Quenching, binding and conformational aspects of interactions

Transcription

1 Quenching, binding and conformational aspects of interactions of antidepressants with bovine serum albumin A B S T R A C T The interaction of antidepressants, BZP and NTPH with BSA was investigated based on Trp fluorescence quenching method. The characteristics of binding were described by fluorescence, UV-vis absorption, FTIR and CD studies. Quenching constants were determined using the Stern-Volmer equation. The values of K for BZP-BSA and NTPH-BSA systems were observed to be 3.73 x 0 3 and.46 x 0 4 M - at 298 K, respectively, while the values of n for both BZP-BSA and NTPH-BSA were observed to be close to unity at 298 K. The distance, r between the donor (BSA) and acceptor (BZP/NTPH) was calculated based on the Förster s theory of non-radiation energy transfer and found to be 2.73 and 2.5 nm for BZP-BSA and NTPH-BSA, respectively. The results of CD and FTIR experiments indicated the conformational changes in secondary structure of protein upon interaction with both drugs. Based on the results of displacement experiments with site probes, it was proposed that both BZP and NTPH bound to site I (subdomain IIA) of BSA. Further, the effects of some common ions have been investigated on drug-protein interactions. 75

2 Buzepide methiodide (BZP) Chemical name diphenylbutyl)hexahydro-- H-azepinium iodide -(4-amino-4-oxo-3,3- methyl- Structure Molecular formula C 23 H IN 2 O Molecular weight Description Solubility Category Pale yellow coloured solid Soluble in water and in common organic solvents Anti-depressant 76

3 Nortryptiline hydrochloride (NTPH) Chemical name 3-(0,-dihydro-5H-dibenzo[a,d] cyclohepten-5-ylidene)- N-methyl-- propanamine Structure Molecular formula C 9 H 22 ClN Molecular weight Description Solubility Category White coloured solid Soluble in water and in common organic solvents Anti-depressant 77

4 INTRODUCTION BSA transports a variety of endogenous and exogenous substances in the body and plays an important role in the distribution and deposition of these substances []. It is widely accepted in pharmaceutical industry that the overall distribution, metabolism and efficacy of many drugs depend on their binding affinity to serum albumin [2]. In addition, many promising new drugs are rendered ineffective because of their unusually high affinity for this abundant protein [3]. Different methods have been developed to evaluate drug-protein interactions viz., equilibrium dialysis [4], ultrafiltration [5], chromatographic methods [6] and capillary electrophoresis [7]. Though these methods provide useful information on binding, they are constrained by one or more of the factors like Donnan effect, sieve effect, non specific adsorption, leakage through membranes, long analysis time and large sample size requirements [8]. However, the fluorescence spectroscopy is a sensitive technique for studying the drug-protein interaction [9]. This technique coupled with CD and FTIR can provide better way of understanding the binding characteristics and conformational aspects of protein. BZP, an anti-depressant [0], is used in the treatment of functional intestinal disorders in combination with haloperidol []. In a meta-analysis of Randomized Clinical Trials (RCT), it was concluded that the antidepressants might be effective in reducing Irritable Bowel Syndrome (IBS) symptoms in about one-third of patients [2]. Since BZP is an antidepressant, attempts have been made to assess its effect to treat the IBS [3]. NTPH is a tricyclic anti-depressant and is widely used in the treatment of unipolar depression [4, 5]. Besides this, there is growing evidence of its efficacy for smoking cessation pharmacological therapy [6]. It has been widely employed in the treatment of major depressive disorders, pain of varying ethiology, pathological crying or laughing [7]. The objective of the proposed work is to determine the affinity of BZP and NTPH towards BSA and to determine the binding and thermodynamic characteristics of interaction by multi-spectroscopic methods. 78

