Appl Biochem Biotechnol DOI 10.1007/s12010-014-1423-z
Mechanistic and Conformational Studies on the Interaction of a Platinum(II) Complex Containing an Antiepileptic Drug, Levetiracetam, With Bovine Serum Albumin by Optical Spectroscopic Techniques in Aqueous Solution Nahid Shahabadi & Saba Hadidi
Received: 22 May 2014 / Accepted: 18 November 2014 # Springer Science+Business Media New York 2014
Abstract Fluorescence spectroscopy in combination with circular dichroism (CD) and ultraviolet-visible (UV–vis) absorption spectroscopy were employed to investigate the binding of a new platinum(II) complex containing an antiepileptic drug “Levetiracetam” to bovine serum albumin (BSA) under the physiological conditions. In the mechanism discussion, it was proved that the fluorescence quenching of BSA by Pt(II) complex is a result of the formation of Pt(II) complex–BSA complex. The thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures (283, 298, and 310 K) were calculated, and the negative value for ΔH and ΔS indicate that the hydrogen bonds and van der Waals interactions play major roles in Pt(II) complex–BSA association. Binding studies concerning the number of binding sites (n~1) and apparent binding constant Kb were performed by fluorescence quenching method. The site marker competitive experiments indicated that the binding of Pt(II) complex to BSA primarily took place in site II. Based on the Förster’s theory, the average binding distance between Pt(II) complex and BSA was obtained (r=5.29 nm). Furthermore, UV–vis, CD, and synchronous fluorescence spectrum were used to investigate the structural change of BSA molecules with addition of Pt(II) complex. These results indicate that the binding of Pt(II) complex to BSA causes apparent change in the secondary structure of BSA and do affect the microenvironment around the tryptophan residue. Keywords Pyrrolidine-platinum(II) complex . Bovine serum albumin binding . Aqueous solution . Spectroscopic techniques . Subdomain IIIA
Introduction Levetiracetam ((-)-(s)-α-ethyl-2-oxo-1-pyrrolidine acetamide) (Fig. 1a) is an antiepileptic drug belonging to the pyrrolidine family. The pyrrolidine derivatives fall into an important N. Shahabadi (*) : S. Hadidi Inorganic Chemistry Department, Faculty of Chemistry, Razi University, Kermanshah, Iran e-mail: [email protected]
Appl Biochem Biotechnol
class of organic compounds, and they have received attention of biochemists because of their biochemical activities [1–3]. Furthermore, a great many pyrrolidine complexes have also provoked a great interest in their diverse spectra of biological and pharmaceutical activities, such as antibacterial and antitumor activities [4–8]. Coordination compounds of platinum have an especially rich history due to the clinical success of cisplatin and related anticancer drugs [9, 10]. In previous study [11], we synthesized a new platinum(II) complex (Fig. 1b) containing an antiepileptic drug “Levetiracetam” belonging to the pyrrolidine family and studied the binding properties of that Pt(II) complex to calf thymus DNA in physiological conditions. Metal complexes that bind and cleave DNA or proteins under physiological conditions are of current interests for their varied applications in nucleic acid and protein chemistry [12–16]. It is known that the distribution, free concentration, and the metabolism of various drugs are strongly affected by drug–protein interactions in the bloodstream [17–20]. This type of interaction can also influence the drug stability and toxicity during the chemotherapeutic process [17]. Serum albumin is the major soluble protein in circulatory system, which has many physiological functions, such as maintaining the osmotic pressure and pH of blood and as carriers transporting a great number of endogenous and exogenous compounds such as fatty acids, amino acids, drugs, and pharmaceuticals [21]. It is of great necessity to investigate the interaction of chemicals with serum albumin, which will help to explain the metabolism and transport process of the chemicals. Bovine serum albumin (BSA) is one of the major components in blood plasma of bovine species, accounting for about 60 % of the total protein, corresponding to a concentration of 43 mg mL−1. It has been widely studied because of its stability and low cost, unusual ligand-binding properties and particularly its structural homology with human serum albumin (HSA) [22]. The molecular interactions are often monitored by spectroscopic techniques because these methods are sensitive and relatively easy to use. In this work, the investigation of bovine serum albumin (BSA) with Pt(II) complex was carried out in aqueous solution at physiological conditions using fluorescence, UV–vis, and circular dichroism approaches. Spectroscopic evidences regarding the drug-binding mode, the association constant, and the change of protein secondary structure are provided. The aim of this work is to clarify the binding mechanism of Pt(II) complex with BSA and provide important information for the study of the structural–activity relationship of the drug and BSA.
