Study on the interaction of sodium morin-5-sulfonate with bovine serum albumin by spectroscopic techniques

Spectrochimica Acta Part A 86 (2012) 191–195

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Study on the interaction of sodium morin-5-sulfonate with bovine serum albumin by spectroscopic techniques Nahid Shahabadi ∗ , Mahnaz Mohammadpour Department of Chemistry, Faculty of Science, Razi University, Kermanshah, Iran

a r t i c l e

i n f o

Article history: Received 9 September 2011 Received in revised form 13 October 2011 Accepted 14 October 2011 Keywords: BSA Morin Fluorescence Circular dichroism

a b s t r a c t In the present investigation, an attempt has been made to study the interaction of sodium morin5-sulfonate (NaMSA) with the transport proteins, bovine serum albumin (BSA) employing UV-vis, fluorometric and circular dichroism (CD) techniques. The experimental results indicated that the quenching mechanism of BSA by the compound was a static procedure. Various binding parameters were evaluated. The negative value of H, positive value of S and the negative value of G indicated that electrostatic interactions and hydrogen bonding play major roles in the binding of the NaMSA and BSA. Based on the Forster’s theory of non-radiation energy transfer, the binding distance, r, between the donor (BSA) and acceptor (NaMSA) was evaluated. The results of CD and UV-vis spectroscopy showed that the binding of this complex to BSA induces some conformational changes in BSA. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Flavonol morin belongs to the flavonoid family and is an important plant pigment in herbal medicine. Recent interest in flavonols stems from their broad-spectrum pharmacological and therapeutic activities. The basic structure of flavonols is usually characterized by two aromatic rings, ring A and ring B, which are joined by a three-carbon linked ␥-pyrone ring (ring C), forming a C6–C3–C6 skeleton unity where polar groups, usually hydroxyl, methoxyl or glycosyl appended at various positions. (Fig. 1a, [1]). Morin (3,5,7,2 ,4 -pentahydroxyflavone) is found in almonds, milk, fig and other moraceae which are used as dietary agents and also as herbal medicines [2]. A light yellowish pigment found in the wood of old fustic Chlorophora tinctori [3]. Morin hydrate is known to have a broad pharmacological activity, such as antitumour, antioxidation, cardiovascular protection and possibly even protective effects against chronic diseases. Each component of flavonols has its specific biological function and activity, and the pharmacological actions of morin are different from those of other flavonols. Morin exerts its functions usually by regulating the activation or modifying the structure of enzymes [1]. The interaction between bio-macromolecules and flavonoids has attracted great interest among researchers for several decades [4]. In this paper, bovine serum albumin (BSA) was selected as the protein model because of its low cost, ready availability, and unusual ligand-binding properties. Bovine serum albumin (BSA) is

∗ Corresponding author. Tel.: +98 831 8360795; fax: +98 831 8360795. E-mail address: [email protected] (N. Shahabadi). 1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2011.10.023

constituted of 582 amino acid residues and based on the distribution of the disulphide bridges and of the amino acid sequence, it seems possible to regard BSA as composed of three homologous domains linked together. The domains can all be subdivided into two sub-domains. The binding sites of BSA for endogenous and exogenous ligands may be in these domains and the principal regions of drugs binding sites of albumin are often located in hydrophobic cavities in sub-domains IIA and IIIA. So called sites I and II are located in subdomain IIA and IIIA of albumin, respectively. Many ligands bind specifically to serum albumin. As proposed by Kragh-Hansen [5], there are at least six binding regions, one or two high affinity binding sites (primary sites) and a number of sites with lower affinity [6]. In this study, we have reported the synthesis of NaMSA watersoluble compound and the binding properties of this compound with BSA has been carried out using different instrumental methods and the binding mode is discussed. Some techniques commonly used to detect interaction between drugs and serum albumin includes fluorescence, UV-vis, and circular dichroism techniques [7]. 2. Experimental 2.1. Materials Bovine serum albumin (BSA) was purchased from Sigma–Aldrich. All BSA solutions were prepared in the Tris–HCl 0.05 M and 0.1 M NaCl buffer to keep pH value constant (pH 7.40), and to maintain the ionic strength of all solutions in experiments. BSA solutions stocked in the dark at 4 ◦ C. Morin was purchased

