Molecular Aspects of the Interaction of Iminium and Alkanolamine

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Molecular Aspects on the Interaction of Iminium and Alkanolamine Forms of the Anticancer Alkaloid Chelerythrine with Plasma Protein Bovine Serum Albumin Sutanwi Bhuiya, Ankur Bikash Pradhan, Lucy Haque, and Suman Das J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b07818 • Publication Date (Web): 10 Dec 2015 Downloaded from http://pubs.acs.org on December 10, 2015

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The Journal of Physical Chemistry

Molecular Aspects on the Interaction of Iminium and Alkanolamine Forms of the Anticancer Alkaloid Chelerythrine with Plasma Protein Bovine Serum Albumin

Sutanwi Bhuiya§, Ankur Bikash Pradhan§, Lucy Haque and Suman Das*

Department of Chemistry Jadavpur University Raja S. C. Mullick Road, Jadavpur Kolkata 700 032 India

*To whom all correspondence should be addressed. Tel.: +91 94 3437 3164, +91033 2457 2349 Fax: +91 33 2414 6266 E-mail: [email protected]; [email protected] §

These authors contributed equally to this work.

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ABSTRACT The interaction between a quaternary benzophenanthridine alkaloid chelerythrine (herein after, CHL) and bovine serum albumin (herein after, BSA) was probed by employing various spectroscopic tools and Isothermal Titration Calorimetry (ITC). Fluorescence studies revealed that the binding affinity of the alkanolamine form of the CHL is higher compared to the iminium counterpart. This was further established by fluorescence polarization anisotropy measurement and ITC. Fluorescence quenching study along with time resolved fluorescence measurements establish that both forms of CHL quenched the fluorescence intensity of BSA through the mechanism of static quenching. Site selective binding and molecular modeling studies revealed that the alkaloid binds predominantly in the BSA sub domain IIA by electrostatic and hydrophobic forces. From Forster resonance energy transfer (FRET) studies, the average distances between the protein donor and the alkaloid acceptor were found to be 2.71 and 2.30 nm between Tryptophan (Trp) 212 (donor) and iminium and alkanolamine forms (acceptor) respectively. Circular dichroism (CD) study demonstrated that the α-helical organization of the protein reduces due to binding with CHL along with an increase in the coiled structure. This is indicative of a small but definitive partial unfolding of the protein. Thermodynamic parameters obtained from ITC experiments revealed that the interaction is favoured by negative enthalpy change and positive entropy change. KEYWORDS: Benzophenanthridine alkaloid, BSA-alkaloid interaction, site selective binding, Isothermal titration calorimetry, Forster resonance energy transfer.


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INTRODUCTION Natural products have long been of interest to researchers not only for their high abundance and low toxicity but also for their diverse range of pharmacological effects. Alkaloids, a class of naturally occurring plant secondary metabolites, are familiar for their versatile and essential physiological effects on both human and animal cell types. Due to its wide-spread biological activity, it often serves as an active ingredient in medications. Among these alkaloids, benzophenanthridine is a class of specific isoquinoline compounds that occurs only in higher plants and show a wide spectrum of non-specific biological activities as well as multiple pharmacological properties. The aspect that makes this class of alkaloid exceptional from others is the existence of characteristic equilibrium between the charged iminium and the neutral alkanolamine form depending on pH of the medium.1-4 Sanguinarine is the most extensively studied alkaloid of this group that exhibits various biological effects. Nucleic acid and protein binding of sanguinarine have been exploited widely.5-8 Chelerythrine

(C21H18NO4)