Fig. 1 Molecular structure of a Levetiracetam and b platinum(II) complex
Appl Biochem Biotechnol
Materials and Methods Materials Bovine serum albumin (BSA) and warfarin were obtained from Sigma-Aldrich; Ibuprofen was obtained from Abidi Pharmaceutical Co. All stock solutions were prepared in the buffer solution adjusted to pH 7.4 with 0.1 M Na2HPO4 and NaH2PO4 in pure aqueous medium. BSA stock solution (1×10−3 M, based on its molecular weight of 66,000 g mol−1) was prepared in 0.1 M phosphate buffer of pH 7.4 and was kept in the dark at 277 K. Triple distilled water was used throughout the experiment. Apparatus Fluorescence measurements were performed with a JASCO spectrofluorimeter Model FP6200 equipped with a thermostat bath, using a 1.0-cm quartz cell. UV–vis absorption spectra were measured on a Perkin Elmer UV–vis spectrophotometer Model Lambda 25 using a 1.0cm cell. CD measurements were recorded on a JASCO spectropolarimeter Model J-810, using a 0.1-cm quartz cell. pH measurements were carried out with a digital pH meter with a combined glass–calomel electrode. Experimental Procedures Fluorescence Quenching Studies The maximal fluorescence emission of BSA at λex =295 nm was located at 345 nm. Fluorescence measurements were performed by keeping the concentration of BSA constant (5×10−6 M) while varying the Pt(II) complex concentration from 0.0 to 9.1×10−5 M at different temperatures (283, 298, and 310 K). Since the drug has very little absorption at 295 nm, the fluorescence intensities in this paper were corrected for absorption of the exciting light and reabsorption of the emitted light to decrease the inner filter effect using the following relationship [23]: F cor ¼ F obs eðAexþAemÞ=2 That Fcor and Fobs are the fluorescence intensities corrected and observed, respectively, and Aex and Aem are the absorption of the system at the excitation and the emission wavelength, respectively. The fluorescence intensity utilized in this paper is the corrected intensity. Warfarin and Ibuprofen Displacement Experiments The displacement experiments were performed using the site probes including warfarin and ibuprofen by keeping the concentration of protein and probe constant (5×10−6 and 5×10−5 M, respectively). The fluorescence quenching titration was used to determine the binding constants of Pt(II) complex–BSA systems in the presence of the above site probes for sites I and II. Synchronous Fluorescence Quenching of BSA by Pt(II) Complex Synchronous fluorescence spectra of BSA (5×10−6 M) were recorded with increasing concentrations of Pt(II) complex (0.0 to 9.1×10−5 M), by setting Δλ=60 nm and Δλ=15 nm for tryptophan and tyrosine residues, respectively.
Appl Biochem Biotechnol
UV–Visible Absorption Studies The absorbance measurements were performed by keeping the BSA concentration constant (1×10−5 M) while varying the Pt(II) complex concentration from 0 to 1.3×10−5 M (ri=[Pt(II) complex]/[BSA]=0.0, 0.3, 0.5, 0.7, 0.9, 1, and 1.3). Equal small aliquots of Pt(II) complex stock solutions were added to both BSA and reference solutions to eliminate the effect of Pt(II) complex absorbance. The samples were incubated at room temperature for 10 min, and the spectra were recorded in the range of 200–400 nm. Circular Dichroism Measurements Circular dichroism (CD) measurements were performed by keeping the concentration of BSA constant (3×10-6 M) while varying the Pt(II) complex concentration from 0 to 3×10-6 M (ri =[Pt(II) complex] /[BSA])=0.0, 0.7, and 1). The CD results were expressed in terms of mean residue ellipticity (MRE) in deg cm2 dmol-1 according to the following equation: MRE¼
Observed CD ðmdegÞ C p nl10
where Cp is the molar concentration of the protein, n is the number of amino acid residues (583 for BSA) and l the path-length (0.1 cm). The α-helical contents of free and combined BSA were calculated from MRE values at 208 nm using the following equation: α−helix ð%Þ ¼
‐MRE208 −4000 33000−4000
Results and Discussion Fluorescence Quenching Spectral Studies Fluorescence emission spectrum of BSA shows a maximum at 345 nm when the exciting wavelength is 295 nm. BSA has three intrinsic fluorophores: tryptophan, tyrosine, and phenylalanine that can be quenched. In fact, as Sulkowska [24] said, because phenylalanine has a very low quantum yield and the fluorescence of tyrosine is almost totally quenched if it is ionized, or near an amino group, a carboxyl group, or a tryptophan, the intrinsic fluorescence of BSA is attributed to the two tryptophans of bovine serum albumin, Trp-213 conserved in all albumins and Trp-134, characteristic of the bovine albumin. A useful feature of the intrinsic fluorescence of proteins is the high sensitivity of tryptophan and its local environment. Some small molecules can change the microenvironment of tryptophan residues, which can generate