence quenching and absorption spectroscopic techniques. The results obtained revealed that there are relatively strong binding affinity for the four anthraquinones with HSA and BSA and the binding constants for the interactions of anthraquinones with HSA or BSA at 20 ◦ C were obtained. Anthraquinone–albumin interactions were studied at different temperatures and in the presence of some metal ions. And the competition binding of anthraquinones with serum albumins was also discussed. The Stern–Volmer curves suggested that the quenching occurring in the reactions was the static quenching process. The binding distances and transfer efficiencies for each binding reactions were calculated according to the F¨oster theory of non-radiation energy transfer. Using thermodynamic equations, the main action forces of these reactions were also obtained. The reasons of the different binding affinities for different anthraquinone–albumin reactions were probed from the point of view of molecular structures. © 2005 Elsevier B.V. All rights reserved. Keywords: Emodin; Rhein; Aloe-emodin; Aloin; Serum albumin; Flurorescence quenching

1. Introduction Fluorescence spectroscopy is an important tool to probe the structure and dynamics of biomacromolecules. The utility of fluorescence techniques stems from the ability of fluorophores to reflect changes in their molecular environment through measurable alterations in emission properties. The major protein in plasma is serum albumin. Serum albumin has been one of the most studied proteins for over 40 years because its primary structure has been well known for a long time and its tertiary structure was determined a few years ago by X-ray crystallography [1,2]. Human serum albumin (HSA) has two fluorophores, namely tryptophan (Trp 214) and tyrosine (Tyr). Bovine serum albumin (BSA) and ∗

Corresponding author. Tel.: +86 4315168352; fax: +86 4315635389. E-mail address: [email protected] (H. Zhang).

1386-1425/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2004.12.049

HSA displays approximately 76% sequence homology [3] and from the spectroscopic point of view the main difference between the two proteins is that BSA has two tryptophan that are tryptophan 135 (Trp 135) and tryptophan 214 (Trp 214) [1]. Tryptophan and tyrosine present in the proteins act as intrinsic fluorescence probes [4]. Many endogenic or xenogenic substants can bind to serum albumins. It is well known that the serum albumin can reversibly bind drug molecules to affect the drugs distribution. The strong fluorescence characteristics of the serum albumins provide a sensitive spectroscopic method to study their interactions with various drug molecules. The changes in the fluorescence intensity of these spectra can be used to probe the nature of the binding reactions of serum albumins and drug molecules. Emodin, rhein, aloe-emodin and aloin are the effective herbal components traditionally used in China for treating various ailments. Emodin (1,3,8-trihydroxy-6-methylan-

204

S. Bi et al. / Spectrochimica Acta Part A 62 (2005) 203–212

thraquinone) is an active constituent of Rheum palmatum herb [5,6]. Pharmacological studies have demonstrated that emodin possesses antibacterial, diuretic and vasorelaxant effects [7–9]. Rhein (1,8-dihydroxy-3-carboxyl-9,10anthraquinone) enriched in the rhizome of rhubarb, a traditional Chinese medicine showing antitumor promotion function [10]. Aloe-emodin (1,8-dihydroxy-3-hydroxymethyl9,10-anthraquinone) also present in Rhei Rhizoma and many studies have indicated that aloe-emodin has laxative, antibacterial, antiviral and hepatoprotective effects [11–15]. Aloin (10-glucopyranosyl-1,8-dihydroxy-3(hydroxy methyl)-9(10H)-anthracenone) is the best known active ingredient in aloe, a folk remedy to strengthen the stomach and to relieve constipation for 3000 years [16]. From their molecular structures given in Scheme 1, emodin, rhein, aloe-emodin and aloin belong to anthraquinone compounds. Because of their pharmacological activity, the investigation of the interactions between these compounds and serum albumins are very important. Generally, the drug molecule interacting biomacromolecule forms a kind of supramolecular complex by virtue of intermolecular acting force. In recent years, the study in different directions on the small molecules binding to protein or DNA is an active field [17–19]. The

deep research on anthraquinones such as emodin, rhein, aloeemodin and aloin binding properties with HSA or BSA was carried out in this paper. Especially, an improved data analysis method based on the previous paper [20] was developed. Compared with the former method appeared in the previous paper, the data process in this paper was more universal. Using the improved data analysis method, the values of binding constants and binding sites were found. In addition, the energy transfer efficiency, the binding distances, the binding forces and the effects of some metal ions on the reactions of these anthraquinones and serum albumins were all obtained. It is worthy of note that the competition binding with serum albumins among these drugs, which was very important for traditional Chinese medicine, was investigated and few reports about this work were carried out. The regularity of the relationship between the binding constants and the molecular structures was explored. The results of these various binding studies may be useful in designing new and promising drugs for a variety of medical conditions in clinic.