(1,2-dimethoxy-12-methyl[1,3]benzo-dioxolo[5,6-

c]phenanthridin-12-ium, herein after CHL, Figure 1), another important quaternary benzophenanthridine alkaloid, has got the spotlight of research interest because of its extensive and distinct physiological effects mainly antimicrobial, antifungal and antiinflammatory which arises due to its interaction with varieties of proteins and DNA.9 It has a selective inhibitory effect against the cell permeable enzyme protein Kinase C (PKC) in comparison

with

tyrosine

protein

kinase,

cAMP-dependent

protein

kinase

Figure 1. Chemical structure of iminium and alkanolamine forms of CHL. 3 ACS Paragon Plus Environment

and


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Ca2+/calmodulin-dependent protein kinase (IC50=0.66 µM in a permeabilized cell system) where it remains reluctant from its role as an inhibitor.10 CHL effectively inhibits the function of BcL-XL proteins (IC50=1.5 µM) and H9c2 cardiomyoblastoma cells resulting apoptosis of the affected cells.11,12 It is established that presence of CHL causes inhibition to the respiration in yeast affecting the mitochondria.13 Keeping in view of the aforesaid cytotoxic nature of CHL, nowadays it is in a way to grab the attention in cancer therapy research.14-16 Structurally, CHL is similar with sanguinarine having a charged iminium form (CHL (I)) and neutral alkanolamine (pseudobase, CHL (A)) form depending on pH with a pKa of 8.58 as revealed from spectrophotometric studies.3 Recently it has been reported that CHL binds strongly to nucleic acids through intercalation.3,4,17 However the study on the interaction of CHL with protein is scanty. A detail insight into the protein-CHL interaction may bring new information which would be helpful in exploring the benzophenanthridine group of alkaloids as effective drugs. Binding of different drugs to plasma proteins such as serum albumin is a field of interest in biomedical and pharmaceutical sciences and also helpful in drug discovery since strong binding between the drug and albumin decreases the bioavailability increasing the half life of the drug in vivo.13 Serum albumin has been most extensively studied being most abundant multifunctional protein in blood plasma with a concentration of 50 g/L. It is the carrier of a number of exogenous and endogenous compounds to blood including fatty acids, metals, amino acids, steroids etc as well as helps in transport of different drugs and ligands in

vivo.18-20 Vespalec and his group have reported a detail study about the interaction of sanguinarine and CHL with the mercapto group bearing molecules.21,22 Their studies revealed that only the pseudobase form of CHL i.e. CHL (A) interacts with the mercapto group of albumins. Hydrophobic as well as the electrostatic contributions have been shown to be the driving force for the binding of CHL (A) with the positively charged sites in the protein.


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Bovine serum albumin (herein after, BSA) is one of the most extensively studied member of this protein family particularly because of its repeating pattern of disulphide linkages and structural homology of about 76% with Human serum albumin.23 It is a heart shaped globular protein with molecular weight of 66.4 kDa. The structural features of BSA illustrate that it is composed of 583 amino acid residues in a single polypeptide chain and contains three structurally similar domains (I, II and III). Each of which is further divided into two sub-domains (A and B) and these sub-domains are cross linked through 17 pairs of disulphide bridges resulting nine loops as evidenced from X-ray crystallography.20, 23-25 BSA is sensitive towards intrinsic fluorescence due to presence of two Tryptophan (Trp) residues, Trp 134 and Trp 212.25 Trp 134 is present at the surface of the molecule in the first domain whereas Trp 212 is underlied within a hydrophobic (non-polar) binding pocket present at the second domain of BSA.26 Therefore, fluorescence can be considered as a technique for measuring binding affinities. In the present study we performed a detail investigation on the binding of CHL (both iminium and alkanolamine forms) to BSA using different spectroscopic techniques namely UV-Vis absorption spectrophotometry, fluorimetry along with calorimetric study. We have also carried out circular dichroism (CD) study to find out the consequences of conformational changes upon binding of CHL to the protein. Two site marker fluorescence probes warfarin and ibuprofen were used for monitoring site I and site II, respectively of BSA.27-29 Moreover, molecular modeling study was exploited to find out the most probable binding site of CHL to BSA. MATERIALS AND EXPERIMENTAL METHODS Materials. CHL, BSA, warfarin and ibuprofen were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Since no impurities were found, they were used without further purification. All buffer salts used were of analytical grade.