5 EXPERIMENTAL Reagents Stock solutions of BSA (250 μm) and drugs (000 μm) were prepared in 0. M phosphate buffer of ph 7.4 containing 0.5 M NaCl and milli pore water was used throughout. Apparatus The details of spectroscopic techniques employed in the present study are given on page number 22. Procedures BZP/NTPH-BSA interactions Fluorescence spectra were recorded at different temperatures (288, 298 and 308 K) in the range of / nm upon excitation at 296 nm for BZP-BSA/NTPH-BSA by keeping the concentration of BSA fixed (5 μm) and varying the concentration of BZP/NTPH (0 to 20 μm). Displacement experiments Displacement experiments were performed using different site probes viz., warfarin, ibuprofen and digitoxin for site I, II and III, respectively by keeping the concentration of BSA (5 µm) and the probe (2.5 µm) constant. The fluorescence quenching titration was carried out as before to determine the binding constant of s in the presence of above site probes. FRET studies The distance of separation and the efficiency of energy transfer were calculated based on the FRET theory by overlapping the absorption spectrum of BZP/NTPH separately with the emission spectrum of BSA at 298 K. 79

6 Circular dichroism studies CD spectra of BSA (0 μm) were recorded in the absence and presence of BZP/NTPH (0-30 μm) in the range of nm at 298 K. FTIR spectra The FTIR spectra of free and bound form of BSA were obtained at 298 K by employing the procedure mentioned on page number 30. UV-vis absorption measurements Absorption spectra of BSA (5 μm) in the absence and presence of BZP/NTPH (0-5 μm) were recorded in the range of nm for BZP-BSA and nm for NTPH-BSA systems, respectively at 298 K. Effects of some common ions The fluorescence spectra of BZP/NTPH (0-20 μm)-bsa (5 μm) were recorded in the presence and absence of common ions (2.5 μm) viz., Fe 3+, Co 2+, K +, Ca 2+, Mn 2+, Ni 2+, Zn 2+ and Cu 2+ in the range of nm upon excitation at 296 nm. RESULTS AND DISCUSSION Intrinsic fluorescence studies The measurement of intrinsic fluorescence of protein is a sensitive tool to investigate the quenching mechanism. Fig. shows the emission spectra of BSA in the presence of different amounts of BZP (Fig. a) and NTPH (Fig. b). BZP/NTPH reduced the fluorescence intensity of BSA and induced an apparent blue shift in fluorescence band maximum. The blue shift observed indicated that the conformational changes induced by the interaction led to a change in the microenvironment around the Trp residues of BSA. These observations also revealed the strong interaction between BZP/NTPH and BSA, and also a radiationless energy transfer between BZP/NTPH and BSA [8]. 80

7 In order to know the type of quenching mechanism, we have carried out the fluorescence studies at different temperatures. The fluorescence quenching data were subjected to Stern-Volmer analysis using the equation [9] shown on page number 32 and calculated the K SV values from Stern-Volmer plots (Fig. 2a and 2b). The corresponding values are given in Table along with K q values. The K q values for BZP-BSA and NTPH-BSA systems were noticed to be higher than the reported value (of the order of 0 0 ). The decreased K SV values with increase in temperature revealed the presence of static quenching mechanism. This also indicated that the static quenching was caused by the formation of a non-fluorescent ground state fluorophore-quencher complex. The static quenching mechanism was further confirmed by analyzing the fluorescence data using the modified Stern-Volmer equation shown on page number 33. The plot of [F 0 /(F 0 -F)] vs /[Q] (Figure not shown) yielded /f a as the intercept on y axis and /(f a K a ) as the slope. The values of f a were noticed to be.24 and.26, respectively showing that only 80.65% and 79.37% of the initial fluorescence of BSA were accessible for BZP and NTPH. Binding characteristics We have calculated the values of K and n using the equation 4, recorded on page number 33 [20] from the plot of log [(F 0 -F)/F] vs log [Q] (Figure not shown). The corresponding results are presented in Table. It is evident from the Table that the binding constant values decreased with increase in temperature resulting in the reduction of the stability of drug-bsa complex at higher temperatures. The values of n close to unity for both BZP-BSA and NTPH-BSA systems indicated that the binding ratio of drug to protein was :. Evaluation of thermodynamic parameters The ligand binding process during ligand-protein complex formation is mainly governed by weak and non-covalent forces. In order to characterize the acting forces between the drug and BSA, the thermodynamic parameters were calculated from the van t Hoff s plot (Fig. 3). The Gibb s free energy change, 8