2. Experimental 2.1. Chemicals HSA and BSA were obtained from Shanghai Biological Products Institute and Jilin Bote Biotechnological Corporation, respectively. Emodin, rhein, aloe-emodin and aloin were purchased from China Drug Biological Products Qualifying Institute. All other chemicals used were of analytical reagent grade and doubly deionized water was used throughout. 2.2. Apparatus Fluorescence measurements were made on a Shimadzu RF-5301PC spectrofluorophotometer, using a quartz cell of 1 cm path length. Both the excitation and emission slits were set at 5 nm. Fluorescence spectra were recorded in the wavelength range of 290–500 nm after exiting HSA or BSA at 280 nm. An electronic thermostat water-bath from Tianjin Taisite Instrument Company in China was used for controlling the temperature. UV spectra measurements were made on an Australian GBC Cintra 10e UV–vis spectrometer equipped with a quartz cell of 1 cm path length. 2.3. General procedures

Scheme 1. Structures of (a) emodin, (b) rhein, (c) aloe-emodin and (d) aloin.

The fluorescence measurements were carried out in 0.05 mol l−1 Tris–HCl buffer of pH 7.4 containing 0.1 mol l−1 sodium chloride by adding appropriate amount of ethanol solutions of anthraquinones to a fix amount of albumin in each test tube. The final volume was made up to 2.5 ml with Tris–HCl buffer selected by this experiment. Thus, a series of tubes containing various amounts of anthraquinones and a definite amount of serum albumins (1.0 × 10−5 mol l−1 ) were obtained. Before determination, the tubes should be shaken up and placed into the thermostat water-bath for 5 min.

S. Bi et al. / Spectrochimica Acta Part A 62 (2005) 203–212

Then the assay solutions were transferred into the quartz cell and the fluorescence measurement was performed at the above selected parameters.

3. Results and discussion 3.1. Binding characteristics When HSA or BSA without adding the anthraquinones was exited at 280 nm, an emission spectrum appeared in the wavelength range of 290–500 nm and the emission maximum was at about 340 nm. Then the other assay solution containing HSA or BSA (same concentration as the above) and a fix amount of anthraquinones in each tube was measured under the selected experiment conditions. The amount of anthraquinones was increased in succession but the concentration of serum albumins was constant all along. The fluorescence spectra of emodin, rhein, aloe-emodin and aloin binding to the serum albumins were observed in Fig. 1. The additions of anthraquinones have significant effects on the fluorescence of both proteins. The emission spectra showed that emodin, rhein, aloe-emodin and aloin could act as quenchers to decrease the fluorescence intensity of HSA or BSA. The peak wavelength shifted when anthraquinones was added into the serum albumins solutions. These changes of spectra suggested that a new complex formed between the anthraquinones and serum albumins. For BSA, the fluorophores Trp 135 and Trp 214 were located subdomains IA and IIA, respectively [1]. Crystal structure analyses revealed that the main HSA binding region was located in subdomains IIA and IIIA for several ligands such as drug molecules [1]. Because of the similarity of spectra for HSA and that for BSA, the conclusion that subdomain IIA was the binding sites for the interaction of anthraquinones and serum albumins was drawn and Trp 135 of BSA is not the target binding site for anthraquinones. Fluorescence quenching technique can reveal the nature of binding reaction by the fluorescence properties changes of chromophore. These changes reflect the environmental alteration around the fluorophore. Two quenching processes are known: static and dynamic quenching. Static quenching refers to formation of a non-fluorescencent fluorophore–quencher complex. Dynamic quenching refers to the quencher diffuse to the fluorophore during the lifetime of the exited state and upon contact, the fluorophore returned to ground state without emission of a photon [21]. The quenching nature of albumin–anthraquione can be analyzed by the Stern–Volmer equation [22] F0 = 1 + Kq τ0 [Q] = 1 + Ksv [Q] F