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Experimental Solutions. Concentration of CHL was determined by UV absorbance study using known molar absorption coefficient value of 37060 M-1cm-1 at 316 nm.4 Fresh solution of CHL was prepared everyday and the prepared solutions were kept in dark to prevent any light induced change. Concentration of BSA solution was calculated by using a molar absorption coefficient value of 43824 M-1cm-1 at 280 nm.8 All the experiments were performed using 10 mM citrate-phosphate (CP) buffer of pH 6.0 and 10 mM carbonate-bicarbonate (CB) buffer of pH 10.2. CP buffer of pH 6.0 was used to perform the experiments with CHL (I) and CB buffer of pH 10.2 was used to conduct the experiments with CHL (A). Under our buffer conditions, CHL remained almost 100% in the iminium and alkanolamine form in pH 6.0 and in pH 10.2 respectively.3 All the buffer solutions were prepared in quartz-distilled deionized water from a Milli-Q source (Millipore, USA) and were filtered through millipore filters of 0.45 µm pore size before use to eliminate the impurities. Methods UV-Vis Spectroscopy. All the UV-Vis absorbance spectra were recorded on a Shimadzu (model UV-1800) spectrophotometer (Shimadzu Corporation, Japan) and during the study matched quartz cuvettes used were of 1 cm path-length. In the meantime, temperatures of the sample and reference

cuvettes

were

maintained

by

a

thermoprogrammer

(attached

to

the

spectrophotometer) by peltier effect. Spectrophotometric titrations were performed keeping CHL (I) concentration fixed at 3.6 µM with increasing the concentration of BSA until saturation (125.6 µM) was reached. Whereas the protein concentration was varied within the range 0-30.2 µM during the titration of fixed concentration of 3.6 µM of CHL (A) to elucidate the effect of addition of protein on the alkaloid absorbance. During the titration equal amount of BSA was also added to the reference cell. The changes in the absorption at the λmax were noted at each protein to alkaloid (P/D) molar ratio till saturation was obtained.


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Spectrofluorimetric Studies.

Fluorescence Spectral Analysis. Entire experiment regarding steady-state fluorescence spectral analysis was performed on a Shimadzu

RF-5301PC

spectrofluorimeter

(Shimadzu

Corporation,

Kyoto,

Japan).

Measurements were carried out in fluorescence free quartz cell of 1 cm path length using an excitation and emission band pass of 3 nm and 5 nm respectively. A fixed amount of CHL (1.8 µM) was titrated with increasing the concentration of BSA from 0 µM to 49.6 µM for the iminium form and from 0 µM to 29.3 µM for the alkanolamine form under constant stirring condition where CHL (I) and CHL (A) were excited at 338 nm and 318 nm respectively. While in another experiment the protein molecule BSA was excited at 295 nm to measure the intrinsic fluorescence of BSA in presence of each form of CHL. Here the protein concentration was kept constant at 1 µM. CHL concentration was varied from 0 µM to 17.8 µM and from 0 µM to 6.6 µM for the iminium and alkanolamine form respectively.

Determination of Fluorescence Polarization Anisotropy. Fluorescence anisotropy values offer vital information about the nature of the surroundings of fluorophores. Factors causing any alteration in size, shape and rigidity of a probe change the anisotropy value. Increase in the rigidity of the environment of fluorophore causes an enhancement in the fluorescence anisotropy. Fluorescence depolarization results due to the rotational diffusion of the fluorophore molecule in the excited state. Hence fluorescence polarization measurements can be employed to assess the rotational mobility of the fluorophore.30 Fluorescence depolarization correlates with the experimental measurement of fluorescence anisotropy (r′). Lower anisotropy value is indicative of faster rotational diffusion. Therefore, the rotational diffusion should decrease on binding of the large protein molecule BSA with CHL and consequently the anisotropy should increase. During anisotropy measurements, the sample is excited with vertically polarized light and the vertical and



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horizontal emission components (IVV and IVH) are recorded. Anisotropy (r′) is given by the following equation described by Larsson et al.31

r'=

(IVV − G ⋅ IVH ) ......................(1) (IVV + 2G .IVH )

where the G-factor (G) is the intensity ratio of the vertical to horizontal components of the emission when the sample is excited with horizontally polarized light (G=IVH/IHH). G depends on monochromator wavelength and slit widths; as long as the experimental conditions remain the same, the G-factor only needs to be measured once.