8 ΔG 0 was calculated using the equation 6, given on page number 34. The negative ΔG 0 values for both BZP-BSA and NTPH-BSA systems suggested the spontaneity of the binding process. For BZP-BSA system, the value of ΔH 0 was found to be negative ( kj mol - ) while ΔS 0 was noticed to be positive (53.6 J mol - K - ). Hence, it was proposed that the formation of BZP-BSA complex was a spontaneous exothermic reaction accompanied by a positive entropy change. In case of NTPH-BSA system, the values of ΔH 0 and ΔS 0 were noticed to be kj mol - and -0.6 J mol - K -. The positive ΔS 0 for BZP-BSA indicated that the hydrophobic interaction played a major role in the binding process [2] while the negative value of it for NTPH-BSA revealed the presence of hydrogen bond and van der Waals forces in the interaction. Displacement experiments with site markers BSA is composed of three domains viz., I, II and III which confer to the protein a heart shaped molecule form [22]. Each domain is constituted by two subdomains. The subdomains share a number of common features such as the hydrophobic face, the cluster of basic amino acid residues and proline residues at the tips of the long loops. However, it is known that each subdomain is also unique and probably exhibits a certain degree of binding specificity [22-24]. In order to determine the specificity of BZP/NTPH binding, displacement experiments were performed with different site competitors like warfarin, ibuprofen and digitoxin. For this, the fluorescence intensity of probe-bsa was recorded in the presence of different amounts of drug, separately and the binding constant, K was evaluated. The corresponding values of K are given in Table 2. These values were found to be decreased significantly in the presence of warfarin. This indicated that there was competition between warfarin and BZP/NTPH for site I of sub-domain IIA in BSA. Further, no significant effect on the binding of BZP/NTPH to BSA was observed in the presence of both ibuprofen and digitoxin thereby indicating that the two site probes did not share a common binding site on BSA. Hence, it was concluded that the site I located 82

9 in subdomain II A near Trp-24 was the main binding site for BZP/NTPH in BSA. Energy transfer between BSA and BZP/NTPH Information concerning the molecular details of donor-acceptor pair can be obtained from non-radiative energy transfer [25]. A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore (in close proximity, typically < 0 nm) through non-radiative dipole-dipole coupling. This mechanism is termed as "FRET". The dependence of energy transfer rate on interaction distance has been widely used to measure the distance between the donor and acceptor. For this, we have recorded the absorption spectrum of the drug and overlapped it with emission spectrum of the protein and the same is shown in Fig. 4a and 4b. We obtained that R 0 = 2.39 nm; J =.466 x 0-4 cm 3 L mol - ; E = and r = 2.73 nm for BZP-BSA system while for NTPH-BSA, R 0 = 2.47 nm; J =.506 x 0-4 cm 3 L mol - ; E = and r = 2.5 nm. The average distance, r < 8 nm [26] between the bound drug and Trp(s) indicated a non-radiative energy transfer mechanism for quenching [9]. The higher value of r compared to that of R 0 revealed the presence of static quenching mechanism [23]. The shorter distance between the bound ligand and Trp residues in the proposed study suggested that there was significant interaction between the drug and protein. Conformational studies. CD Studies The primary structure of BSA is constituted by a single chain of about 582 amino acid residues [27]. Its secondary structure is formed by 67% of α-helix of six turns and 7 disulphide bridges [28]. When small molecules bind to BSA, the intramolecular forces responsible for maintaining the secondary structure can be altered, producing conformational changes in the protein [29]. CD is one of the strong and sensitive spectroscopic techniques to explore the 83