(1)

In this equation, F0 and F are the fluorescence intensities in the absence and presence of quencher, [Q] the quencher concentration, Kq the biomolecular quenching rate constant, τ 0 the average lifetime of molecule in the absence of quencher

205

and its value is 10−8 s [23] and Ksv is the Stern–Volmer dynamic quenching constant. The Stern–Volmer graphs are shown in Fig. 2 at various temperatures. For emodin, rhein and aloe-emodin, the values of Ksv at 37 ◦ C were all lower than the ones at 20 ◦ C and the values of Ksv for aloin were almost equal at the both temperatures. This demonstrated that the quenching occurring in albumin–anthraquinone was not a dynamic quenching process. Moreover, the values of Kq , which were in the range of 2.5 × 1012 to 1.4 × 1013 l s−1 mol−1 for all reactions of anthraquinone– albumin and were far greater than 2.0 × 1010 l s mol−1 , the maximum diffusion collision quenching rate constant of various quenchers with the biopolymer [24]. Therefore, the point that anthraquinones binding proteins was a static quenching process proved to be true. An improved data analysis method based on the method of the previous paper [20] was put forward. For this quenching process, suppose there is the same and independent binding site n in the protein, namely the binding capacity of serum albumins at each binding site is equal. The reaction of drug molecules and protein can be expressed as P + nD = Dn P

(2)

where P is protein, D the drug molecule, Dn P the new forming n where K is the bindcomplex and its binding constant is KA A ing constant for the reaction P + D = PD. From the previous n has been found paper [20], the expression of KA n KA =

[Dn P] [Df ]n [Pf ]

(3)

where [Df ] and [Pf ] are the free concentration of drug and protein, respectively. If the overall concentration of the protein and that of the drug are [Pt ] and [Dt ], respectively, the following equations can be obtained: [Dt ] = [Df ] + n[Dn P]

(4)

[Pt ] = [Pf ] + [Dn P]

(5)

[Pf ] F = [Pt ] F0

(6)

Based on Eqs. (4)–(6), the following relationship is found:
 F0 − F n F0 − F n KA [Dt ] − n[Pt ] (7) = F F0 or


F0 − F F0 − F log = n log KA + n log [Dt ] − n[Pt ] F F0 (8) Suppose n in the parentheses equals one for its slightly effect on Eq. (8), then 
F0 − F F0 − F log = n log KA + n log [Dt ] − [Pt ] F F0 (9)

206

S. Bi et al. / Spectrochimica Acta Part A 62 (2005) 203–212

Fig. 1. Fluorescence emission spectra of (a) HSA and (b) BSA of (1) emodin, (2) rhein, (3) aloe-emodin and (4) rhein in 0.05 mol l−1 Tris–HCl buffer of pH 7.4 containing 0.1 mol l−1 NaCl at 20 ◦ C. Both the total concentrations of HSA and BSA are 1.0 × 10−5 mol l−1 . Total concentrations of emodin either in HSA or BSA (from top to bottom): 0, 0.8, 1.6, 2.4, 3.2, 4.0, 4.8, 5.6 × 10−6 mol l−1 ; total concentrations of rhein either in HSA or BSA (from top to bottom): 0, 1.5, 3.0, 4.5, 6.0, 7.5, 9.0, 10.5 × 10−6 mol l−1 ; total concentrations of aloe-emodin either in HSA or BSA (from top to bottom): 0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 14.0 × 10−6 mol l−1 ; total concentrations of aloin (from top to bottom):0, 0.6, 1.2, 1.8, 2.4, 3.0, 3.6, 4.2 × 10−5 mol l−1 and 0, 0.8, 1.6, 2.4, 3.2, 4.0, 4.8, 5.6 × 10−5 mol l−1 in HSA and BSA, respectively. The excitation wavelength was 280 nm. Both excitation and emission slits were 5 nm and sensitivity was low.

