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Spectrochimica Acta Part A 92 (2012) 164–174

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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa

Nickel(II)-Schiff base complex recognizing domain II of bovine and human serum albumin: Spectroscopic and docking studies Aurkie Ray 1 , Banabithi Koley Seth 1 , Uttam Pal, Samita Basu ∗ Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700 064, India

a r t i c l e

i n f o

Article history: Received 20 September 2011 Received in revised form 13 February 2012 Accepted 17 February 2012 Keywords: Nickel complex Serum albumins Fluorescence quenching Protein denaturation Docking

a b s t r a c t It has been spectroscopically monitored that a mononuclear nickel(II)-Schiff base complex {[NiL]·CH3 OH = NSC} exhibits greater binding affinity for bovine serum albumin (BSA) than that of its human counterpart (HSA). Moreover the modes of binding of NSC with the two serum albumins also differ significantly. Docking studies predict a relatively rare type of ‘superficial binding’ of NSC at domain IIB of HSA with certain mobility whereas for BSA such phenomena has not been detected. The mobile nature of NSC at domain IIB of HSA has been well correlated with the spectroscopic results. It is to be noted that thermodynamic parameters for the NSC interaction also differ for the two serum albumins. Occurrence of energy transfer between the donor (Trp of BSA and HSA) and acceptor (NSC) has been obtained by means of Förster resonance energy transfer (FRET). The protein stability on NSC binding has also been experimented by the GuHCl-induced protein unfolding studies. Interestingly it has been found that NSC–HSA interaction enhances the protein stability whereas NSC–BSA binding has no such impact. Such observations are indicative of the fact that the conformation of NSC is responsible in recognizing the two serum albumins and selectively enhancing protein stability. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The applications of Schiff base-metal complexes in biology have gained importance as they show antimicrobial [1], antifungal, antibacterial [2], antiviral, antipyretic, antidiabetic [3] and antitumor activities. Recent review by Sakurai unveils that, transition metal complexes act as potential therapeutic agents against several human diseases like carcinomas, lymphomas, infection control, anti-inflammatory, diabetes and neurological disorders [4]. They also serve the purpose as free radical scavengers and thereby gain importance as antioxidant agents [5]. Although metal complexes of platinum and ruthenium (anticancer agent), vanadium and zinc (antidiabetic agents), copper, manganese and iron (antioxidant and SOD mimetic agents) [6] have played a major role in development of metallopharmaceutics but there has been a scarcity of nickel complexes in medicinal biochemistry. The biochemical activity of nickel is not much explored due to its nonfunctional nature yet it known to play an essential role in enzyme like urease and presumed to function in association with some molecules in the biological systems. Nickel has also been distinguished as an essential trace element for bacteria, plants, animals and humans [7].

∗ Corresponding author. Tel.: +91 33 2337 5345; fax: +91 33 2337 4637. E-mail address: [email protected] (S. Basu). 1 These authors have equal contribution. 1386-1425/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2012.02.060

The role of nickel complexes have gained importance as several tetraazamacrocylic complexes of nickel(II) with relatively low NiIII /NiII reduction potentials (≤0.86 V) bear the ability to act as a powerful inhibitor of the free-radical autoxidation of aldehydes [8]. Recently such inhibitory actions of nickel complexes have inspired authors to investigate its role in vitro as potential antioxidant of the highly reactive oxygen derived free radicals that are generated by the metabolic processes in human body [9]. Moreover such antioxidant property acquired by some nickel complexes may also help to cure variety of pathophysiological abnormalities such as inflammation, diabetes, genotoxicity and cancer that mainly result from reactive oxygen species (ROS). Such potential applications of nickel complexes can be more specifically studied if certain nickel assisted drug leads can be generated that are not readily achieved by other means [10]. Schiff base complexes as analytical reagents are greatly gaining importance for their versatile metal coordination behaviors and easy determination of different organic and inorganic substances [11]. Binding of Schiff base metal complexes with the most abundant carrier proteins (serum albumins) have also been a subject of interest as such drug–protein binding greatly influences absorption, drug transport, storage, metabolism and excretion properties of typical drugs in vertebrates [12]. In addition, it has been shown that binding of Schiff-base metal complexes to bovine serum albumin (BSA) have enhanced the antioxidant capacity of BSA in ROSs scavenging for about more than 10-times [13]. To gain further

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sodium chloride and hydrochloric acid (AR) were purchased from Merck and were used to prepare Tris–HCl buffer (0.01 M) of pH 7.4. The Fourier Transform Infrared spectra (4000–400 cm−1 ) of the ligands and the complex have been recorded on a Perkin–Elmer Spectrum RX I FT-IR system with solid KBr disk and the C, H, N microanalyses are carried out with a Perkin–Elmer 2400 II elemental analyzer. UV–Vis absorption spectra have been recorded on a JascoV-650 absorption spectrophotometer over a wavelength range from 250 to 500 nm at 298 K. The steady-state fluorescence measurements have been made in a Spex vluoromax-3 spectrofluorimeter. A pair of 1 cm × 1 cm path length quartz cuvettes is used for absorption and emission experiments. Singlet-state fluorescence lifetime studies have been carried out with a time-correlated single-photon-counting (TCSPC) spectrophotometer (Edinburgh) and during lifetime study the samples are excited at 295 nm. CD measurements have been performed on a Jasco-720 automatic recording spectrophotometer using a 0.1 cm path length quartz cuvette. 2.2. Syntheses of the Schiff base ligand and the complex Fig. 1. Perspective view of nickel(II)-Schiff base complex (NSC) generated by Hg software. Hydrogen atoms connected to carbon frame constituting the Schiff base ligand are not shown for clarity.

