Study on the interaction of silver(I) complex with bovine serum albumin by spectroscopic techniques

Spectrochimica Acta Part A 92 (2012) 184–188

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Study on the interaction of silver(I) complex with bovine serum albumin by spectroscopic techniques Nahid Shahabadi ∗ , Maryam Maghsudi, Zeinab Ahmadipour Department of Chemistry, Faculty of Science, Razi University, Kermanshah, Iran

a r t i c l e

i n f o

Article history: Received 22 November 2011 Received in revised form 15 February 2012 Accepted 17 February 2012 Keywords: BSA Ag(I) complex Fluorescence Circular dichroism Phenanthroline

a b s t r a c t The interaction of silver(I) complex, [Ag (2,9-dimethyl-1,10-phenanthroline)2 ](NO3 )·H2 O, and bovine serum albumin (BSA) was investigated by spectrophotometry, spectrofluorimetry and circular dichroism (CD) techniques. The experimental results indicated that the quenching mechanism of BSA by the complex was a static procedure. Various binding parameters were evaluated. The negative value of H, negative value of S and the negative value of G indicated that van der Waals force and hydrogen bonding play major roles in the binding of the complex and BSA. Based on Forster’s theory of non-radiation energy transfer, the binding distance, r, between the donor (BSA) and acceptor (Ag(I) complex) was evaluated. The results of CD and UV–vis spectroscopy showed that the binding of this complex could bind to BSA and be effectively transported and eliminated in the body. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Silver is a metal of interest in cancer therapy because its toxicity in humans is believed to be quite low [1]. The biocidal effect of silver, with its broad spectrum of activity including bactericidal action, is particularly well known. Silver ions induce errors in DNA transcription processes, which may cause disturbance of normal functionality of nucleic acids [2,3]. The pharmaceutical design problems are avoiding precipitation of silver(I) as AgCl when it contacts physiological fluids and preventing the composition of the chosen compound through photo-activated processes. 1,10-Phenanthroline (phen) and substituted derivatives, both in the metal-free state and as ligands coordinated to transition metals, disturb the functioning of a wide variety of biological systems [4]. The 3,8- and 4,7-substituted phenanthrolines were less effective than 1,10-phenanthroline at preventing fungal growth, whereas 2,9-dimethyl-1,10-phenanthroline (2,9-dmp) was the most potent inhibitor [5]. As the metal-free N,Nchelating bases are found to be bioactive [6], it seems likely that the combination of Ag(I) and phenanthrolines may possess potent anticancer activity. Among the various of silver complexes so far investigated, those of phenanthroline and its derivatives have attracted much attention for their various functions. Previous work has demonstrated that the metal-based drugs such as [Ag2 (phen)3 (mal)]·2H2 O (phen = 1,10-phenanthroline; mal H2 = malonic acid) inhibit the growth of Candida albicans

by around 95% in a concentration range of 1.25–5.0 mg/mL [7,8]. [Ag(phendion)2 ]ClO4 (phendion = 1,10-phenanthroline-5,6dione) causes gross distortions in fungal cell morphology and there is evidence for disruption of cell division [9]. [Ag (bpy) (Hdahmp)]NO3 (bpy = bipyridine; Hdahmp = protonated neutral 4,6-diamino-5-hydroxy-2-mercaptopyrimidine) shows remarkable efficacy against Ehrlich ascites tumor cells [10]. These complexes induced extensive changes in the internal structure of cells, including retraction of the cytoplasm, nuclear fragmentation, and disruption of the mitochondria [11]. Treatment of fungal cells with the Cu(II) and Ag(I) complexes resulted in a reduced amount of ergosterol in the cell membrane and subsequent increase in its permeability [8]. Therefore, to the best of our knowledge, there is not any of research about the interaction of this kind of complex and BSA. 2. Materials and methods 2.1. Chemicals and materials Bovine serum albumin (BSA) was purchased from Sigma–Aldrich. Silver nitrate, absolute ethanol, diethyl ether, acetone, NaH2 PO4 , Na2 HPO4 , dimethyl sulfoxide (DMSO), KI, and MeOH were purchased from Merck. 2,9-Dimethyl-1,10phenanthroline (2,9-dmp) was purchased from Riedel-deHa˘en. 2.2. Reagents

∗ Corresponding author. Tel.: +98 831 8360795; fax: +98 831 8360795. E-mail address: [email protected] (N. Shahabadi). 1386-1425/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2012.02.071

Silver complex and BSA solutions were prepared in the buffer solution adjusted to pH 7.2 with 0.01 M Na2 HPO4 and NaH2 PO4 in