S. Bi et al. / Spectrochimica Acta Part A 62 (2005) 203–212

207

Fig. 2. The Stern–Volmer graphs of (1) emodin, (2) rhein, (3) aloe-emodin and (4) aloin binding to HSA (
) and BSA ().

Eq. (9) is the method described in the previous paper [20], but it is not exact because n in the parentheses was supposed to be one. An improved method on Eq. (7) follows that:


1/n F0 F KA [Dt ] − n[Pt ] 1 − (10) = −1 F F0 If

F = u, then F0

KA ([Dt ] − n[Pt ](1 − u)) =




1/n

1 −1 u

The following form:
 
(1/n)−1
d[Dt ] 1 1 1 − 2 KA + n[Pt ] = −1 du n u u can be obtained. It follows that:
(1/n)−1 d[Dt ] 1 1 1 − n[Pt ] =− −1 du nKA u u2

(11)

(12)

(13)

Based on the experimental data, the curve of u (= FF0 ) versus [Dt ] canbe plotted and fitted and based on the curve, the value t]  of d[D du u=1 can be obtained. From Eq. (13) when u = 1, the following relationship is easy to know, that is:  d[Dt ]  = −n[Pt ] = m (14) du u=1 Therefore, substituting m obtained from the curve of u verF0 −F sus [Dt ] into Eq. (7),  then the plot of log F versus log [Dt ] + m F0F−F can be obtained. The slope of the plot 0 gave the value of n. If the value of n from the slope of m the plot was not equal to the value of −[P obtained from t] m the fitting curve of u versus [Dt ], that is n = −[P , then n t ]  F0 −F obtained from the slope of the plot of log versus F   log [Dt ] + m F0F−F was substituted into Eq. (14) and the 0

S. Bi et al. / Spectrochimica Acta Part A 62 (2005) 203–212

208

  value of m was calculated again and a new plot of log F0F−F   versus log [Dt ] + m F0F−F was obtained and also a new 0 value of n was obtained from the slope of the plot. Such the above process is repeated again and again by the method of approaching step by step using the software of origin 6.0, till the slope n is equal to the n calculated by Eq. (14). In the end, an only value of n can be got and the value of KA obtained by the intercept on Y-axis. According described  above, the plots of   to the method  F0 −F F0 −F log versus log [Dt ] + m F0 were depicted in F Fig. 3 at 20 ◦ C. Both the results got by the improved method and those obtained from Eq. (9) are listed in Table 1 in order to make a comparison. The binding constants of the reactions of the albumin– anthraquinone decreased in the following order: emodin–albumin > rhein–albumin > aloe-emodin–albumin > aloin–albumin. Both binding constants of HSA–anthraquinone and that of BSA–anthraquinone confirmed to such an order. The estimation of the intermolecular acting force existed in the reaction of albumin–anthraquinone helps us to understand the nature of the binding reaction. The following thermodynamics formulae:
ln

KA2 KA1




=

1 1 − T1 T2



H R

(15)

G = H − TS = −RT ln KA

(16)

can be used to attain the aim mentioned above if the reaction enthalpy change, H, is a constant. The binding constant KA at two different temperatures, T1 and T2 , were all obtained by fluorescence quenching method; accordingly the free energy change G, the entropy change S and enthalpy change H were calculated based on these equations. The results of the parameters H, G and S are listed in Table 2. For these reactions, H < 0 and S > 0, the acting force was mainly electrostatic force [25].

Fig. 3. The plots of log((F0 − F)/F) vs. log([Dt ] + m(F0 − F)/F0 ) (m = −n[Pt ]) for (1) emodin, (2) rhein, (3) aloe-emodin and (4) aloin at 20 ◦ C. Plot (a) is for HSA–anthraquinones and plot (b) is for BSA–anthraquinones.