Fluorescence Lifetime Measurements. Time correlated single photon counting (TCSPC) measurements were performed in 10 mM CP buffer of pH 6.0 and in 10 mM CB buffer of pH 10.2 in the absence and in the presence of increasing concentration of BSA for the fluorescence decay of CHL (I) and CHL (A) respectively at 25 ºC. Further the fluorescence decay behaviour of BSA was measured in absence and presence of CHL using the aforesaid two different buffer solutions to evaluate the interaction with each form of CHL. During the TCSPC measurements the photoexcitation were made at 330 nm for each form (iminium and alkanolamine) of CHL and 300 nm for BSA using a picosecond diode laser (IBH Nanoled-07) in an IBH fluorocube apparatus while monitoring the intensity at 564 nm and 420 nm respectively for the charged CHL (I) and neutral CHL (A) form. Fluorescence decay data were recorded on a Hamamatsu MCP photo multiplier (R3809) and were analyzed by using IBH DAS6 software using the equation,

 t F ( t ) = ∑ α i e  − .....................(2)  τ i where αi denotes the ith pre-exponential factor and τ represents the decay time. The decay time is referred to as the life time of the excited species.


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Spectropolarimetric Studies.

Circular Dichroism (CD) Spectra. Circular dichroism (CD) spectra were recorded on a PC-driven JASCO J815 spectropolarimeter (Jasco International Co., Japan) equipped with a thermal programmer (model PFD-425L/15) and temperature controller interfaced in a rectangular quartz cuvette of 1 cm path length. All the CD spectra were recorded with a scan speed of 100 nm/min within the wavelength range of 195-300 nm. Here, a fixed concentration of BSA (0.28 µM) was titrated with increasing the concentration of CHL from 0 µM to saturation concentration 28.7 µM (both iminium and alkanolamine forms). All the measurements were performed in 10 mM CP buffer (pH 6.0) for CHL (I) and 10 mM CB buffer (pH 10.2) for CHL (A) at 25 °C. Each spectrum was averaged from five scans and smoothed to enhance signal-to-noise ratio. The obtained CD spectra were expressed in terms of molar ellipticity ([θ], in units of deg cm2 dmol-l), based on the protein (BSA) concentration, by using the software provided with the instrument. Isothermal Titration Calorimetric (ITC) Study. ITC studies were carried out on a Microcal LLC VP-ITC microcalorimeter (Microcal, Inc., Northampton, MA, USA) at 25 °C. Data were interpreted by using the dedicated origin 7.0 software provided with the instrument. The calorimetric syringe (40 µL) was filled with 440 µM of either CHL (I) or CHL (A). Aliquots of CHL (2 µL) were injected from the syringe, rotating in 290 rpm to 200 µL 25 µM of BSA solutions kept in the isothermal chamber. In our CHL-protein interaction study, duration of each injection was 10 s and the delay time between the two successive injections was 120 s. Each injection produced a heat burst curve with time and the area under each peak was integrated to get the associated heat of each 9 ACS Paragon Plus Environment


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injection. Actual heat change associated with the CHL-protein interaction was obtained by subtracting the heat change of appropriate control experiment from the heat of CHL-protein mixing. In our experimental condition the heat of dilution was negligibly small. The resulting data were analyzed to obtain the value of binding stoichiometry (N), binding constant (Kb), enthalpy of complexation (∆H°) and free energy change (∆G°) of binding interaction. Molecular Docking Simulation Study. Binding interaction and the probable binding site of CHL with BSA were obtained with the help of the docking program AutoDock (version 4.2). The X-ray crystal structure of BSA was taken from RCS Protein Data Bank having PDB ID: 4F5S and the 3D structure of CHL were created in Chem3D Ultra 8.0 and required modification was performed using two wellknown software packages Gaussian 09W and AutoDock 4.2. The optimized geometry of CHL was obtained from the DFT//B3LYP/6-31G level of theory using the Gaussian 09W set of programs and the resultant optimized geometry was exploited in the Gauss view 5.0 software in a compatible file format. This was further used to generate the required file in AutoDock 4.2. Polar hydrogen atoms and Gasteiger charges were added to the protein and ligand. A grid box with dimensions 120, 120 and 120 Å and a grid spacing of 0.403 Å were assigned to perform the docking simulations. Other set of parameters were taken the default values shown by the AutoDock program. The grid maps for energy were deliberated using AutoGrid and Lamarckian genetic algorithm (LGA) was used to perform the docking calculations.32,33 The best optimised docked model having lowest docking energy was chosen for further analysis of docking simulations which was best viewed in PyMOL and Mercury softwares.