insights about how metal complexes can be administered intravenously by primary target molecules like serum albumins, authors have shown considerable interest in the study of the biochemical behavior of these complexes using spectroscopic tools [14,15]. Inspired from the earlier works we have classified a new bioactive nickel(II)-Schiff base complex (NSC) which was earlier synthesized by Ray et al. [16] (Fig. 1) and have investigated the binding interactions of NSC with transport proteins (bovine serum albumin and human serum albumin) by conventional spectroscopic techniques. The purpose of our investigation is to explore whether NSC acquire a suitable conformation that could recognize the two serum albumins. Recognition is a very first and a very crucial step for a metallodrug concept. For metallodrug activity it is very crucial that two proteins, A and B, are recognized by the targeting domain of the drug and only one protein is selectively inactivated depending on its chemical environment [17]. For NSC the absorption spectra support the ground state interactions with the two serum albumins whereas excited state interactions have been inferred from the fluorescence spectra. Changes in protein conformations due to NSC interaction have also been confirmed from the CD spectra. In silico, molecular docking simulations predict the site(s) and mode of NSC binding. Moreover GuHCl-induced protein unfolding studies infer the effect on the protein stability upon NSC binding with serum albumins. Such spectroscopic and docking studies are indeed helpful in detecting whether NSC has a potential ability to recognize the two serum albumins and can selectively induce protein stability. 2. Experimental

The Schiff base ligand and the nickel complex were synthesized earlier by Ray et al. [16]. In this paper the ligand and the complex have been resynthesized for the spectroscopic investigations with the biological macromolecules. The crystalline products obtained on resyntheses have been characterized by elemental analysis and infrared spectroscopy and the results are in good agreement to that of the literature values (vide supplementary information for details). 2.3. Structural aspects of NSC The reddish brown crystalline product of NSC has not been further subjected to single crystal X-ray diffraction study and the structure of characterized product is expected to remain the same. For docking studies that propose possible interactions of NSC with biological macromolecules, the authors have considered the earlier crystallographic information with CCDC number 633804 [16]. This data can be obtained free of charge on request at http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033; e-mail: [email protected]). Supplementary information contains a summarized description of the structural aspects of NSC. 2.4. Preparation of serum albumin solutions The concentrations of the stock solutions of BSA and HSA (CBSA/HSA ∼ 2.25 × 10−4 M) in tris buffer are determined from absorption spectroscopy. The concentrations are calculated by dividing absorbances at 280 nm by the molar extinction coefficients of the respective proteins (ε280 = 44,300 M−1 cm−1 for BSA and ε280 = 35,353 M−1 cm−1 for HSA) [18]. Stock solutions of the serum albumins are stored at 4 ◦ C and used in 4 days.

2.1. Materials and methods 2.5. Binding studies of NSC All chemicals and solvents used for the syntheses were of analytical grade. For the synthesis of nickel(II)-Schiff base complex, 1,3-diamino propane, 2-hydroxyacetophenone, nickel nitrate hexahydrate were purchased from Aldrich Chemical Co. and were used without further purivication. Fatty acid and globulin-free bovine serum albumin (BSA) and human serum albumin (HSA) were obtained from Sigma Chemical Co. Guanidine hydrochloride (GuHCl) was also purchased from Sigma Chemical Co. Triple distilled water was used for the preparation of all solutions. Tris buffer,

2.5.1. Absorption measurements The presence of ground state interactions between the biological marcromolecules (BSA, HSA) and NSC have been detected from absorption spectroscopy. The absorption titration experiments for serum albumins have been performed by maintaining the metal complex concentration fixed to (2 ml, 30 ␮M) and varying the BSA and HSA concentration from 0 to 15 ␮M. Titrations are done manually using micropipette and the change in absorbance data for the

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characteristic band of the complex at max = 380 nm is recorded each time during successive addition of the titrant. 2.5.2. Fluorescence measurements NSC is nonfluorescent in tris–HCl buffer at biological pH 7.4, hence NSC binding to serum albumins has been supported through the emission quenching experiment. Quenching of the intrinsic tryptophan fluorescence of BSA and HSA has been used as a tool to study the interaction of NSC with the transport proteins. For fluorescence titration experiments of serum albumins, BSA or HSA solution (2 ml, 10 ␮M) is placed in a quartz cuvette and titrated with various amounts of a concentrated solution of the complex such that the final concentration of NSC ranged from 0 to 90 ␮M (ex = 295 nm, em = 310–580 nm, slit 4). After each addition, the solution is mixed for 30 s and allowed to sit at the appropriate temperature for 5 min before measurement. The quenching of fluorescence for serum albumins has been analyzed using Stern–Volmer equation (1). F0 = 1 + KSV [Q ], F

(1)

where F0 and F (after inner filter effect correction [19]) are the relative fluorescence intensities in the absence and the presence of quencher, respectively, KSV is the Stern–Volmer constant and [Q] is the concentration of the quencher (NSC).

2.5.5. Docking experiment The crystal structure of HSA is obtained from Protein Data Bank (PDB ID: 1E78) and BSA (SP: P02769) structure is modeled on SWISS-MODEL server [23] (http://swissmodel.expasy.org) using HSA as the template. It is mentioned that the nickel complex (NSC) structure has been obtained from Cambridge Crystallographic Data Center quoting CCDC number 633804 as .cif file. The ligand .cif and sdf file types are converted to pdb file type using OpenBabel to use it as an input to AutoDock Vina. AutoDock Vina [24] (http://vina.scripps.edu/) and MGLTools [25] (http://mgltools.scripps.edu/) of The Scripps Research Institute are mainly used to perform the docking calculations (vide supplementary information for details). The results are also verified with AutoDock 4 [25] and on PatchDock [26] server. The later uses a different algorithm from that of AutoDock. The PyMOL [27] molecular viewer and the MGLTools are used to render the output and to calculate the distance between nearest atoms. The accessible surface area (ASA) of the unbound and ligand bound receptors are calculated by Mark Gerstein’s calc-surface program [28] on Helix Systems server at NIH (http://helixweb.nih.gov/structbio/basic.html) using 1.4 A˚ probe size. The minimum energy docked conformations are chosen. The changes in ASA upon ligand binding are calculated using ASA = ASAreceptor − ASAcomplex and the per-residue-change for the proteins has been graphically represented. 3. Results and discussion