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pure aqueous medium. BSA stock solution (3 × 10−5 M, based on its Molecular weight of 66,000) was prepared in 0.01 M phosphate buffer of pH 7.2 and was kept in the dark at 277 K. Triple distilled water was used throughout the experiment. 2.2.1. Synthesis of [Ag (2,9-dmp)2 ](NO3 ) The complex was synthesized according to the previous work [12]. 2.3. Instrumentation Absorbance spectra were recorded using an HP spectrophotometer (Agilent 8453), equipped with a thermostatted bath (Huber polysat cc1). Absorption titration experiments were carried out by keeping the concentration of BSA constant (3 × 10−5 M) while varying the complex concentration from 0 to 0.7 × 10−5 M (ri = [Ag complex]/[BSA] = 0.0, 0.1, 0.2, 0.3, 0.5, 0.7). Absorbance values were recorded after each successive addition of BSA solution and equilibration (ca. 20 min). Circular dichroism (CD) measurements were recorded on a JASCO (J-810) spectropolarimeter by keeping the concentration of BSA constant (3 × 10−5 M) while varying the complex concentration from 0 to 0.03 × 10−5 M (ri = [Complex]/[BSA]) = 0.0, 0.01 and 0.03). The data were recorded after each successive addition of BSA solution and equilibration (ca. 20 min). Fluorescence measurements were carried out with a JASCO spectrofluorimeter (FP 6200) by keeping the concentration of BSA constant (3 × 10−5 M) while varying the complex concentration from 0 to 1.2 × 10−5 M (ri = [complex]/[BSA]) = 0.0, 0.1, 0.2, 0.3, 0.5, 0.7, 1, 1.2) at different temperatures (288, 298, 310, 318 K). Fluorescence intensities were recorded after each successive addition of complex solution and equilibration (ca. 20 min). The solutions of complex and buffer were also measured under the same conditions and were used to correct the observed fluorescence. 3. Results and discussion 3.1. General Bovine serum albumin (BSA) is 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. As proposed by Kragh Hansen [13], there are at least six binding regions and, another characteristic feature of albumin–ligand interactions, one or two high affinity binding sites (primary sites) and a number of sites with lower affinity.

Fig. 1. Fluorescence spectra of BSA in the various of concentrations of Ag(I) complex, C (BSA) = 3 × 10−5 : C (complex)/C (BSA); 0, 0.1, 0.2, 0.3, 0.5, 0.7, 1, 1.2, respectively.

amounts of complex a remarkable intrinsic fluorescence decrease of BSA was observed. Fluorescence quenching is described by the Stern–Volmer equation [15]: F0 = 1 + Kq 0 [Q] = 1 + KSV [Q] F

(1)

where F0 and F represent the fluorescence intensities in the absence and in the presence of quencher, respectively. Kq is the quenching rate constant of the biomolecule, Ksv the dynamic quenching constant,  0 the average life-time of the biomolecule without quencher  0 = 6.2 ns) [16] and [Q] the concentration of quencher. Fig. 2 displays the Stern–Volmer plots of the quenching of BSA fluorescence by the complex at different temperatures. As seen, the plot of F0 /F for BSA vs. [complex], ranging from 0 to 1.2 × 10−5 M is linear. One way to distinguish static quenching from dynamic quenching is to examine their different dependence on temperature [17]. Dynamic quenching depends upon diffusion: higher temperatures result in larger diffusion coefficients. As a result, the bimolecular quenching constants are expected to increase with temperature rising. In contrast, increased temperature is likely to result in decreasing stability of complexes, and thus lower values of the static quenching constants. The results in Table 1 indicate that the probable quenching mechanism of fluorescence of BSA by the complex is a static quenching procedure, because Ksv is decreased with increasing temperature [18]. In addition, according to Kq = KSV / 0 the quenching rate constant, Kq , can be calculated. The values of Kq are of the order of 1013 M−1 s−1 (Table 1).

3.2. Fluorescence spectroscopy Fluorescence quenching is the decrease of the quantum yield of fluorescence from a fluorophore induced by a variety of molecular interactions with quencher molecule. Under the conditions of fixed pH, temperature and ionic strength, fluorescence quenching may result from ground state complex formation, energy transfer and dynamic quenching processes [14]. Dynamic quenching refers to a process that the fluorophore and the quencher come into contact during the lifetime of the excited state, whereas static quenching refers to fluorophore–quencher complex formation. The effect of Ag(I) complex on BSA fluorescence intensity is shown in Fig. 1. The concentration of BSA was stabilized at 3 × 10−5 M while the concentration of the complex was varied from 0 to 1.2 × 10−5 M. It is obvious that BSA has a strong fluorescence emission peaked at 343 nm. When BSA was titrated with different

Fig. 2. Stern–Volmer plots for the interaction of the complex with BSA at different temperatures (T = 288, 298, 310, 318 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–Ag complex system at different temperatures. T (K)

Ksv × 105 (L mol−1 )