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2.2.3. Fluorescence spectra All fluorescence spectra were recorded with a LS-55 Spectrofluorimeter (Perkin-Elmer corporate, UK) equipped with quartz cells (1.0 cm) and a thermostat bath. The maximal fluorescence emission of BSA at ex = 289 nm was located at 346 nm. Fluorescence spectra were recorded from 300 to 500 nm with the excitation wavelength at 289 nm. First 3 mL of solution containing constant concentration of BSA (6 × 10−6 ) was titrated by the successive addition of NaMSA solution (concentration was varied from 0 to 20 × 10−6 mol L−1 ). Titration was done manually via a microsyringe and the fluorescence intensity of BSA in the absence and the presence of the NaMSA was measured. All experiments were measured at three temperatures (295, 298, and 303 K). For quenching property, the mechanism can be described by the Stern–Volmer equation (Eq. (3)). F0 = 1 + Ksv [Q ] F

(3)

where F0 and F are the fluorescence intensities in the absence and presence of a quencher, KSV is the Stern–Volmer quenching constant and [Q] is the concentration of a quencher. The plot of F0 /F vs. [Q] shows the value of KSV (KSV = Kq  0 ).where Kq is the quenching rate constant and  0 is the fluorescence lifetime of protein in the absence of quencher, the value of  0 is considered to be 10−8 s [9]. Fig. 1. (a) The structure of 2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4one (morin); (b) the basic structure of the sodium salt of morin-5 -sulfonic acid (NaMSA).

from Merck, dry EtOH, DMSO, methanol and H2 SO4 were obtained from Fluka Company. Doubly distilled water was used to prepare the buffers. 2.2. Methods 2.2.1. UV-spectrophotometry Absorbance spectra were recorded using an hp spectrophotometer (agilent 8453), equipped with a thermostated bath (Huber polysat cc1). Absorption titration experiments were carried out by keeping the concentration of BSA constant (3 × 10−5 M) while varying the NaMSA concentration from 0 to 4.5 × 10−5 M (ri = [morin]/[BSA] = 0.0, 0.2, 0.4, 0.6, 0.8, 1, 1.2,1.5). Absorbance values were recorded after each successive addition of BSA solution and equilibration (ca. 10 min). 2.2.2. CD studies Circular dichroism (CD) measurements were recorded on a JASCO (J-810) spectropolarimeter (between 200 and 250 nm and cell length path was 0.1 cm) by keeping the concentration of BSA constant (3 × 10−6 M) while varying the NaMSA concentration from 0 to 6 × 10−6 M (ri = [morin]/[BSA]) = 0.0, 0.3, 0.5, 1, 1.5, 2). The CD results were expressed in terms of mean residue ellipticity (MRE) in degcm2 dmol−1 according to the following equation: MRE =

observed CD (mdeg) Cpnl × 10

(1)

where Cp is the molar concentration of the protein, n the number of amino acid residues (583 for BSA) and l the path-length (0.1 cm). The ␣-helical contents of free and combined BSA were calculated from MRE values at 208 nm using the Eq. (2): ˛-helix(%) =

−MRE280 − 4000 33, 000 − 4000

(2)

The observed MRE value at 208 nm, MRE208 , of ␤-form and random coil conformation cross in total and a pure ␣-helix were 4000 and 33000, respectively. From the above equation, the ␣-helicity in the secondary structure of BSA was determined [8].