2.5.3. Analysis of thermodynamic parameters The thermodynamic parameters H◦ and S◦ for NSC binding with BSA and HSA are determined from the van’t Hoff equation: ln Ka = −

H ◦ S ◦ + , RT R

(2)

where Ka is the binding affinity constant, R and T are the universal gas constant and absolute temperature, respectively. van’t Hoff plot of ln Ka vs. 1/T gives a straight line. The standard enthalpy change (H◦ ) and standard entropy change (S◦ ) values have been obtained from the slope (−H◦ /R) and intercept (S◦ /R) of the van’t Hoff plot [20]. Knowing these two values free energy change (G◦ ) is calculated from the following standard equation (3), G◦ = H ◦ − TS ◦

(3)

For all cases G◦ values are calculated at 298 K. 2.5.4. Circular dichroism measurements The CD spectra of serum albumins are finally obtained by averaging four successive scans recorded at a scan speed of 50 nm min−1 and subtracting the appropriate blanks (tris buffer) from these spectra. For serum albumins (BSA and HSA) the changes in the far UV-CD spectra (200–250 nm) provide insights about the changes in the secondary structure of the proteins. The results are expressed in terms of mean residual ellipticity (MRE) in◦ cm2 dmol−1 according to Eq. (4) [21]. MRE =

observedCD(mdeg) , Cp nl × 10

(4)

where Cp is the molar concentration of the protein, n is the number of amino acid residues (n = 583 for BSA and 585 for HSA [21]) and l is the path length (here l = 0.1 cm, i.e. the path length of the cuvette used). ˛-Helical content of free and bound protein has been eventually evaluated from the MRE values at 208 nm using Eq. (5) [22]. ␣-helix % =

 −MRE



− 4000 × 100 33000 − 4000 208

(5)

Prior to performing the desired spectroscopic experiments, the characterization of NSC in buffer medium and its stability within a certain temperature range have been carefully checked using UV/vis spectroscopy (vide supplementary information). 3.1. Interaction of NSC with serum albumins by absorption studies Ground state interactions of NSC with the serum albumins have been inferred from the changes in the absorption spectra of NSC (in the range 300–450 nm) on gradual addition of BSA or HSA at 298 K (Fig. 2) (for details vide supplementary informations). The spectral changes acquired for BSA and HSA are quiet alike. Hypochromism with no apparent shift has been detected for the characteristic absorption band of NSC at 380 nm. An isosbestic point at 338 nm (for BSA) and 341 nm (for HSA) has been detected in their respective absorption spectra. However in case of NSC–HSA the isosbestic point does not include the spectrum of pure HSA which signifies that the preliminary complexation between NSC and HSA is responsible for maintaining the equilibrium for further complexation with higher concentration of HSA. Such changes in the absorption spectra with a prominent isosbestic point support the ground state interaction of NSC with both the serum albumins. 3.2. Interaction of NSC with serum albumins by fluorescence quenching study The steady-state quenching of the intrinsic fluorescence intensity of BSA or HSA (for the tryptophan residue) on gradual increase in the concentration of nickel complex ascertain changes in the local environment of tryptophan caused by binding of NSC molecule near the residue. Moreover blue shifts from 345 nm and 344 nm ( = 5 nm and 6 nm) for BSA and HSA, respectively, indicate that the microenvironment around tryptophan becomes a bit hydrophobic on the addition of NSC to the respective serum albumins (Figs. 3 and 4). To analyze the quenching mechanism for both the systems, and to compare the strength and mode of interactions, the quenching constants for the NSC–BSA/HSA interactions

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Fig. 2. Absorption spectra affirming the ground state interactions of NSC (2 ml 30 ␮M) with (a) BSA and (b) HSA (0–15 ␮M) at 298 K. Dotted lines in the spectra indicate the spectrum for free NSC and the arrows indicate the changes in the absorption bands of NSC on the addition of the serum albumins. Isosbestic point has been encircled in the respective figures (a, b).

(6)

8

F0 / F

Fluorescence Intensity (a.u.)

where F0 and F are the fluorescence intensities in the absence and the presence of a quencher, kq is the bimolecular quenching rate constant,  0 is the average life time of fluorophore in the absence of a quencher and [Q] is the concentration of a quencher (NSC). KSV is the Stern–Volmer quenching constant in M−1 and it is the slope of the Stern–Volmer plot (F0 /F vs. [Q]) when the straight line makes an intercept close to one. It has been experimentally found that for 10 ␮M BSA or HSA the average life time is 6.18 ns and 6.17 ns, respectively. Quenching of emission spectra can result from variety of molecular interactions including excited-sate reactions through collisional quenching, energy transfer, ground state complex formation and molecular rearrangements. The different mechanisms of quenching are usually classified as either static quenching (the formation of a complex between quencher and fluorophore) or dynamic quenching (a collisional process). The type of quenching mainly operating in the systems can be speculated from the nature of the Stern–Volmer plots and the dependence of KSV on temperature. Linear Stern–Volmer plots represent a single quenching mechanism, either static or dynamic [29]. If the slope (KSV ) of the

6

1.5 1.2 0.9 0 15 30 45 60 [NSC]

4 2 0

350

400

450

Wavelength (nm) Fig. 3. Fluorescence quenching spectra of BSA (2 ml 10 ␮M) with increasing concentration of NSC (0–90 ␮M) in Tris–HCl/NaCl buffer (pH 7.4) at 298 K. Arrow shows the change in the emission intensity of BSA on increasing NSC concentration. Inset: Stern–Volmer plot for the quenching of BSA fluorescence by NSC; KSV = 1.03 × 104 M−1 at 298 K.