Because the main intermolecular acting force of anthaquinones binding to HSA (or BSA) was electrostatic force, the polarity of the drug molecules became one of the domination factors and it determined the binding capacity. Studying

Table 1 Binding constants and binding sites of anthraquinones to HSA and BSA at 20 ◦ C Compound

Albumin

Method Ia

Method IIb

KA (l mol−1 )

n

r

KA (l mol−1 )

n

r

Emodin

HSA BSA

3.18 × 105 2.03 × 105

0.66 0.95

0.990 0.993

2.94 × 105 1.93 × 105

0.75 0.94

0.999 0.996

Rhein

HSA BSA

2.20 × 105 1.16 × 105

0.72 0.60

0.996 0.999

2.05 × 105 1.09 × 105

0.65 0.62

0.994 0.994

Aloe-emodin

HSA BSA

1.14 × 105 3.84 × 104

0.59 0.98

0.999 0.996

1.03 × 105 3.82 × 104

0.66 0.98

0.996 0.996

Aloin

HSA BSA

2.77 × 104 3.10 × 104

1.14 0.95

0.999 0.994

2.83 × 104 3.07 × 104

1.14 0.96

0.999 0.994

r is the regression coefficient. a Fluorescence method of using Eq. (9) in the text. b Improving fluorescence method.

S. Bi et al. / Spectrochimica Acta Part A 62 (2005) 203–212

209

Table 2 Thermodynamic parameters for anthraquiones binding to HSA and BSA obtained at 20 ◦ C Compound

Albumin

H (kJ mol−1 )

G (kJ mol−1 )

S (J mol−1 K−1 )

Emodin

HSA BSA

−4.95 −7.51

−30.69 −29.66

87.78 75.56

Rhein

HSA BSA

−7.80 −22.04

−29.81 −28.27

75.06 21.25

Aloe-emodin

HSA BSA

−15.19 −14.45

−28.13 −25.71

44.16 38.42

Aloin

HSA BSA

−6.23 −11.32

−24.98 −25.18

63.97 47.29

the molecular structures of emodin, rhein, aloe-emodin and aloin seen in Scheme 1, 1,8-dihydroxyanthraquinone is their common part in these anthraquinones molecules. But the groups attaching C(3) are different for each compound and they are C(3)–OH, C(3)–COOH for emodin and rhein, respectively, as well as C(3)–CH2 OH for both aloeemodin and aloin. The electron-attracting capacity is decreasing in the following order as: –OH > –COOH > –CH2 OH and there is –CH3 attaching C(6) in emodin. Considering these structure characteristics, the largest molecule polarity is emodin, then is rhein and aloe-emodin and the lowest is aloin because of its steric hindrance resulting from C(10)–glucopyranosyl. Therefore, the values of the binding constants KA were in the same order as the polarity of these molecules, that is, emodin–albumin > rhein–albumin > aloeemodin–albumin > aloin–albumin.

concentration of one drug in a series of test tubes containing BSA and varing the concentrations of another drug, the ap of the latter was obtained. Such parent binding constant KA a determination process was performed in various concentrations of the former. Then the effects of aloe-emodin or rhein on the studied reactions such as emodin–BSA as well as the effect of emodin on the reaction of aloe-emodin and BSA were explored, respectively. The results are shown in Fig. 4. For emodin–BSA, aloe-emodin–BSA and rhein–BSA reac-

3.2. Influence of foreign ions The influences of various ions on the binding reactions were tested. The concentrations of Cu2+ , Zn2+ , Mg2+ , Ca2+ and Al3+ from CuCl2 , ZnCl2 , MgCl2 , CaCl2 and AlCl3 were all 5.0 × 10−5 mol l−1 . For a system in which serums, anthraquinones and metal ions coexist, the addition order of the substances has no influence upon the experiments results. According to the fluorescence measurement procedure and the improved analysis method mentioned in the text, the results obtained are shown in Table 3. The binding constants of the reaction of anthraquinone–albumin decreased in various degrees. The ultraviolet absorption spectra revealed that the presence of these metal ions did not affect the absorption properties of anthraquinones but affect the absorption properties of serum albumins. This implied that the metal ions entered into rivalry with anthraquinones when both of them were binding with HSA or BSA. Such a rivalry resulted in the decrease in the binding capacity of the anthraquinones and serum albumins. 3.3. Competition binding of these anthraquinones with serum albumins The mutual influences on the interactions of anthraquinone–BSA for these drugs were studied. Keeping the fixed

Fig. 4. The competition binding curves. (a) The curve of the apparent binding constants of emodin in the presence of (夽) aloe-emodin and () rhein and (b) the curve of apparent binding constants of aloe-emodin in the presence of emodin.