2.3. Synthesis 2.3.1. Synthesis of NaMSA Sulfonate derivative of morin, sodium morin-5-sulfonat (NaMSA) was obtained by the method described [10]. 40 mL of concentrated sulfuric acid was added to 10 g of morin. The mixture was heated for 4 h at 80 ◦ C with occasional stirring. The pH of the cold reaction mixture was adjusted to 3 with 20% aqueous NaOH, and then 50 mL of saturated aqueous NaCl was added. The precipitated yellow solid was separated and the isolated yield was 50%. Elemental analysis gave the formula C15 H9 O10 SNa·2H2 O. 3. Results and discussion 3.1. General In this study, we eliminated the inner filter effect for all of the fluorescence results to obtain accurate data. For the BSA–drug system, the inner filter effect is caused by the absorption of the excitation and emission wavelengths by drug and BSA in fluorescence experiments, which change the intensity of fluorescence spectra of BSA, affecting the binding parameters calculated from the fluorescence data [11]. The fluorescence intensities were corrected for absorption of exciting light and reabsorption of the emitted light to decrease the inner filter effect using the relationship [12]: FCOR = Fobs × e(Aex +Aem /2)

(4)

where Fcor and Fobs are the fluorescence intensities corrected and observed, respectively, and Aex and Aem are the absorption of the systems at the excitation and the emission wavelength, respectively. The intensity of fluorescence used in this paper is the corrected fluorescence intensity Fluorescence spectroscopy has been usually used in the study of molecular interactions between ligands and proteins, mainly due to the high sensitivity of this methodological approach and the variety of parameters related with the interaction molecular that can be obtained. Fluorescence quenching refers to any process which decreases the fluorescence intensity of a fluorophore. A variety of molecular interactions can result in fluorescence quenching, including excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation and collisional

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Fig. 2. Fluorescence spectra of BSA in the absence and the presence of NaMSA, [BSA] = 6 × 10−6 M and [NaMSA] = 0, 2, 4, 6, 10, 15, 20 × 10−6 , ex = 289 nm and em = 346 nm, silt width = 10 nm. Insets in: Stern–Volmer curve for the binding of NaMSA with BSA.

quenching [13]. The maximal fluorescence emission of BSA at ex = 289 nm was located at 346 nm (Fig. 2) The fluorescence intensity of BSA decreased in the presence of NaMSA and the maximum emission wavelengths was shifted from 346 to 345.07 nm. This suggested that the microenvironment around BSA was changed after the addition of the compound. The results indicate that there is strong interaction and energy transfer between BSA and sulfonated morin [14].

3.2. Quenching constants Fluorescence quenching is the decrease of the quantum yield of fluorescence from a fluorophore induced by a variety of molecular interactions with a quencher molecule. Fluorescence quenching can occur via different mechanisms, which are usually classified as either static quenching or dynamic quenching. Static quenching, resulting from the formation of a ground-state complex between the fluorophore and a quencher, dynamic quenching, resulting from collisional encounters between the fluorophore and quencher. In both cases, molecular contact is required between the fluorophore and the quencher for fluorescence quenching to occur [15]. Fig. 2 displays the Stern–Volmer plots of the quenching of BSA fluorescence by NaMSA. The data are shown in Table 1. Using the approximate fluorescence lifetime of BSA (10 ns) [16], the bimolecular quenching constant (kq ) was determined from Kq = KSV / 0 . The values of Kq obtained for the binding of BSA to the compound are of the order of 1013 L mol −1 s−1 (Table 1). The quench constants are greater than those in the biopolymer (2.0 × 1010 L mol−1 s−1 ) by the maximum scatter collision mechanism. This means that the possible quenching mechanism of fluorescence of BSA by NaMSA is not initiated by dynamic collision but from the formation of a complex. The quenching procedure can be further confirmed by temperature dependence of the quenching: KSV values decrease with an increase in temperature for static quenching and the reverse will be observed for dynamic quenching [16]. From Table 1, it can be found that the Ksv decreasing with increasing temperature. So the quenching is a static quenching procedure [16].

determined according to the method described by Jiang et al. [17] using the following equation: log

F − F  0 F

= log Kb + n log[Q ]