2.5

3.00

2.0

F0 / F

F0 = 1 + kq 0 [Q ] = 1 + KSV [Q ], F

plot is inversely correlated with temperature then the quenching mechanism is predicted to be a static one as increased temperature is likely to result in decreased stability of complexes. On the other hand an increase in the magnitude of KSV with increase temperature supports the play role of dynamic quenching as higher temperatures result in larger diffusion coefficients and kq are expected to increase with increasing temperature. Sometimes a nonlinear Stern–Volmer plot with an upward curvature, concave toward the y-axis at high [Q], may result if both static and dynamic quenching processes [19] operate simultaneously in an interacting system. The Stern–Volmer plots for the present interactive systems (NSC–BSA and NSC–HSA) are shown as insets in Figs. 3 and 4. It is now clear from the plots that two different types of quenching mechanisms operate for BSA and HSA with NSC. The NSC–BSA interactions at three different temperatures follow Stern–Volmer linear dependence of F0 /F upon the total concentration range of [Q] (Eq. (6)) and the calculated KSV values are inversely correlated with temperatures (Table 1). Such inverse relation of KSV with increasing temperatures indicates that the probable quenching mechanism of NSC–BSA binding reaction has been initiated by complex formation. Unlike the NSC–BSA system, the Stern–Volmer plot of NSC–HSA interaction is not purely linear. At lower concentration range of the quencher (NSC) the Stern–Volmer plot shows linearity but at higher concentration range of NSC the plot shows positive deviation from linearity (Fig. 4 inset). The absorption study has already supported

Fluorescence Intensity (a.u.)

have been calculated using Stern–Volmer equation, which again can be expressed in terms of bimolecular quenching rate constant and average life time of the fluorophore as shown in Eq. (6) [19].

2.25 1.50 0.75

1.5

0

40

80

[NSC]

1.0 0.5 0.0

350

400

450

Wavelength (nm) Fig. 4. Fluorescence quenching spectra of HSA (2 ml 10 ␮M) with increasing concentration of NSC (0–90 ␮M) in Tris–HCl/NaCl buffer (pH 7.4) at 298 K. Arrow shows the change in the emission intensity of HSA on increasing NSC concentration. Inset: Stern–Volmer plot for the quenching of HSA fluorescence by NSC; KSV = 2.42 × 104 M−1 at 298 K.

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Table 1 Stern–Volmer quenching constants, binding constants and binding sites for NSC–BSA interaction at different temperatures. pH

7.4 a b

T (K)

KSV (M−1 )

kq (M−1 s−1 )

293 298 303

1.07 × 10 1.03 × 104 0.93 × 104

1.74 × 10 1.67 × 1012 1.50 × 1012

4

12

Ra

Ka (M−1 )Double-logarithm equation

nb

Ra

0.99669 0.99688 0.99848

12.75 × 10 2.87 × 104 0.996 × 104

1.25 1.11 1.01

0.99922 0.99838 0.99766

R is the correlation coefficient. The binding site number (n) approximated to 1.

the formation of more than one ground-state complex for NSC–HSA system, so it can be predicted that at lower concentration of NSC the quenching could be initiated by a stable ground-state complex formation (1:1 type, like BSA). In accordance to the prediction, the Stern–Volmer plot at lower concentration range of NSC (0–32 ␮M) shows linearity and the KSV decreases with increasing temperature (KSV = 2.42 × 104 M−1 at 298 K) but at much higher concentration range of NSC (32–90 ␮M) the Stern–Volmer plot no longer remains linear and an upward bending toward the F0 /F axis indicates the formation of the second (1:2 type) NSC–HSA complex. Such 1:2 type complex formation for NSC–HSA system may have aroused due to the more flexible nature of HSA (than that of BSA) that have favored to form some loose binding interaction of NSC with HSA [19]. 3.3. Quantitative analyses of binding constants for BSA It has been already discussed that the static quenching mechanism is solely operative in NSC–BSA interaction. From the literature survey it has been found that for static quenching process, when small molecules are bound independently to a set of equivalent sites on a macromolecule, the equilibrium between free and bound molecules is given by double-logarithm equation (7) [30]. This equation has been employed in order to determine the binding constant (Ka ) and the number of binding sites (n) for NSC–BSA interaction. log

4

F − F  0 F

= log Ka + n log[Q ],

(7)

where F0 and F are the fluorescence intensities in the absence and the presence of quencher, and [Q] is the concentration of quencher (NSC). The plot of log [(F0 − F)/F] vs. log [Q] for the present system is linear (Fig. 5) and the values of Ka and n have been obtained from the intercept and slope, respectively. The calculated binding constants and binding sites for NSC–BSA at three different temperatures have been presented in Table 1. It has been found that the magnitude of the binding constants decrease with increasing temperature, which is again in accordance to the static quenching mechanism. At 298 K the number of binding sites has been found to be 1.11 and in the

3.4. Quantitative analyses of binding constants for HSA The upward bended nonlinear nature of the Stern–Volmer plot for the fluorescence quenching study of NSC–HSA interaction abstains us from calculating the Stern–Volmer quenching constant (KSV ) for the entire concentration range of the quencher (NSC). In order to calculate the effective quenching constant for the accessible fluorophores, which is also analogous to the associative binding constants for the quencher–acceptor system, three different analyses approaches have been considered. In these analyses the total concentration range of the quencher can be taken into account. The results obtained have been tabulated in Table 2 and they show good parity.

3.4.1. Modified Stern–Volmer analysis The nonlinearity in the Stern–Volmer plot (Fig. 4 inset) suggests that fluorophores can be quenched by both diffusion and complex formation with the same quencher. Moreover the magnitude of kq (1012 M−1 s−1 , where kq = KSV / 0 , and  0 for biopolymers is generally 10−8 s [31]) for NSC–HSA interaction is greater than the maximum diffusion collision quenching rate constant that has been obtained for a variety of quenchers with biopolymers (2.0 × 1010 M−1 s−1 ) [32]. Therefore it suggests that the fluorescence quenching process of HSA might be governed initially by a static quenching mechanism arising from a system formation rather than a dynamic quenching mechanism [22,32]. Assuming, the quenching process is predominantly static, the effective quenching constant or the binding constant can be analyzed from modified Stern–Volmer (MSV) analysis, Eq. (8) [33]. 1 F0 1 1 + = fa Ka [Q ] fa F

(8)

In the present case, F is the difference in fluorescence (i.e. F0 − F) in the absence (F0 ) and the presence (F) of the quencher (NSC) at concentration [Q], fa is the fraction of the fluorophore that is initially accessible to NSC and Ka is the effective quenching constant or the binding constant; by assuming that quenching of fluorescence intensity results due to the binding of NSC with HSA. The linearity in the modified Stern–Volmer plot (F0 /F0 − F vs. 1/[Q]) for the present system supports NSC–HSA ground state complex formation (Fig. 6). The binding constants at three different temperatures (Table 2) have been found from the ratio of the intercept to the slope of the MSV plot (Ka = 1.06 × 104 M−1 at 298 K). The fractional accessibility at 298 K has been found to be 1.68 and this implies that there might be more than one binding site in the protein environment for NSC.