Kq × 1013 (L mol −1 s−1 )

Kb × 106 (mol L−1 )

n

G (kJ mol−1 )

288 298 310 318

1.7 1.6 1.5 1.4

2.74 2.58 2.41 2.25

6.2 2.7 0.91 0.31

1.02 1.02 0.939 1.09

−37.40 −36.57 −34.77 −33.82

According to the literatures [19,20], for dynamic quenching, the maximum collision-quenching constant of various quenchers with the biopolymer is 2.0 × 1010 L mol−1 s−1 and the Ksv increases with increasing temperature. Considering that in our experiment the rate constant (Kq ) of the protein quenching procedure initiated by Ag complex is much greater than 2.0 × 1010 L mol−1 s−1 and that the Ksv decreased with increasing temperature, it can be concluded that the quenching is not initiated by dynamic quenching, but probably by static quenching procedure resulted from the formation of BSA–Ag(I) complex adduct. 3.2.1. Binding constant When small molecules bind independently to a set of equivalent sites on a macromolecule, the binding constant (Kb ) and the numbers of binding sites (n) can be determined by the following equation [20]: Log

F − F  0 F

= Log Kb + n Log[Q]

(2)

where in the present case, Kb is the binding constant for the complex-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 [complex] based on the Eq. (2) (Table 1). The correlation coefficients are larger than 0.99, indicating that the assumption underlying the deviation of Eq. (2) is satisfactory. Crystal structure of BSA shows that BSA is a heart-shaped helical monomer composed of three homologous domains named I, II, III, and each domain includes two sub-domains called A and B to form a cylinder. The principal regions of ligand-binding sites on albumin are located in hydrophobic cavities in sub-domains IIA and IIIA, which exhibit similar chemistry properties [21]. Hence it is suggested that the complex most likely binds to hydrophobic pocket located in subdomain IIA, that is to say, Trp-212 is near or within the binding site [22]. 3.2.2. Binding mode The interaction forces between small molecule and biological macromolecule include hydrophobic force, hydrogen bond, van der Waals force and electrostatic interactions, etc. [23]. The signs and magnitudes of the thermodynamic parameters (H and S) can account for the main forces involved in the binding reaction. For this reason, the temperature-dependent thermodynamic parameters were studied. In order to elucidate the interaction of the complex with BSA, the thermodynamic parameters were calculated. The plot of log Kb versus 1/T (Eq. (3)), 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. (4) (Table 1). Ln Kb = −

H S + RT R

G = H − T S

H (kJ mol−1 )

S (J mol−1 K−1 )

−19.18

−48.93

Ross and Subramanian [24] 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 [25], and from the structure of Ag(I) complex, H-bond can be formed in the nitrogen atom. Thus, from the thermodynamic characteristics summarized above, the negative H and S values indicate that van der Waals force and hydrogen bond played major roles in the Ag(I) complex–BSA binding reaction and contributed to the stability of the complex. 3.2.3. Energy transfer The overlap of the UV–vis absorption spectrum of the complex with the fluorescence emission spectrum of BSA is shown in Fig. 3. The importance of the energy transfer in biochemistry is that the efficiency of transfer can be used to evaluate the distance between the ligand and the tryptophan residues in the protein. According to the theory of Forster non-radiative energy transfer [26], the efficiency of energy transfer mainly depends on the following factors (1) the donor can produce fluorescence, (2) fluorescence emission spectrum of the donor and UV–vis absorbance spectrum of the acceptor have more overlap and (3) the distance between the donor and the acceptor is approached and lower than 8 nm. The energy transfer effect is related, not only to the distance between the donor and acceptor, but also to the critical energy transfer distance, that is calculated by the following equation: E =1−

R6 F = 6 0 F0 (R0 + r 6 )

(5)

where r0 represents the distance between the donor and the acceptor and R0 is the critical distance at which transfer efficiency equals 50%. The value of R0 is calculated using the following equation: R06 = 8.79 × 10−25 K 2 n−4 ϕJ

(6)

(3) (4)

Fig. 3. Spectral overlap of Ag(I) complex absorption (b) with BSA fluorescence (a); C (BSA) = C (complex) = 3 × 10−5 M (T = 298 K).