changes of intrinsic fluorescence intensity of BSA. Changes in the emission spectra of tryptophan are common in response to protein conformational transitions, subunit associations, substrate binding, or denaturation [24, 25]. Therefore, the intrinsic fluorescence of proteins can provide considerable information on their structure and dynamics and is often utilized in the study of protein folding and association reactions. The fluorescence emission spectra of BSA in the absence and presence of Pt(II) complex with an excitation wavelength at 295 nm are shown in Fig. 2. As shown in Fig. 2, BSA exhibited a strong fluorescence emission peak at 345 nm, while Pt(II) complex did not show intrinsic fluorescence under the same experimental conditions. Furthermore, the fluorescence
Appl Biochem Biotechnol
Fig. 2 Fluorescence spectra of BSA in the absence and the presence of Pt(II) complex, [BSA]=5.0×10−6 M and [Pt(II) complex]=from 0.0 to 9.10×10−5 M; (λex =295 nm, T=310 K, pH 7.4)
intensity of BSA remarkably decreased with increasing the concentration of Pt(II) complex. This indicated that Pt(II) complex interacted with BSA and quenched its intrinsic fluorescence. Quenching Mechanism Fluorescence quenching refers to any process which decreases the fluorescence intensity of a fluorophore. Fluorescence quenching could proceed via different mechanisms, usually classified as dynamic quenching and static quenching. Dynamic and static quenching can be distinguished by their different dependence on temperature and excited-state lifetime [26]. For the dynamic quenching, higher temperatures will result in faster diffusion and larger amounts of collisional quenching; hence, the quenching constant values will increase with increasing temperature, but the reverse effect would be observed for static quenching. In order to study the quenching mechanism between Pt(II) complex and BSA, the fluorescence quenching data were analysed using the Stern–Volmer equation [27]: F0 ¼ 1 þ K sv ½Q ¼ 1 þ K q τ 0 ½Q F where Fo and F are the fluorescence intensities in the absence and presence of quencher, respectively. KSV is the Stern–Volmer quenching constant, which was determined by linear regression of a plot of Fo/F against [Q]. Kq is the quenching rate constant of biomolecule, τo is the average lifetime of the fluorophore without quencher, the value of τo of the biopolymer is 10−8 s [26], and [Q] is the concentration of quencher. KSV and Kq obtained from the Stern−Volmer equation are presented in Table 1. It is seen that KSV and Kq increase as the temperature increases, indicating that the mechanism of the quenching may be a dynamic quenching. For dynamic quenching, the maximum scattering
Appl Biochem Biotechnol Table 1 The quenching constants (KSV), number of binding sites (n), and binding constants (Kb) for the interaction of Pt(II) complex with BSA at different temperatures T (K)
Ksv ×103
Kq ×1011
R2
n
Log Kb
Kb
R2
283
6.29
6.29
0.9925
1.12
4.36
2.29×104
0.9809
298
6.31
6.31
0.9946
1.05
4.05
1.12×104
0.9940
310
7.67
7.67
0.9937
1.01
3.98
9.55×103
0.9959
collisional quenching constant of various quenchers is 2.0×1010 L mol−1 s−1. However, Kq is much larger than 2.0×1010 L mo−1 s−1, suggesting that the quenching process may be a static quenching. As described, the quenching mechanism can be classified as static quenching and dynamic quenching, and they represent two totally different quenching processes. These two kinds of quenching mechanism demonstrate some differences that can be distinguished experimentally, such as the change in the UV–visible spectra of the BSA and the temperature dependence of the quenching constant [28]. A complex of BSA and drug forms in static quenching, so there will be some changes in the UV–visible spectra of the BSA, whereas dynamic quenching has no such change. As to the temperature dependence of the quenching constant, diffusion is the control step for dynamic quenching, so the quenching constant will increase with increasing temperature [29]. As reported by our group and other research groups, most of the quenching is in good accordance with this theory and fits the results in the distinguishing experiments [30]. Thus, we employed UV–visible absorption spectra to give some more evidence for the actual quenching process. The UV–visible absorption spectra of BSA and the Pt(II) complexBSA system were measured, according to the theory mentioned; the UV spectra of BSA would have no detectable changes if the quenching was a dynamic mechanism [31]. On the other hand, ground-state Pt(II) complex-BSA complex forms in the static quenching, and the UV spectrum of BSA changes as a direct consequence [32]. Therefore, according to the UV spectra, the fluorescence quenching of BSA in our case seems to be primarily caused by complex formation between BSA and Pt(II) complex.