0.0

log [(F0- F) / F ]

studied temperature range the n averages to 1.12 which suggests that there is only a single binding site on the protein.

-0.4

-0.8

-1.2 -5.1

-4.8

-4.5

log [NSC]

-4.2

Fig. 5. Double-logarithm plot determining binding constant and number of binding sites for NSC–BSA interaction (T = 298 K).

3.4.2. Lineweaver–Burk analysis This analysis is based on a double reciprocal plot from which one can easily obtain the binding constant for the interacting system.

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Table 2 Binding constants and binding sites for the interaction of NSC with HSA. pH

T (K)

Ka (M−1 ) Modified Stern–Volmer

Ra

Ka (M−1 ) Lineweaver–Burk

Ra

Ka (M−1 ) Scatchard

nb

Ra

7.4

293 298 303

0.579 × 104 1.06 × 104 1.51 × 104

0.99801 0.99834 0.99721

0.606 × 104 1.08 × 104 1.53 × 104

0.99791 0.99802 0.99753

0.882 × 104 1.31 × 104 1.38 × 104

1.90 1.43 1.35

−0.90706 −0.96574 −0.98152

a b

R is the correlation coefficient. The binding site number (n) approximated to 1.56.

The plot is a graphical representation of Lineweaver–Burk (LWB) equation (9) that is somewhat similar to the MSV-equation. 1 1 1 = , + F0 − F F0 F0 Ka [Q ]

(9)

here F0 and F are the fluorescence intensities in the absence and in the presence of the quencher (NSC) at concentration [Q]. Ka is the binding constant and is calculated from the ratio of the intercept to the slope of the straight line obtained from the plot of 1/(F0 − F) vs. 1/[Q]. This analysis provides binding constant for NSC–HSA interaction (Fig. 6 inset) and the magnitude is almost same as that obtained from modified Stern–Volmer analysis for three different temperatures (Table 2). 3.4.3. Scatchard analysis The method of analysis is based on Scatchard’s equation (10) from which we can calculate the association binding constant and number of binding sites for NSC–HSA interactive system by measuring the change in intrinsic fluorescence of the single tryptophan in HSA on successive addition of NSC. r = nKa − rKa , (10) Cf

6

3.5. Determination of thermodynamic parameters The binding constants calculated from double-logarithm equation (for BSA) and modified Stern–Volmer equation (for HSA) at three different temperatures have been used to obtain the van’t Hoff plot (ln Ka vs. 1/T, Fig. 8) for determination of the thermodynamic parameters for NSC–BSA or HSA interactions. The nature of the van’t Hoff plot for BSA significantly differs from that of HSA and the thermodynamic parameters (H◦ and S◦ ) for the two interactive systems differs in signs (Table 3) which again suggest that the forces involved in the NSC–BSA or HSA interactions are of different types. The four types of noncovalent interactions, e.g. van der Waals forces, hydrogen bonding, electrostatic and hydrophobic, that are responsible for binding of small molecules to proteins are mainly governed by the signs of the thermodynamic parameters [22]. Negative enthalpy (H◦ ) favors predominance of van der Waals forces and hydrogen bonding in the interactive system

0.4

18000

0.2 0.0

16000

0.1 1/[NSC]

r / Cf

F0 / (F0- F)

9

1 / (F-0 F)

where r (r = F/F0 ) is the moles of drug bound per mole of protein, Cf is the molar concentration of the free metal complex, n is number of binding sites and Ka is the association binding constant [34]. Unlike the MSV and LWB plots the Scatchard plot (r/Cf vs. r, Fig. 7) shows a negative slope as indicated by the sign in Eq. (10). The association binding constant at three different temperatures for NSC–HSA interaction is the slope obtained from the respective Scatchard plots. The magnitude of different Ka values are comparable with that of the effective quenching constant and binding constant obtained from MSV and LWB analyses (Table 2). The number of binding sites has been calculated from the ratio of the intercept to the slope obtained from the plot of r/Cf vs. r. It has been observed that on increasing temperature the number of binding sites decreases (Table 2) which again supports that NSC–HSA

binding interaction is governed by both compound formation and some diffusion mechanism. At 298 K the number of binding sites has been found to be 1.43 and for the studied temperature range the n averages to 1.56, which suggests that there are more than a single binding site (or shared site) on the protein. In contrary to Table 1, it is now clear from Table 2 that unlike BSA, for all three analyses, the magnitude of the binding constants of NSC–HSA interactions increases with temperature (in the range 293–303 K). Such trend can be justified only if some diffusion mechanism is taken into account. Such diffusion can only occur when some loose binding interactions are considered for NSC–HSA interaction. Thus it can be predicted that at low metal-complex concentration, 1:1 type ground state complex is formed between NSC and HSA but at higher metal-complex concentration HSA forms two types of complexes: one is due to normal binding like that of BSA and other is loose bound complex. Since the flexibility of HSA is more than that of BSA so such loose binding is possible for HSA. This kind of loose binding increases the mobility of NSC toward HSA with increasing temperatures.

3

14000

12000

0

0.03

0.06

0.09

0.12

1/[NSC] Fig. 6. Determination of associative binding constant for NSC–HSA interaction from modified Stern–Volmer plot. Inset: Lineweaver–Burk plot determining the binding constant for NSC–HSA interaction (T = 298 K).