92.2 96.7 0.999 0.998 2.61 × 104 2.97 × 104 Aloin

HSA BSA

74.7 83.8 0.990 0.985 7.69 × 104 3.20 × 104 Aloe-emodin

HSA BSA

80.5 79.4 0.992 0.999 1.65 × 105 8.66 × 104 HSA BSA Rhein

 is the value of binding constants in the presence of metal ions and its units is l mol−1 , r the regression coefficient, P = K  /K and K is the value of binding constants in the absence of metal ions. KA A A A

0.999 0.999 2.65 × 104 2.44 × 104 0.998 0.998 0.999 0.999 2.58 × 104 2.59 × 104 0.999 0.999 2.57 × 104 2.61 × 104

90.8 85.0

91.2 84.4

2.71 × 104 2.48 × 104

95.8 80.8

94.3 98.7 0.996 0.993 9.71 × 104 3.77 × 104 0.996 0.995 0.998 0.994 9.89 × 104 3.00 × 104 0.998 0.996 1.01 × 105 3.37 × 104

98.1 88.2

96.0 78.5

9.87 × 104 3.48 × 104

95.8 91.1

81.5 79.5 0.993 0.992 0.994 0.996 0.992 0.993 1.64 × 105 8.80 × 104 0.997 0.996 2.00 × 105 9.34 × 104

97.6 85.7

80.0 80.7

1.51 × 105 9.33 × 104

73.7 85.6

1.67 × 105 8.67 × 104

92.9 92.2 0.999 0.998 1.78 × 105

98.3 93.3 0.991 0.991 1.80 × 105

98.6 99.0 0.996 0.990 1.91 × 105

69.0 93.8 0.996 0.991 62.9 84.5 Emodin

HSA BSA

1.63 × 105

0.990 0.995

1.81 × 105

P (%) r

2.73 × 105

 KA

P (%) r

2.89 × 105

 KA

P (%) r

2.90 × 105

 KA

P (%) r P (%) r  KA

1.85 × 105

 KA

2.03 × 105

Al3+ Ca2+ Mg2+ Zn2+ Cu2+ Protein Compound

Table 3 The effects of metal ions on anthraquiones binding to HSA and BSA (20 ◦ C)

93.6 79.5

S. Bi et al. / Spectrochimica Acta Part A 62 (2005) 203–212

210

tions, the binding constants were 1.93 × 105 , 3.82 × 104 and 3.07 × 104 l mol−1 (using the improved method) at 20 ◦ C, respectively, when the other drugs were absent. If there was aloe-emodin or rhein in the BSA solution, the apparent bind of emodin and BSA decreased with the coning constant KA centration of aloe-emodin or rhein increasing. Both the binding constants of aloe-emodin–BSA and rhein–BSA were less than that of emodin–BSA, so the value of apparent binding  of emodin–BSA in the presence of aloe-emodin constant KA or rhein dropped relatively slowly. However, the values of  of aloe-emodin–BSA dropped apparent binding constant KA rapidly when there was a fixed concentration of emodin in the BSA solution. This was also because of the much greater binding capacity of emodin than that of aloe-emodin. The conclusion that emodin, aloe-emodin and rhein possessed a common binding site in BSA was drawn from the results shown in Fig. 4 and there was a competition binding with BSA between emodin and rhein or aloe-emodin. 3.4. Energy transfer between serum albumins and anthraquinones The fluorescence of the serum albumin was quenched by the anthraquinone and formed a non-fluorescence albumin–anthraquinone complex. This quenching was a static quenching process. As this in the case, the serum albumin could be regarded as a donor and anthraquinone as an acceptor of the fluorescence. The energy transfer efficiency E is E =1−

F F0

(17)