Fig. 3 Stern–Volmer plots for the Pt(II) complex–BSA system at different concentrations of Pt(II) complex at 310 K and pH=7.40: a [BSA]=2.50×10−6 M, b [BSA]=7.50×10−6 M
Appl Biochem Biotechnol
In order to further determine the quenching mechanism, the analysis of the Stern–Volmer plots at various concentrations of BSA was carried out. Fig. 3 shows plots of F0/F for BSA versus [Q] of Pt(II) complex at different concentrations of BSA. It could be found that the KSV value decreased with the concentration of BSA increasing. It suggested that the fluorescence quenching process was static quenching rather than dynamic quenching mechanism [33]. Binding Constant Measurements For static quenching, the relationship between fluorescence intensity and concentration of a quencher can be described by the equation shown below [34]: log
F 0 −F F
¼ logK b þ nlog ½Q
where Kb is the binding constant and n is the number of binding sites per BSA. The values of Kb and n (Table 1) are evaluated from the plot of log(F0–F)/F versus log[Q] [34]. The larger values of Kb observed in the present study indicated the presence of strong binding between Pt(II) complex and protein. Further, the binding constant values decreased with increase in temperature suggesting the reduction in stability of Pt(II) complex–BSA complex [17]. The values of n are observed to be close to unity indicating that there is one independent class of binding sites on BSA for Pt(II) complex. Identification of Binding Location of Pt(II) Complex on BSA There are two major specific ligand-binding sites in BSA and the principal regions usually located in hydrophobic cavities in subdomains IIA and IIIA, which are called site I and site II, respectively [35]. Sudlow et al. have suggested that site I of serum albumin showed affinity for warfarin, phenylbutazone, etc. [36], site II for ibuprofen, flufenamic acid, etc., and site III for digitoxin [37–39]. In order to determine the location of Pt(II) complex-binding site on BSA, the competitive displacement experiments were carried out using different site probes, viz., warfarin (for site I) and ibuprofen (for site II). For this, varied amounts of the Pt(II) complex were added to a solution containing fixed amounts of BSA and site probe, and fluorescence intensities were noted down upon excitation at 295 nm (Figs. 4 and 5). The binding constant values were evaluated. From the results shown in Table 2, it is evident that the warfarin is not significantly displaced by Pt(II) complex. However, Pt(II) complex exhibited significant displacement of ibuprofen suggesting that the site for Pt(II) complex and ibuprofen is same. This means that the binding site for Pt(II) complex on BSA is site II located in subdomain IIIA [27]. Thermodynamic Parameters and Nature of the Binding Forces Generally, the acting forces contributing to the macromolecule interactions with small ligands include hydrogen bond, van der Waals force, electrostatic interaction, and hydrophobic force [40]. The force acting may be predicted by knowing the value of the enthalpy change (ΔH) and entropy change (ΔS), which can be evaluated using the following van’t Hoff’s equation: lnK b ¼ −
ΔH ΔS þ RT R
Appl Biochem Biotechnol
Fig. 4 Effect of site marker to Pt(II) complex–BSA system; [warfarin]=5.0×10−5, [BSA]=5.0×10−6 M, and [Pt(II) complex]=from 0.0 to 9.10×10−5 M at 310 K
where Kb is the binding constant at corresponding temperature and R is the gas constant. The enthalpy change (ΔH) and the entropy change (ΔS) were obtained from the slope and intercept of the fitted curve of ln Kb against 1/T respectively. From the thermodynamic standpoint, ΔH>0 and ΔS>0 implies a hydrophobic interaction, ΔH
Mechanistic and Conformational Studies on the Interaction of a Platinum(II) Complex Containing an Antiepileptic Drug, Levetiracetam, With Bovine Serum Albumin by Optical Spectroscopic Techniques in Aqueous Solution Nahid Shahabadi & Saba Hadidi
Received: 22 May 2014 / Accepted: 18 November 2014 # Springer Science+Business Media New York 2014