10000 0.2

0.4

0.6

r Fig. 7. Scatchard plot determining association binding constant and number of binding sites for NSC–HSA interaction (T = 298 K).

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Table 3 Determination of thermodynamic parameters for NSC–BSA and NSC–HSA systems at different temperatures. Serum albumin

pH

BSA 7.4 HSA

T (K)

Ka a (M−1 )

293 298 303 293 298 303

12.75 × 10 2.87 × 104 0.996 × 104 0.579 × 104 1.06 × 104 1.51 × 104 4

ln Ka

G◦ (kJ mol−1 )

11.76 10.26 9.21 8.66 9.27 9.63

−29.45 −26.64 −23.84 −20.97 −22.38 −24.03

H◦ (kJ mol−1 )

S◦ (J mol−1 K−1 )

Rb

−193.91

−561.29

0.99898

77.58

336.51

−0.96569

a The Ka values considered for BSA are obtained from the double logarithm equation whereas Ka calculated from the modified Stern–Volmer analysis at different temperatures has been considered for HSA. b R is the correlation coefficient.

8.4 0.0033

0.0034 -1

1/T (K )

10

9

0.00332

0.00336

0.00340

-1

1/T (K ) Fig. 8. van’t Hoff plot of ln Ka vs. 1/T for the binding of the NSC with BSA. Inset: van’t Hoff plot for the binding interaction of NSC with HSA. For both the systems three different temperatures have been considered (293 K, 298 K and 303 K).

R06

(11)

R06 + r 6

R06 = 8.79 × 10−25 k2 −4 J()˚

(12)

In Eq. (12),  is the orientation factor of the donor and acceptor and for random orientation of donor and acceptor a value of 2/3 is assumed for 2 . The refractive index of the medium,  is equal to 1.34 for BSA and 1.36 for HSA. The value of quantum yield ˚ of BSA and HSA has been found to be 0.15 and 0.074, respectively [37]. J is the overlap integral for the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor and is given by Eq. (13):



J() =

FD ()εA ()4 d



FD ()d

(13)

,

where FD () is the corrected fluorescence intensity of BSA or HSA (donor) at wavelength  to  +  and εA () is the molar extinction coefficient of NSC (acceptor) at wavelength . The unit of εA () is normally taken to be M−1 cm−1 . The FRET results affirm the energy transfer between NSC and serum albumins (Table 4). The results indicate that the energy transfer from the serum albumins to NSC occur with high possibility as average distance between donor fluorophore and an acceptor fluorophore is 3.70 nm for BSA and 3.27 nm

(b)

0.010

0.008

(a)

0.8 0.010

0.4 0.0

0.008

1.0 1.2 1.4

r/R0

0.006

-1

11

9.1

F0

=

0.006

-1

lnKa

12

9.8

ln Ka

13

F

0.004

0.004

0.002

0.002

330

360

390

420

450

ε (M cm )

A variety of information that include precision in location of NSC in BSA or HSA, energy transfer processes and efficiency of energy transfer have been studied according to Förster’s theory of nonradiative energy transfer (FRET) [35]. According to the theory the rate of energy transfer that could occur through a direct electrodynamic interaction between primarily excited donor molecule (BSA or HSA) and its neighbor acceptor molecule (NSC) depends on: (i) the relative orientation of the donor and acceptor dipoles, (ii) the ability of the donor to produce fluorescence light; (iii) the extent of overlap of

E =1−

Efficiency

3.6. Energy transfer between NSC and serum albumins

the emission spectrum of the donor with the absorption spectrum of the acceptor and (iv) the distance between the donor and the acceptor and is desired to be lower than 8 nm [36]. An overlapping region in the wavelength range 310–422 nm has been obtained for the emission spectra of both BSA and HSA (1 × 10−5 M) with the absorption spectrum of NSC (1 × 10−5 M) and considered to examine FRET (Fig. 9). The efficiency of energy transfer (E) is not only related to the actual distance between the donor and the acceptor (r), but also to the critical energy transfer distance (R0 ) which is also known as the Förster distance where efficiency is just 50%. These parameters could be calculated using FRET equations (11) and (12).

Fuorescence Intensity (a.u)

whereas positive entropy (S◦ ) values have been encountered for the systems where ionic and hydrophobic interactions play a major role. For NSC–BSA interaction, negative changes in both enthalpy and entropy values have been observed but much greater negative magnitude of H◦ values compared to S◦ assures that binding reaction is driven by van der Waals forces and hydrogen bonding interactions and H◦ governs the spontaneity of NSC–BSA binding process reaction by resulting negative change in free energy values (G◦ = −26.64 kJ mol−1 at 298 K). The spontaneity of NSC–HSA interaction is almost comparable to NSC–BSA interaction with a bit lesser negative change in free energy values (G◦ = −22.38 kJ mol−1 at 298 K). Unlike NSC–BSA, the positive values of H◦ and S◦ have been calculated for NSC–HSA interaction. Here TS◦ is greater than H◦ which indicates that the binding of NSC with HSA is entropy driven process where ionic interactions as well as hydrophobic interactions are the key forces of association and TS◦ governs the spontaneity of the reaction. Moreover the trend in the changes of Ka and G◦ values from Table 3 indicate that the spontaneity of NSC–BSA binding interactions decreases whereas for NSC–HSA interactions spontaneity increases on increasing the temperature from 293 K to 303 K.

480

Wavelength (nm) Fig. 9. Spectral overlap of the emission spectrum of the donor (HSA: (a)) with the absorption spectrum of the acceptor (NSC: (b)). c (HSA) = c (NSC) = 1 × 10−5 M (T = 298 K). Inset shows the efficiency (E) vs. r/R0 plot.