F is the fluorescence intensity of serum albumins in the presence of anthraquinones and the concentration ratio of serum albumins to anthraquinones is 1:1. Using Eq. (17), the energy transfer efficiencies E between donors and acceptors in our experiments were obtained. They were 11.3, 58.1, 45.0 and 15.9% as well as 5.9, 47.7, 30.7 and 15.3% for the systems of anthraquinone–HSA and anthrquinone–BSA, respectively, in the order of emodin, rhein, aloe-emodin and aloin. According to the F¨orster non-radiation energy transfer theory [26,27], E is also expressed as E=

R60 R60 + r06

(18)

where r0 is the acting distance between the donor and acceptor and R0 is a characteristic distance, called the F¨orster distance or critical distance, at which the efficiency of transfer is 50%, R60 = 8.8 × 10−25 K2 N −4 φJ

(19)

Here, K2 is the spatial orientation factor describing the relative orientation in space of the transition dipoles of the donor and acceptor, K2 = 2/3, N the refraction index for the medium, φ the fluorescence quantum yield of the donor in the absence of the acceptor, N = 1.33, φ = 0.13 [28], and J is the overlap

S. Bi et al. / Spectrochimica Acta Part A 62 (2005) 203–212

integral between the donor fluorescence emission spectrum and the acceptor absorption spectrum. J can be calculated by the expression  F (λ)ε(λ)λ4 λ  (20) J= F (λ) λ In Eq. (20), F(λ) is the fluorescence intensity of the fluorescence donor at wavelength λ, ε(λ) the molar absorption coefficient of the acceptor at wavelength λ and its unit is l mol−1 cm−1 . The overlap of the absorption spectra of these anthraquinones with the serum fluorescence emission spectra of serum albumins is shown in Fig. 5. Then J could be calculated by integrating the overlap spectra in Fig. 4 in the wavelength range of 290–500 nm. For emodin, rhein, aloe-emodin and aloin interacting with HSA, the values

211

of J were 8.98 × 10−15 , 6.03 × 10−15 , 1.45 × 10−14 and 1.12 × 10−14 cm3 l mol−1 and for these anthraquinones interacting with BSA, the values of J were 8.54 × 10−15 , 6.30 × 10−15 , 1.39 × 10−14 and 1.18 × 10−14 cm3 l mol−1 , respectively. By Eq. (19), the critical distance R0 could be calculated accordingly; they were 2.13, 1.99, 2.31 and 2.21 nm for emodin, rhein, aloe-emodin and aloin interacting with HSA, respectively, as well as 2.11, 2.01, 2.29 and 2.23 nm for emodin, rhein, aloe-emodin and aloin interacting with BSA, respectively. Finally, the distance between the anthraquinones and serum albumins could be obtained from Eq. (18). Consequently, the values of r0 are 3.01, 1.89, 2.39 and 2.92 nm for emodin, rhein, aloe-emodin and aloin for interacting with HSA, respectively, and 3.35, 2.04, 2.63 and 2.97 nm for emodin, rhein, aloe-emodin and aloin interacting with BSA, respectively. The values of R0 and r0

Fig. 5. Overlap of the fluorescence spectra (a) of HSA and the fluorescence spectra (b) of BSA with the absorption spectra (c) of such anthraquinones as (1) emodin, (2) rhein, (3) aloe-emodin and (4) aloin.

212

S. Bi et al. / Spectrochimica Acta Part A 62 (2005) 203–212

for the anthraquinones are all in the academic values range (R0 = 5–10 nm, r0 = 7–10 nm). These results indicated that the fluorescence quenching arose from the interaction of serum albumins and anthraquinone was a non-radiation transfer process (Fig. 5).

4. Conclusions The anthraquinones acting as quenchers changed the fluorescence properties of serum albumins (HSA and BSA). Trp 214 is an intrinsic fluorescence probe of HSA and BSA. The interactions between anthraquiones and serum albumins were discussed in detail. The Stern–Volmer analysis revealed the quenching process of anthraquinones binding to serum albumins was a static quenching. The binding constants KA and binding sites n were obtained by the improved method data analysis method present in this paper. Based on the F¨oster non-radiation energy transfer theory, the energy transfer efficiency and the binding acting distance between the anthraquinones and HSA or BSA were got. In addition, the binding constants of serum albumins and anthraquinones in the presence of metal ions were also found in this work and the results suggested that there was a competition between anthraquinones and metal ions. The effect of one drug on the other drug binding with serum albumins was discussed and the results revealed that there was a competition binding in the solutions containing serum albumins and two or more different drugs. The research on the interaction of the drug molecules binding to protein may give us a better understanding of the pharmacokinetics such as drug metabolism and distribution. The knowledge of the relationship of the drug molecule structure and binding capacity to protein can provide the theory basis for the new drug designing.