Abstract Fluorescence spectroscopy in combination with circular dichroism (CD) and ultraviolet-visible (UV–vis) absorption spectroscopy were employed to investigate the binding of a new platinum(II) complex containing an antiepileptic drug “Levetiracetam” to bovine serum albumin (BSA) under the physiological conditions. In the mechanism discussion, it was proved that the fluorescence quenching of BSA by Pt(II) complex is a result of the formation of Pt(II) complex–BSA complex. The thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures (283, 298, and 310 K) were calculated, and the negative value for ΔH and ΔS indicate that the hydrogen bonds and van der Waals interactions play major roles in Pt(II) complex–BSA association. Binding studies concerning the number of binding sites (n~1) and apparent binding constant Kb were performed by fluorescence quenching method. The site marker competitive experiments indicated that the binding of Pt(II) complex to BSA primarily took place in site II. Based on the Förster’s theory, the average binding distance between Pt(II) complex and BSA was obtained (r=5.29 nm). Furthermore, UV–vis, CD, and synchronous fluorescence spectrum were used to investigate the structural change of BSA molecules with addition of Pt(II) complex. These results indicate that the binding of Pt(II) complex to BSA causes apparent change in the secondary structure of BSA and do affect the microenvironment around the tryptophan residue. Keywords Pyrrolidine-platinum(II) complex . Bovine serum albumin binding . Aqueous solution . Spectroscopic techniques . Subdomain IIIA
Introduction Levetiracetam ((-)-(s)-α-ethyl-2-oxo-1-pyrrolidine acetamide) (Fig. 1a) is an antiepileptic drug belonging to the pyrrolidine family. The pyrrolidine derivatives fall into an important N. Shahabadi (*) : S. Hadidi Inorganic Chemistry Department, Faculty of Chemistry, Razi University, Kermanshah, Iran e-mail: [email protected]
Appl Biochem Biotechnol
class of organic compounds, and they have received attention of biochemists because of their biochemical activities [1–3]. Furthermore, a great many pyrrolidine complexes have also provoked a great interest in their diverse spectra of biological and pharmaceutical activities, such as antibacterial and antitumor activities [4–8]. Coordination compounds of platinum have an especially rich history due to the clinical success of cisplatin and related anticancer drugs [9, 10]. In previous study [11], we synthesized a new platinum(II) complex (Fig. 1b) containing an antiepileptic drug “Levetiracetam” belonging to the pyrrolidine family and studied the binding properties of that Pt(II) complex to calf thymus DNA in physiological conditions. Metal complexes that bind and cleave DNA or proteins under physiological conditions are of current interests for their varied applications in nucleic acid and protein chemistry [12–16]. It is known that the distribution, free concentration, and the metabolism of various drugs are strongly affected by drug–protein interactions in the bloodstream [17–20]. This type of interaction can also influence the drug stability and toxicity during the chemotherapeutic process [17]. Serum albumin is the major soluble protein in circulatory system, which has many physiological functions, such as maintaining the osmotic pressure and pH of blood and as carriers transporting a great number of endogenous and exogenous compounds such as fatty acids, amino acids, drugs, and pharmaceuticals [21]. It is of great necessity to investigate the interaction of chemicals with serum albumin, which will help to explain the metabolism and transport process of the chemicals. Bovine serum albumin (BSA) is one of the major components in blood plasma of bovine species, accounting for about 60 % of the total protein, corresponding to a concentration of 43 mg mL−1. It has been widely studied because of its stability and low cost, unusual ligand-binding properties and particularly its structural homology with human serum albumin (HSA) [22]. The molecular interactions are often monitored by spectroscopic techniques because these methods are sensitive and relatively easy to use. In this work, the investigation of bovine serum albumin (BSA) with Pt(II) complex was carried out in aqueous solution at physiological conditions using fluorescence, UV–vis, and circular dichroism approaches. Spectroscopic evidences regarding the drug-binding mode, the association constant, and the change of protein secondary structure are provided. The aim of this work is to clarify the binding mechanism of Pt(II) complex with BSA and provide important information for the study of the structural–activity relationship of the drug and BSA.