A. Ray et al. / Spectrochimica Acta Part A 92 (2012) 164–174

171

Table 4 Tabulation of FRET data for NSC–BSA and NSC–HSA systems. System BSA HSA a b c d

Ea 0.111 0.107

Jb (M−1 cm3 ) −14

1.19 × 10 1.15 × 10−14

R0 c (nm)

rd (nm)

2.61 2.29

3.70 3.27

Efficiency of energy transfer. Spectral overlap integral. Critical energy transfer distance. Distance between the fluorophore and the ligand (NSC).

for HSA which is less than 8 nm and 5R0 < r < 1.5R0 [36]. Furthermore the values of r are higher than R0 in the present study indicating the presence of static quenching interaction between BSA or HSA and NSC [38]. 3.7. Circular dichroism studies CD measurements provide insights about folding and unfolding of serum albumins induced by the ligand binding interactions. For serum albumins (10 ␮M), changes in the CD spectra for the secondary and tertiary structures have been noted upon NSC binding (20 ␮M) but such changes are much greater for BSA than compared to that of HSA. It has been found that binding of NSC to BSA causes a reduction of ␣-helix of the protein secondary structure from 54.48% (free BSA) to 47.24% (NSC–BSA) whereas no detectable reduction of ␣-helix for HSA has been observed upon NSC binding (Figs. S1 and S2, provided as supplementary data) [39]. Such results suggest a partial protein unfolding for NSC–BSA binding interactions [40] but no significant conformational changes in the secondary and tertiary structures have been noted for HSA. 3.8. Docking results As the BSA structure is not available, it has been predicted from its sequence depending on the homology it shares with its human counterpart. The residues 29–605 of SP: P02769 (RefSeq) has been modeled. It shows 75.78% sequence similarity with 1E78:A. The model has a QMEAN4 [41] score of 0.661 on a scale of 0–1 and Zscore QMEAN of −1.56 (|Z-score| < 2); which makes it an acceptable structure. Errors are mainly in the peripheral regions (Fig. S3, provided as supplementary data). HSA has three structurally homologous domains (I–III), each of which are subdivided into subdomains A and B [42]. The binding pocket at domain IIA where lies the sole fluorophore, Trp214, of HSA has been reported to be one of the two primary drug binding sites of HSA. Extensive crystallographic studies identified additional drug binding sites across the molecule, many of which are likely to be the secondary binding pockets that get occupied at high ligand concentrations [43]. Docking results for HSA and BSA from three different software packages, are tabulated in Table 5. The NSC has been predicted to bind to the primary drug binding site (domain IIA) of HSA and also in the similar position of BSA (Figs. 10 and 11). The binding energies calculated by AutoDock 4 are very close to the experimentally obtained values (vide Table 3: experimental data). Bindings are mainly hydrophobic and van der Waals type of in nature. A considerably superficial binding has also been predicted in the domain IIB of HSA (Fig. 10). Although the binding at domain IIB has been predicted to be energetically as favorable as the binding to domain IIA, large portion of the ligand (NSC) remains exposed to the solvent (vide Table 5, exposed surface area of ligand) and due to the lack of hydrogen bonding or ionic interactions it must be mobile in nature. Change in per residue accessible surface area is represented in Fig. 12. The residues which are affected more are involved more in the interaction.

Fig. 10. (a) NSC docked conformation of HSA. NSC binds to the already reported primary drug binding site at domain IIA and it also binds to the domain IIB. (b) Close up view of the binding sites. Nearest distances of the ligand from the Trp214 are shown in Å.

3.9. Correlation among docking analyses and spectroscopic studies The docking results predict the formation of two types of complexes between NSC and HSA, one is through proper binding (like BSA) and the other is through loose binding. Moreover the surface area exposure for the second complex (at domain IIB) is larger than that of first one (at domain IIA), which makes it “mobile” in nature. The formation of two types of complexes is also evident from the absorption spectra of NSC with different concentrations of HSA (Fig. 2b). The isosbestic point actually establishes the equilibrium between the first and second type of complexes. Moreover the Stern–Volmer plot for NSC–HSA is nonlinear in nature, which predicts the formation of more than one type of complexes. In accordance to the above point the time-resolve fluorescence quenching studies cannot signify the dynamic quenching mechanism as the excited state life time of the fluorophore ( = 6.17 ns) remains unchanged in the presence of the quencher. Such  value again supports the formation of NSC–HSA ground state complexes. Further this kind of loose binding NSC–HSA interaction is supported by MSV and Scatchard plots, which estimates the binding sites of HSA for NSC is fractional and is greater than one (average value 1.56). It is also interesting to note that with temperature the value of binding constant for HSA increases which further supports the mobile nature of the NSC. 3.10. GuHCl-induced protein denaturation Guanidine hydrochloride (GuHCl) is a chaotrope like urea, which can also induce protein denaturation. Increase in concentration of GuHCl from 0 to 6 M leads to unfolding of HSA in at least three distinct steps: (i) 1 M GuHCl causes initial, reversible separation of domains I and II, (ii) irreversible unfolding of domain II was attained on the addition of 2 M GuHCl and (iii) finally the irreversible unfolding of domain I caused the total denaturation of HSA in the presence of 6 M GuHCl [44]. Moreover kinetic measurements on GuHCl induced unfolding of HSA had supported that unfolding

Fig. 11. (a) NSC docked conformation of BSA. Like HSA, NSC binds to the domain IIA of BSA. (b) Close up view of the binding site. Nearest distance of the ligand from the Trp237 is shown in Å.