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Fig. 5. CD spectra of BSA in the presence of various concentrations of the complex (T = 298 K). C (BSA) = 3 × 10−5 M. The concentration ratios of BSA to the complex were 1:0, 1:0.01, and 1:0.03, respectively. Fig. 4. The UV–vis absorption spectra of BSA in the presence of various concentrations of the complex, C (BSA) = 3 × 10−5 M: C (complex)/C (BSA): 0, 0.1, 0.2, 0.3, 0.5 and 0.7, respectively.

where K2 is an orientation factor dependent on the alignment of the donor and acceptor dipoles, n is the refractive index of the medium, ϕ is the luminescence quantum yield in the absence of energy transfer and J is the overlap between the luminescence spectrum of the donor and the absorption spectrum of the acceptor; J is given by: [27]. J=

∞ F()ε()4 d 0 ∞ 0

F() d

(7)

where F() is the corrected fluorescence intensity of the donor at the wavelength  to  +  to and ε is the extinction coefficient of the acceptor at . In the present case, n = 1.36 and ϕ = 0.15 [27]. From Eqs. (5)–(7), J = 1.98 × 10−14 cm3 L mol−1 , E = 0.68, R0 = 2.82 nm, and r = 2.48 nm were calculated. As the binding distance r = 2.48 nm is less than 8 nm, and 0.5R0 < r < 1.5R0 , the energy transfer from BSA to Ag complex occurred with high possibility [28]. 3.3. UV–vis spectroscopy UV–vis absorption measurement is a very simple method and applicable to explore the structural changes and to know the complex formation [29]. In the present study, to explore the structural changes of BSA by addition of the complex, we measured UV–vis spectra of BSA with various amounts of complex (Fig. 4). There was a red shift at 240 nm and that the intensity of BSA decreased with the increasing of the concentration of complex. The literature [30] shows that the peak in the 240 nm region in the difference spectra of proteins is related to changes in the conformation of the peptide backbone associated with the helix–coil transformation [31]. 3.4. Circular dichroism CD is a sensitive technique to monitor conformational changes of protein upon interaction with small molecules. BSA has a high percentage of ␣-helical structure which shows a characteristic strong double minimum signals at 222 and 208 nm [32,33] (Fig. 5, curve a). The intensities of two double minimum reflect the amount of helicity of BSA and further these indicate that BSA contains more than 50% of ␣-helical structure. Upon addition of the complex to BSA the extent of ␣-helicity of the protein decreased and hence, the intensity of double minimum was reduced. This is indicative of change in helicity when the Ag complex is completely bound to BSA.

Table 2 Fractional contents of the secondary structure of BSA (3 × 10−5 M) with and without Ag complex. Concentration ratio (BSA/complex)

␣-Helix (f␣ )

0 0.01 0.03

84.5% 81.9% 74.2%

␤-Beta (f␤ ) 0% 0% 0%

Turn (fturn ) 0% 0% 0%

Random (frandom ) 15.5% 18.1% 25.8%

Fractional contents of the secondary structure of BSA (f␣ , f␤ , fturn and frandom ) with and without Ag complex are calculated utilizing circular dichroism spectroscopy and are shown in Table 2. The CD results show that the addition of the Ag complex to BSA caused the reduction of the ␣-helix fraction (f␣ ) from 84.5 to 74.2. Also changes in the fraction of random coil (frandom ) was observed on the addition of the titled complex to BSA (Table 2). These results are in agreement with the previous studies on the interaction of albumin with small molecules [34], which suggests the interaction of Ag complex with BSA. The CD spectra of BSA in the presence and absence of the Ag complex were observed to be similar in shape, which meant that the structure of BSA was also predominantly of ␣-helix [35]. The CD results were expressed in terms of mean residue ellipticity (MRE) in degcm2 dmol−1 according to the following Eq. (8): MRE =

observed CD (m deg) Cp n1 × 10

(8)

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. (9): ␣-helix (%) =

−MRE280 − 4000 33,000 − 4000

(9)

The observed MRE value at 208 nm, MRE208 , of ␤-form and random coil conformation cross in total and a pure ␣-helix were 4000 and 33,000, respectively. From the above equation, the ␣-helicity in the secondary structure of BSA was determined [36]. 4. Conclusions According to the results arising from different spectroscopic methods, we conclude that, the Ag(I) complex binds to BSA with a high affinity through a static mode and transport in the body. The results in Table 1 indicate that the probable quenching mechanism of fluorescence of BSA by the complex is a static quenching procedure, because Ksv is decreased with increasing temperature. The negative value of H, negative value of S and the negative value of G indicate that van der Waals force and hydrogen bonding play

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major roles in the binding of the complex and BSA. According to the FRET theory, the critical distance between BSA and the complex (r0 ) is 2.48 nm, in the 2–8 nm range, and this shows that the energy transfer from BSA to the complex occurs with high probability. Decrease of the absorption intensity of the BSA spectrum and a red shift at 240 nm in the presence of various amounts of Ag complex indicates that the peak in the 240 nm region in the difference spectra of proteins is related to changes in the conformation of the peptide backbone associated with the helix–coil transformation The CD spectra of BSA in the presence and absence of the Ag complex were observed to be similar in shape, which meant that the structure of BSA was also predominantly of ␣-helix. Acknowledgment Financial support from the Razi University Research Center is gratefully acknowledged. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]

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