Fig. 1 Molecular structure of a Levetiracetam and b platinum(II) complex
Appl Biochem Biotechnol
Materials and Methods Materials Bovine serum albumin (BSA) and warfarin were obtained from Sigma-Aldrich; Ibuprofen was obtained from Abidi Pharmaceutical Co. All stock solutions were prepared in the buffer solution adjusted to pH 7.4 with 0.1 M Na2HPO4 and NaH2PO4 in pure aqueous medium. BSA stock solution (1×10−3 M, based on its molecular weight of 66,000 g mol−1) was prepared in 0.1 M phosphate buffer of pH 7.4 and was kept in the dark at 277 K. Triple distilled water was used throughout the experiment. Apparatus Fluorescence measurements were performed with a JASCO spectrofluorimeter Model FP6200 equipped with a thermostat bath, using a 1.0-cm quartz cell. UV–vis absorption spectra were measured on a Perkin Elmer UV–vis spectrophotometer Model Lambda 25 using a 1.0cm cell. CD measurements were recorded on a JASCO spectropolarimeter Model J-810, using a 0.1-cm quartz cell. pH measurements were carried out with a digital pH meter with a combined glass–calomel electrode. Experimental Procedures Fluorescence Quenching Studies The maximal fluorescence emission of BSA at λex =295 nm was located at 345 nm. Fluorescence measurements were performed by keeping the concentration of BSA constant (5×10−6 M) while varying the Pt(II) complex concentration from 0.0 to 9.1×10−5 M at different temperatures (283, 298, and 310 K). Since the drug has very little absorption at 295 nm, the fluorescence intensities in this paper were corrected for absorption of the exciting light and reabsorption of the emitted light to decrease the inner filter effect using the following relationship [23]: F cor ¼ F obs eðAexþAemÞ=2 That Fcor and Fobs are the fluorescence intensities corrected and observed, respectively, and Aex and Aem are the absorption of the system at the excitation and the emission wavelength, respectively. The fluorescence intensity utilized in this paper is the corrected intensity. Warfarin and Ibuprofen Displacement Experiments The displacement experiments were performed using the site probes including warfarin and ibuprofen by keeping the concentration of protein and probe constant (5×10−6 and 5×10−5 M, respectively). The fluorescence quenching titration was used to determine the binding constants of Pt(II) complex–BSA systems in the presence of the above site probes for sites I and II. Synchronous Fluorescence Quenching of BSA by Pt(II) Complex Synchronous fluorescence spectra of BSA (5×10−6 M) were recorded with increasing concentrations of Pt(II) complex (0.0 to 9.1×10−5 M), by setting Δλ=60 nm and Δλ=15 nm for tryptophan and tyrosine residues, respectively.
Appl Biochem Biotechnol
UV–Visible Absorption Studies The absorbance measurements were performed by keeping the BSA concentration constant (1×10−5 M) while varying the Pt(II) complex concentration from 0 to 1.3×10−5 M (ri=[Pt(II) complex]/[BSA]=0.0, 0.3, 0.5, 0.7, 0.9, 1, and 1.3). Equal small aliquots of Pt(II) complex stock solutions were added to both BSA and reference solutions to eliminate the effect of Pt(II) complex absorbance. The samples were incubated at room temperature for 10 min, and the spectra were recorded in the range of 200–400 nm. Circular Dichroism Measurements Circular dichroism (CD) measurements were performed by keeping the concentration of BSA constant (3×10-6 M) while varying the Pt(II) complex concentration from 0 to 3×10-6 M (ri =[Pt(II) complex] /[BSA])=0.0, 0.7, and 1). The CD results were expressed in terms of mean residue ellipticity (MRE) in deg cm2 dmol-1 according to the following equation: MRE¼
Observed CD ðmdegÞ C p nl10
where Cp is the molar concentration of the protein, n is the number of amino acid residues (583 for BSA) and l the path-length (0.1 cm). The α-helical contents of free and combined BSA were calculated from MRE values at 208 nm using the following equation: α−helix ð%Þ ¼
‐MRE208 −4000 33000−4000
Results and Discussion Fluorescence Quenching Spectral Studies Fluorescence emission spectrum of BSA shows a maximum at 345 nm when the exciting wavelength is 295 nm. BSA has three intrinsic fluorophores: tryptophan, tyrosine, and phenylalanine that can be quenched. In fact, as Sulkowska [24] said, because phenylalanine has a very low quantum yield and the fluorescence of tyrosine is almost totally quenched if it is ionized, or near an amino group, a carboxyl group, or a tryptophan, the intrinsic fluorescence of BSA is attributed to the two tryptophans of bovine serum albumin, Trp-213 conserved in all albumins and Trp-134, characteristic of the bovine albumin. A useful feature of the intrinsic fluorescence of proteins is the high sensitivity of tryptophan and its local environment. Some small molecules can change the microenvironment of tryptophan residues, which can generate