172

A. Ray et al. / Spectrochimica Acta Part A 92 (2012) 164–174

Table 5 Summary of the protein–NSC docking results obtained from three different software packages. Binding sites

Binding parameters

AutoDock Vina

AutoDock 4

PatchDock

BSA domain IIA

G◦ (kJ mol−1 ) RMSDa (Å) Exposed surface area of ligand (Å2 ) Nearest distance of NSC from Trp237b (Å) Nearest distance of Ni from Trp237 (Å)

−33.91 0.000 28.84 3.3 5.5

−25.08 5.48 42.53 3.6 6.2

−38.16 4.59 15.59 3.7 5.7

HSA domain IIA

G◦ (kJ mol−1 ) RMSD (Å) Exposed surface area of ligand (Å2 ) Nearest distance of NSC from Trp214 (Å) Nearest distance of Ni from Trp214 (Å)

−33.08 0.000 48.85 3.4 4.9

−26.46 4.62 14.85 3.3 5.8

−24.89 4.65 43.68 2.9 5.6

HSA domain IIB

G◦ (kJ mol−1 ) RMSD (Å) Exposed surface area of ligand (Å2 ) Nearest distance of NSC from Trp214 (Å) Nearest distance of Ni from Trp214 (Å)

−35.169 0.000 107.08 7.1 11.6

−26.628 5.622 105.62 9.9 12.1

−44.20 5.677 114.35 6.7 7.8

a b

Root mean square deviation (RMSD) calculated with reference to the least energy structure predicted by AutoDock Vina. Nearest distance from Trp means (in all cases) the nearest distance from Trp aromatic rings.

of domain II occurred in a stepwise manner which thereby suggest that GuHCl mediated unfolding of HSA occurs in multiple steps [45,46]. The binding interactions of NSC with the serum albumins near domain II (as proposed by docking studies) have inspired us to investigate, whether NSC binding to BSA/HSA shows any stabilizing or destabilizing effect on the protein unfolding at domain II by GuHCl in comparison to that of free serum albumins. To study the GuHCl induced protein unfolding, steady-state tryptophan fluorescence (at 350 nm) of the free (2 ml, 10 ␮M protein) and the bound forms (2 ml, 10 ␮M protein and 30 ␮M NSC) of BSA or HSA in the presence of the various concentrations of the denaturant (GuHCl = 0–8 M) have been monitored. At the beginning when GuHCl concentration is very low, the fluorescence intensity of the protein does not decrease significantly then a sudden large change in intensity has been recorded from 0.8 to 2.1 M GuHCl for the free serum proteins, which is in accordance to the change observed for the irreversible unfolding of domain II of HSA [44]. Similar changes in spectra have been observed for the bound serum proteins but here the concentration of GuHCl responsible for large change in fluorescence intensity differs from that of the free form for only HSA and no significant change has been observed for BSA. This clearly indicates that unlike BSA, NSC binding with HSA has either stabilized or destabilized the protein. In order to specify the GuHCl

Table 6 Tabulation of GuHCl-[den]1/2 for the free and bound forms of BSA and HSA. System

BSA

NSC–BSA

HSA

NSC–HSA

[den]1/2 in (M)

1.72

1.74

1.61

2.16

concentration required to unfold the free and bound form of protein a transition curve using relative fluorescence intensity (i.e. fluorescence intensity of the proteins in the presence of GuHCl (F) to that in its absence (F0 )) has been plotted against concentration of GuHCl for both BSA and HSA system (Fig. 13). The transition curves are sigmoidal in nature with a sudden shoot down in the F/F0 values within a very small concentration range near 2 M GuHCl indicating the formation of unfolded protein intermediate [47]. The GuHCl concentration corresponding to the midpoints of these transition curves has been reported for the free and bound form of the serum proteins (Table 6). Since this concentration indicated as [den]1/2 signifies the intermediate state where one-half of the native state of protein has been denatured [48]. The value of [den]1/2-NSC–HSA is greater than [den]1/2-HSA and [den]1/2-NSC–BSA is almost equal to [den]1/2-BSA . Such results affirm that HSA has been significantly stabilized upon NSC binding whereas no such stabilization effect has been obtained for BSA.

Fig. 12. Per residue change in the accessible surface area plot for HSA (a) and BSA (b). Higher change indicates more involvement in ligand binding. Peaks are tagged with residue code and their position in peptide chain. ––, at domain IIA of HSA and BSA; –䊉–, at domain IIB of HSA.

A. Ray et al. / Spectrochimica Acta Part A 92 (2012) 164–174

1.0

(a)

(b)

1.0

0.8

0.8

F / F0

F / F0

173

0.6

0.6

0.4 0

2

4

6

8

[GuHCl]

0.4

0

2

4

6

8

[GuHCl]

Fig. 13. Plot of (F/F0 ) of the proteins ((a) = BSA and (b) = HSA) against concentration of GuHCl in the absence (black square box) and in the presence (red triangles) of NSC. ex is 295 nm and em is 350 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

4. Conclusion

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In conclusion the spectroscopic results of the paper can be summarized as depending upon the conformation of NSC facilitated by the unequal flexibility of the two serum albumins, it has an ability to recognize the two serum albumins. NSC shows a potential selectivity/versatility in its binding modes with BSA and HSA. It is very sensitive to bind at domain II of two similar types of serum albumins (BSA, HSA). Such selective behavior of NSC can be investigated further, specifying its functional role for development in the fields of metallopharmaceutics in future.

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Supplementary information CD spectra supporting conformational changes acquired by serum albumins on NSC binding (Figs. S1 and S2) have been provided. It also contains the structural model of BSA with an acceptable QMEAN4 scores (Fig. S3). This section incorporates the supplementary data from the experimental discussion such as: (i) syntheses of the Schiff base ligand and the complex (ii) structural aspects of NSC (iii) elaboration of the docking experiment. From the result and discussion part details of the characterization of NSC by UV/vis spectroscopy and the interaction of NSC with serum albumins by absorption studies have been provided as supporting information.

Acknowledgments Dr. A. Ray would like to acknowledge the financial assistance provided by SINP to carry out this study as her post doctoral work and B. Koley Seth would like to thank UGC, New Delhi for financial support. We would also thank Prof. Soumen Basak and Mr. Abhijit Shome for guiding the CD experiments and Mr. Ajay Das for TCSPC measurements (Chemical Science Division, SINP, Kolkata). Last but not the least the authors acknowledge the financial support obtained from Chemical and Biophysical Approaches for Understanding Natural Processes (CBAUNP) and Molecular Mechanism of Diseases and Drug Action (MMDDA) projects of SINP.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.saa.2012.02.060.

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