10 various aspects of protein structure and also to understand the interaction between protein and small molecules. The far-uv region ( nm) can be used to investigate the secondary structure content of the protein. The main absorbing groups in this region are peptide bonds. Because of the asymmetric protein environment and consequently its inherent chirality, the induced asymmetry of some achiral ligand molecules was observed during their reversible binding interactions [30]. The CD spectra (Fig. 5a and 5b) of BSA exhibited two negative bands at 208 and 222 nm, characteristic of α-helical structure of protein. The band intensity at 208 and 222 nm decreased regularly with the addition of BZP/NTPH. The CD results are expressed in terms of MRE by employing equation 0 shown on page number 37. The α-helical contents of free and bound BSA were calculated from MRE value at 208 nm using the equation given on page number 37. It was noticed that the α-helicity values decreased from % in free BSA to % in BZP-BSA and to % in NTPH-BSA thereby revealing the changes in secondary structure of protein upon interaction. Further, the addition of BZP/NTPH to protein led to a significant decrease in intensity for both negative bands. However, the shape of peaks and the peak maximum remained almost same. This indicated that the BSA was predominantly of α-helix nature even after binding to the drug [3]. 2. FTIR studies In order to get more information on the binding of the drug to BSA and on structural changes upon their interaction, FTIR spectra were recorded. The FTIR spectra of drug free and drug bound BSA with its difference spectrum are given in Fig. 6a and 6b, respectively. Hydrogen bonding and the coupling between transition dipoles are amongst the most important factors governing conformational sensitivity of amide bands. It was noticed that the peak position of amide I band was shifted from (in free BSA) to cm - (in BZP-BSA complex) and from (in free BSA) to 84

11 648.5 cm - (in NTPH-BSA complex). The amide II band was also shifted from (in free BSA) to cm - (in BZP-BSA complex) and from (in free BSA) to cm - (in NTPH-BSA complex). These results indicated that the drug has interacted with BSA and resulted in conformational changes of secondary structure of BSA [32]. 3. UV-vis absorption studies In the present study, we have recorded the UV absorption spectra of BZP, NTPH, BSA and their complexes (Fig. 7a and 7b). It was evident from the figure that the UV absorption intensity of BSA increased regularly with the variation of the concentration of BZP/NTPH. Moreover, the maximum peak positions of both BZP-BSA and NTPH-BSA systems were observed to be shifted slightly towards higher wavelength region. This change in λ max indicated the change in polarity around the Trp residue and the change in peptide strand of BSA molecule and hence the change in hydrophobicity [33]. This indicated that the binding between BZP/NTPH and protein molecule has resulted in conformational changes [34, 35]. Effects of cations Trace metal ions play an important structural and functional role in many proteins. So, the effects of some metal salt solutions viz., FeCl 3, NiCl 2, CoCl 2, CaCl 2, MnCl 2, KCl, CuCl 2 and ZnCl 2 on the binding of BZP/NTPH to BSA were investigated in the present study. The binding constants of BZP-BSA and NTPH-BSA systems in the presence of above cations were evaluated and the corresponding results are shown in Table 3. The binding constants of BZP-BSA and NTPH-BSA systems were found to be decreased to various degrees in the presence of above ions. This was probably attributed to conformational changes in the vicinity of the binding site. Further, the decrease in binding constant in the presence of above metal ions has shorten the storage time of drug in blood plasma and hence more amount of free drug would be available in plasma and might be excreted quickly [36]. This might 85

12 lead to the need for more doses of BZP/NTPH to achieve the desired therapeutic effect in the presence of above metal ions. CONCLUSIONS In the present work, we have studied the mechanism of interaction of two antidepressant drugs with BSA by employing different spectroscopic techniques. These studies showed that both BZP and NTPH quenched the intrinsic fluorescence of BSA through static quenching mode. The experimental results also indicated that both BZP and NTPH bound to BSA at site I (subdomain IIA). Secondary structure of BSA was found to be perturbed in the presence of the drug. 86