changes of intrinsic fluorescence intensity of BSA. Changes in the emission spectra of tryptophan are common in response to protein conformational transitions, subunit associations, substrate binding, or denaturation [24, 25]. Therefore, the intrinsic fluorescence of proteins can provide considerable information on their structure and dynamics and is often utilized in the study of protein folding and association reactions. The fluorescence emission spectra of BSA in the absence and presence of Pt(II) complex with an excitation wavelength at 295 nm are shown in Fig. 2. As shown in Fig. 2, BSA exhibited a strong fluorescence emission peak at 345 nm, while Pt(II) complex did not show intrinsic fluorescence under the same experimental conditions. Furthermore, the fluorescence
Appl Biochem Biotechnol
Fig. 2 Fluorescence spectra of BSA in the absence and the presence of Pt(II) complex, [BSA]=5.0×10−6 M and [Pt(II) complex]=from 0.0 to 9.10×10−5 M; (λex =295 nm, T=310 K, pH 7.4)
intensity of BSA remarkably decreased with increasing the concentration of Pt(II) complex. This indicated that Pt(II) complex interacted with BSA and quenched its intrinsic fluorescence. Quenching Mechanism Fluorescence quenching refers to any process which decreases the fluorescence intensity of a fluorophore. Fluorescence quenching could proceed via different mechanisms, usually classified as dynamic quenching and static quenching. Dynamic and static quenching can be distinguished by their different dependence on temperature and excited-state lifetime [26]. For the dynamic quenching, higher temperatures will result in faster diffusion and larger amounts of collisional quenching; hence, the quenching constant values will increase with increasing temperature, but the reverse effect would be observed for static quenching. In order to study the quenching mechanism between Pt(II) complex and BSA, the fluorescence quenching data were analysed using the Stern–Volmer equation [27]: F0 ¼ 1 þ K sv ½Q ¼ 1 þ K q τ 0 ½Q F where Fo and F are the fluorescence intensities in the absence and presence of quencher, respectively. KSV is the Stern–Volmer quenching constant, which was determined by linear regression of a plot of Fo/F against [Q]. Kq is the quenching rate constant of biomolecule, τo is the average lifetime of the fluorophore without quencher, the value of τo of the biopolymer is 10−8 s [26], and [Q] is the concentration of quencher. KSV and Kq obtained from the Stern−Volmer equation are presented in Table 1. It is seen that KSV and Kq increase as the temperature increases, indicating that the mechanism of the quenching may be a dynamic quenching. For dynamic quenching, the maximum scattering
Appl Biochem Biotechnol Table 1 The quenching constants (KSV), number of binding sites (n), and binding constants (Kb) for the interaction of Pt(II) complex with BSA at different temperatures T (K)
Ksv ×103
Kq ×1011
R2
n
Log Kb
Kb
R2
283
6.29
6.29
0.9925
1.12
4.36
2.29×104
0.9809
298
6.31
6.31
0.9946
1.05
4.05
1.12×104
0.9940
310
7.67
7.67
0.9937
1.01
3.98
9.55×103
0.9959
collisional quenching constant of various quenchers is 2.0×1010 L mol−1 s−1. However, Kq is much larger than 2.0×1010 L mo−1 s−1, suggesting that the quenching process may be a static quenching. As described, the quenching mechanism can be classified as static quenching and dynamic quenching, and they represent two totally different quenching processes. These two kinds of quenching mechanism demonstrate some differences that can be distinguished experimentally, such as the change in the UV–visible spectra of the BSA and the temperature dependence of the quenching constant [28]. A complex of BSA and drug forms in static quenching, so there will be some changes in the UV–visible spectra of the BSA, whereas dynamic quenching has no such change. As to the temperature dependence of the quenching constant, diffusion is the control step for dynamic quenching, so the quenching constant will increase with increasing temperature [29]. As reported by our group and other research groups, most of the quenching is in good accordance with this theory and fits the results in the distinguishing experiments [30]. Thus, we employed UV–visible absorption spectra to give some more evidence for the actual quenching process. The UV–visible absorption spectra of BSA and the Pt(II) complexBSA system were measured, according to the theory mentioned; the UV spectra of BSA would have no detectable changes if the quenching was a dynamic mechanism [31]. On the other hand, ground-state Pt(II) complex-BSA complex forms in the static quenching, and the UV spectrum of BSA changes as a direct consequence [32]. Therefore, according to the UV spectra, the fluorescence quenching of BSA in our case seems to be primarily caused by complex formation between BSA and Pt(II) complex.