Acknowledgements This work was supported by the National Natural Science Foundations of China (Grant number 30371757) and Research Fund for the Doctoral Program of Chinese Universities (Grant number 2003183035).

References [1] X.M. He, D.C. Carter, Nature 358 (1992) 209. [2] D. Carter, B. Chang, J.X. Ho, K. Keeling, Z. Krishnassami, Eur. J. Biochem. 226 (1994) 1049. [3] T. Peters, Adv. Protein Chem. 37 (1985) 161. [4] R. Boelens, R.M. Sheek, K. Dijkstra, R. Kaptein, J. Magn. Reson. 62 (1985) 378. [5] T.H. Tsai, C.F. Chen, Asia Pac. J. Pharmacol. 7 (1992) 53. [6] J.W. Liang, S.L. Hsiu, H.C. Huang, P.D. Lee-Chao, J. Food Drug Anal. 1 (1993) 251. [7] M. Koyama, T.R. Kelly, K.A. Watanabe, J. Med. Chem. 31 (1988) 283. [8] X.M. Zhou, Q.H. Chen, Acta Pharmacol. Sin. 23 (1988) 17. [9] H.C. Huang, S.H. Chu, P.D. Lee-Chao, Eur. J. Pharmacol. 198 (1991) 211. [10] S.G. Lin, M. Fujii, D.X. Hou, Arch. Biochem. Biophys. 418 (2003) 99. [11] G. Krumbiegel, H.U. Schulz, Pharmacology 47 (Suppl. 1) (1993) 120. [12] S.K. Agarwal, S.S. Singh, S. Verma, S. Kumar, J. Ethnopharmacol. 72 (2000) 43. [13] T. Hatano, H. Uebayashi, H. Ito, S. Shiota, T. Tsuchiya, T. Yoshida, Chem. Pharm. Bull. 47 (1999) 8. [14] D.O. Andersen, N.D. Weber, S.G. Wood, B.G. Hughes, B.K. Murray, J.A. North, Antivir. Res. 16 (2) (1991) 185. [15] B. Arosio, N. Gagliano, L.M. Fusaro, L. Parmeggian, J. Tagliabue, P. Galetti, D. Castri, C. Moscheni, G. Annoni, Pharmacol. Toxicol. 87 (5) (2000) 229. [16] S.H. John, Acad. Med. 66 (1990) 647. [17] A. Humra, A. Nisar, T. Saad, A.Q. Mohammad, Int. J. Biol. Macromol. 25 (1999) 353. [18] D. Petra, T. Iztok, P.U. Nataˇsa, J. Inorg. Biochem. 96 (2003) 407. [19] C.Q. Jiang, M.X. Gao, X.Z. Meng, Spectrochim. Acta Part A 59 (2003) 1605. [20] S.Y. Bi, D.Q. Song, Y. Tian, Z.Y. Liu, H.Q. Zhang, Spectrochim. Acta Part A 61 (2005) 629. [21] F. Moreno, M. Cortijo, J.G. Jimenez, Photochem. Photobiol. 69 (1999) 8. [22] T.G. Dewey, Biophysical and Biochemical Aspects of Fluorescence Spectroscopy, Plenum, New York, 1991, pp. 1–41. [23] J.R. Lakowica, G. Weber, Biochemistry 12 (1973) 4161. [24] W.R. Ware, J. Phys. Chem. 66 (1962) 455. [25] D.P. Ross, S. Sabramanian, Biochemistry 20 (1981) 3096. [26] L.A. Sklar, B.S. Hudson, R.D. Simoni, Biochemistry 16 (23) (1977) 5100. [27] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum, New York, 1983. [28] S. Margaret, P.L. Hue, S. Fauzia, A.M. Robert, R.B. Edward, W.D. Dennis, Biochemistry 22 (1983) 2415.