(5)

where, in the present case, Kb is the binding constant for the NaMSA with protein interaction and n is the number of binding sites per albumin molecule, which can be determined by the slope and the intercept of the double logarithm regression curve of log(F0 − F/F) versus log [Q] based on the Eq. (5) (Fig. 3 and Table 1). The correlation coefficients are larger than 0.9, indicating that the assumption underlying the deviation of Eq. (5) is satisfactory. The value of n at the experimental condition are approximately equal to 1, which indicates that there is just one single binding site in BSA for the compound. In order to elucidate the interaction of the NaMSA with BSA, the thermodynamic parameters were calculated. The plot of log Kb versus 1/T (Eq. (6)), where Kb is the binding constant at the corresponding temperatures and R is the gas constant, allows the determination of enthalpy change (H) and entropy change (S). If the temperature does not vary significantly, the enthalpy change (H) can be regarded as a constant. Based on the binding constants at different temperatures, the free energy change (G) can be estimated by Eq. (7) (Table 1). Ln Kb =

S −H + RT R

G = H − TS

(6) (7)

3.3. Binding constants and the number of binding sites For the static quenching interaction, if it is assumed that there are similar and independent binding sites in the biomolecule, the binding constant (Kb ) and the number of binding sites (n) can be

Fig. 3. Determination of NaMSA–BSA binding constant and number of binding sites on BSA at 298 K.

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Table 1 The quenching constants (Ksv ) (Kq), binding constants (Kb ), number of binding sites (n) and relative thermodynamic parameters of the BSA–NaMSA system at different temperatures. T (K)

KSV × 10−4 (L mol−1 )

Kq × 1012 (L mol− s−1 )

Kb × 103 (mol L−1 )

n

S (J mol−1 K−1 )

H (kJ mol−1 )

G (kJ mol−1 )

293 298 303

1.30 1.23 1.08

1.3 1.23 1.08

2.37 2.25 2.16

0.98 0.94 0.95

39.73

−7.28

−11.65 −11.85 −12.05

Ross and Subramanian [18] have characterized the sign and magnitude of the thermodynamic parameter associated with various individual kinds of interaction that may take place in protein association process, which can be easily concluded as: (a) H > 0 and S > 0, hydrophobic force; (b) H < 0 and S < 0, van der Waals force and hydrogen bond; (c) H < 0 and S > 0, electrostatic interactions. The negative H value is frequently taken as evidence for hydrogen bond in the binding interaction [19]. Thus, from the thermodynamic characteristics summarized above, the negative H and positive S values indicate that electrostatic force and hydrogen bond played major roles in the NaMSA–BSA binding reaction and contributed to the stability of the complex. 3.4. Energy transfer between NaMSA and BSA The spectral studies suggested that NaMSA form a complex with BSA. To get much more information about NaMSA–BSA system attention was focused on the aspect of energy transfer in the system. Generally speaking, fluorescence resonance energy transfer occurs when the emission spectrum of a fluorophore overlaps the absorption spectrum of another molecule. Fig. 4 shows the spectral overlap between absorption spectrum of NaMSA with fluorescence spectrum of BSA in the wavelength range of 300–500 nm. The importance of the Förster resonance energy transfer in biochemistry is that the efficiency of transfer can be used to evaluate the distance between the ligand and the tryptophan residues responsible of the natural intrinsic fluorescence of the protein [20]. According to Förster’s non-radiative energy transfer theory, the energy transfer between the excited molecule and its neighbours will occur under the following conditions: (i) the donor must fluoresce; (ii) the fluorescence emission spectrum of the donor and the UV–vis absorbance spectrum of the acceptor must overlap significantly; and (iii) the distance between the donor and the acceptor must be lower than approximately 8 nm. The distance, r, between the acceptor (NaMSA) and the donor (BSA) was calculated as follows: [21]. E=

R06 R06

+ r6 =

F0 − F F0

Fig. 4. Spectral overlap of NaMSA absorption with BSA fluorescence.