Fig. 3 Stern–Volmer plots for the Pt(II) complex–BSA system at different concentrations of Pt(II) complex at 310 K and pH=7.40: a [BSA]=2.50×10−6 M, b [BSA]=7.50×10−6 M
Appl Biochem Biotechnol
In order to further determine the quenching mechanism, the analysis of the Stern–Volmer plots at various concentrations of BSA was carried out. Fig. 3 shows plots of F0/F for BSA versus [Q] of Pt(II) complex at different concentrations of BSA. It could be found that the KSV value decreased with the concentration of BSA increasing. It suggested that the fluorescence quenching process was static quenching rather than dynamic quenching mechanism [33]. Binding Constant Measurements For static quenching, the relationship between fluorescence intensity and concentration of a quencher can be described by the equation shown below [34]: log
F 0 −F F
¼ logK b þ nlog ½Q
where Kb is the binding constant and n is the number of binding sites per BSA. The values of Kb and n (Table 1) are evaluated from the plot of log(F0–F)/F versus log[Q] [34]. The larger values of Kb observed in the present study indicated the presence of strong binding between Pt(II) complex and protein. Further, the binding constant values decreased with increase in temperature suggesting the reduction in stability of Pt(II) complex–BSA complex [17]. The values of n are observed to be close to unity indicating that there is one independent class of binding sites on BSA for Pt(II) complex. Identification of Binding Location of Pt(II) Complex on BSA There are two major specific ligand-binding sites in BSA and the principal regions usually located in hydrophobic cavities in subdomains IIA and IIIA, which are called site I and site II, respectively [35]. Sudlow et al. have suggested that site I of serum albumin showed affinity for warfarin, phenylbutazone, etc. [36], site II for ibuprofen, flufenamic acid, etc., and site III for digitoxin [37–39]. In order to determine the location of Pt(II) complex-binding site on BSA, the competitive displacement experiments were carried out using different site probes, viz., warfarin (for site I) and ibuprofen (for site II). For this, varied amounts of the Pt(II) complex were added to a solution containing fixed amounts of BSA and site probe, and fluorescence intensities were noted down upon excitation at 295 nm (Figs. 4 and 5). The binding constant values were evaluated. From the results shown in Table 2, it is evident that the warfarin is not significantly displaced by Pt(II) complex. However, Pt(II) complex exhibited significant displacement of ibuprofen suggesting that the site for Pt(II) complex and ibuprofen is same. This means that the binding site for Pt(II) complex on BSA is site II located in subdomain IIIA [27]. Thermodynamic Parameters and Nature of the Binding Forces Generally, the acting forces contributing to the macromolecule interactions with small ligands include hydrogen bond, van der Waals force, electrostatic interaction, and hydrophobic force [40]. The force acting may be predicted by knowing the value of the enthalpy change (ΔH) and entropy change (ΔS), which can be evaluated using the following van’t Hoff’s equation: lnK b ¼ −
ΔH ΔS þ RT R
Appl Biochem Biotechnol
Fig. 4 Effect of site marker to Pt(II) complex–BSA system; [warfarin]=5.0×10−5, [BSA]=5.0×10−6 M, and [Pt(II) complex]=from 0.0 to 9.10×10−5 M at 310 K
where Kb is the binding constant at corresponding temperature and R is the gas constant. The enthalpy change (ΔH) and the entropy change (ΔS) were obtained from the slope and intercept of the fitted curve of ln Kb against 1/T respectively. From the thermodynamic standpoint, ΔH>0 and ΔS>0 implies a hydrophobic interaction, ΔH