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15 Intensity Intensity Chapter a wavelength(nm) 500 b Wavelength(nm) Fig.. Fluorescence spectra of BSA in the presence of BZP (a) and NTPH (b). Concentration of BSA was kept constant (5 μm) while that of BZP/NTPH was maintained at () 0, (2) 2.5, (3) 5, (4) 7.5, (5) 0, (6) 2.5, (7) 5, (8) 7.5 and (9) 20 μm. 89

16 Fo/F Fo/F Chapter 4.5 a [Q] µm.6.4 b [Q] µm Fig. 2. The Stern-Volmer curves for BZP-BSA (a) and NTPH-BSA (b) at () 288, (2) 298 and (3) 308 K. 90

17 Log K Chapter b a /T Fig. 3. van t Hoff s plot for BZP-BSA (a) and NTPH-BSA (b). 9

18 Intensity Intensity Absorbance Absorbance Chapter a Wavelength, nm b Wavelength, nm Fig. 4. (a): Overlap of the emission spectrum of BSA () with the absorption spectrum of BZP (2); (b): Overlap of the emission spectrum of BSA () with the absorption spectrum of NTPH (2). 92

19 MRE [θ] MRE Chapter a Wavelength (nm) b Wavelength (nm) Fig. 5. CD spectra of the BZP-BSA (a) and NTPH-BSA (b). The concentration of BSA was fixed at 0 μm (). In BZP-BSA system, the concentration of BZP was maintained at 0 (2) and 30 μm (3) and in NTPH-BSA system, the NTPH concentration was kept at 0 (2) and 30 μm (3). 93

20 Absorbance Absorbance Chapter 4 a Wavenumber (cm - ) b Wavenumber (cm - ) Fig. 6. FTIR spectra and difference spectra of BSA. () FTIR spectrum of free BSA (subtracting the spectrum of buffer from that of BSA) and (2) FTIR difference spectrum of BSA (subtracting the spectrum of the drug-free form from that of drug-bsa bound form). BZP-BSA (a) and NTPH-BSA (b) systems. 94

21 Absorbance Absorbance Chapter a X Wavelength (nm) 0.5 b x Wavelength (nm) Fig. 7. Absorption spectra of (a) BSA, BZP and BZP-BSA systems and (b) BSA, NTPH and NTPH-BSA systems. Concentration of BSA was fixed at 5 μm () while that of BZP/NTPH in the system was maintained at 2.5 (2), 5 (3), 7.5 (4), 0 (5), 2.5 (6), and 5 μm (7). Concentration of 2.5 μm (x) was used for BZP/NTPH only. 95

22 Table. Stern-Volmer quenching constant, binding sites and binding constant values of drug-bsa systems at different temperatures. Temperature (K) K SV (L mol - ) K q (L mol - s - ) K (M - ) n BZP-BSA system x x x x x x x x x NTPH-BSA system x x x x x 0.46 x x x 0.09 x Table 2. Comparison of binding constants of BZP/NTPH-BSA before and after the addition of the site probe at 298 K. System Binding constant for BZP-BSA, M - Binding constant for NTPH-BSA, M - without site probe 3.73 x x 0 4 with warfarin (site I) 2.88 x x 0 2 with ibuprofen (site II) 3.80 x x 0 4 with digitoxin (site III) 3.72 x x

23 Table 3. Effects of common ions on binding constant of BZP-BSA and NTPH-BSA. System Binding constant for BZP-BSA, M - Binding constant for NTPH-BSA, M - In the absence of cation 3.73 x x 0 4 K x x 0 2 Ni 2+.3 x x 0 2 Co x x 0 2 Cu x x 0 2 Fe x x 0 2 Zn x x 0 2 Ca x x 0 3 Mn x x