(8)

where R0 is the critical distance when the transfer efficiency, E, is 50%. It can be estimated from Eq. (9): R06 = 8.8 × 10−25 K 2 n−4 J˚

(9)

where K2 is the spatial orientation factor of the dipole, n the refractive index of the medium, ˚ the fluorescence quantum yield of the donor, J the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor. Therefore, Eq. (10) J=

˙F0 ()ε()4  F()

(10)

where F() is the fluorescence intensity of the fluorescent donor at wavelength , and ε() is the molar absorptivity of the acceptor at wavelength . J can be obtained by integrating the overlap in the spectra (Fig. 4). The required constants in the above equations for BSA are: k2 = 2/3,  = 0.118, n = 1.336 [21] and with these values, it was found that r = 2.38 nm for NaMSA, the donor–acceptor distance, r < 8 indicates that the energy transfer from BSA to NaMSA occurs with high possibility [22]. 3.5. Conformation investigation 3.5.1. UV-vis spectroscopy Spectroscopy techniques can be used to explore the structural changes of protein and to investigate protein–ligand complex formation. BSA has two absorption peaks. The strong absorption peak at about 208 nm reflects the framework conformation of the protein. The weak absorption peak at about 279 nm appears to be due to the aromatic amino acids (Trp, Tyr, and Phe) [9]. It is well known that the absorption of a chromophore is shifted in directions and magnitudes that depend on whether it is transferred to a more hydrophilic or more hydrophobic environment. These shifts are ascribed to a change of ␲–␲* transition brought about by changes in the polarizability of the solvent [23]. In order to obtain more information on NaMSA–BSA interaction, UV–vis absorption spectrum of system was recorded at 298 K. It was observed that the absorption of BSA increased upon the addition of drug (Fig. 5). The maximum peak position of NaMSA–BSA was shifted slightly towards longer wavelength region. This indicated the change in polarity around the tryptophan residue and the change in peptide strand of BSA molecules and hence the change in hydrophobicity. So, the binding of NaMSA to protein molecule might lead to change in protein conformation [24]. 3.5.2. Circular dichroism CD is a sensitive technique for monitoring conformational changes of protein upon interaction with small molecules. Bovine serum albumin has a high percentage of ␣-helical structure which shows characteristic strong double minimum signals at 222 and 208 nm [8]. The reasonable explanation may be that the negative peaks between 208 and 222 nm are both contributed to n–␲* transfer for the peptide bond of ␣-helix [25]. The CD spectra of BSA in the absence and the presence of NaMSA are shown in Fig. 6. The addition of increasing amount of NaMSA causes the distortion in the CD spectra. This indicates the disordered form of ␣-helical structure.

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4. Conclusion In this paper, we studied the interaction of the NaMSA with BSA via fluorescence, UV–vis and CD spectrum. The results of fluorescence, UV–vis and CD spectra indicate that the secondary structure of BSA molecules changes in the presence of NaMSA. The experimental results also indicate that the quenching of fluorescence of BSA by NaMSA is a static quenching process. The negative H and positive S values indicate that electrostatic force and hydrogen bond played major roles in the NaMSA–BSA binding reaction and contributed to the stability of the complex. This work provides some valuable information for the transportation and distribution of NaMSA in vivo, and is helpful for clarifying toxicity and dynamics of NaMSA. References

Fig. 5. UV–vis spectra of BSA in the absence and the presence of NaMSA. [BSA] = 3 × 10−5 M [NaMSA]/[BSA] = 0.0, 0.2, 0.4, 0.4, 0.6, 0.8.

Fig. 6. CD spectra of BSA in the absence and the presence of NaMSA.

The percentage of helicity of BSA is 87.8% in pure BSA (Fig. 6) and in the presence of NaMSA it becomes 95.6% which shows that binding of NaMSA to BSA may induce some conformational changes. It can be deduced that the ␣-helical structure is affected probably due to insertion of some of NaMSA residues into the hydrophobic surface of BSA. So when NaMSA bounds to BSA the ␣-helicity increases. This observation strongly indicates that the binding of NaMSA to BSA induces some conformational changes in BSA but the protein retains its secondary structure and helicity when interact with NaMSA. This phenomenon is important for biomedical applications [26].

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