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RESULTS AND DISCUSSION Spectral Characteristics of Iminium and Alkanolamine Forms of CHL. As mentioned earlier, like other quaternary benzophenanthridine alkaloid, CHL also exists between the cationic CHL (I) and the neutral CHL (A) form with a pKa of 8.58.3 The C(6)– N(5) double bond of CHL (I) is prone to be attacked by nucleophiles and this generates remarkable changes in both absorbance spectra as well as fluorescence spectra. The spectral characteristics of the two forms are presented in Figure 2. The UV spectrum of CHL (I) is

Figure 2. Absorption and emission spectra of CHL: (A) Absorption spectra of (3.6 µM) iminium and alkanolamine forms of CHL and (B) emission spectra of (1.8 µM) iminium and alkanolamine forms of CHL in CP buffer (pH 6.0) and CB buffer (pH 10.2) at 25 °C respectively. λex=338 nm and 318 nm for CHL (I) and CHL (A) respectively.

characterized by two well distinguished bands at 267 nm and 316 nm followed by a hump at 338 nm. CHL (I) has an absorption band in the region 370-460 nm which is absent in case of CHL (A) (Figure 2A). For CHL (A), the absorbance spectrum shows two notable peaks, one 11 ACS Paragon Plus Environment


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at 227 nm and the other one at 280 nm, respectively, along with a hump at 318 nm (Figure 2A). On the other hand the fluorescence spectrum of each form of CHL is presented in Figure 2B. The former (CHL (I)) is characterized by small emission band intensity with maximum at 564 nm while the latter (CHL (A)) has a maximum at 420 nm. At any particular concentration, the fluorescence intensity of CHL (A) is several folds higher than that of the CHL (I) form. UV-Vis Absorption Study. Study of the spectral changes and complex formation using UV-Vis absorption method is a simple but potential method in the field of protein-ligand interaction. CHL has characteristic absorption spectra in the wavelength range of 250-500 nm for both the iminium and alkanolamine forms depending on pH of the solution. The effect of addition of aliquots of

Figure 3. Spectrophotometric titration of CHL in presence of BSA at 25 °C. (A) Curves 1-4 denote the titration of CHL (I) (3.6 µM) treated with 0, 26.5, 63.1, 91.4, 112.1 and 125.6 µM of BSA in CP buffer of pH 6.0 and (B) curves 1-9 denote the titration of CHL (A) (3.6 µM) treated with 0, 1.9, 3.8, 5.7, 7.6, 11.4, 15.2, 22.8 and 30.2 µM of BSA in CB buffer of pH 10.2.


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BSA solution to the absorption spectra of CHL (I) and CHL (A) are presented in Figure 3A and 3B. Both the results show hypochromic effect on the intensity maxima of CHL with increasing concentration of BSA until saturation is reached. The extent of hypochromicity is more in case of CHL (A) (Figure 3B) compared to that of CHL (I) (Figure 3A). In addition, no significant shift of λmax of CHL (I) and ~2 nm bathochromic shift in case of CHL (A) are observed in presence of the protein molecule BSA. This clear observation of hypochromic and bathochromic effect on the UV-Vis spectrum of CHL can be attributed to the aromatic π– π stacking interactions.34,35 The decrease in intensity of absorbance may be ascribed to the penetration of the ligand into the protein molecule which restricts the mobility of the ligand resulting the complex formation. We have not observed any isosbestic point in the resulting spectra and have not evaluate the binding parameters from this study. Fluorescence Emission Study. Fluorescence spectroscopy has been considered to be the most comprehensive method to explore protein-ligand interactions. Thus we employed fluorescence spectroscopy as a major technique to study the interaction between BSA and CHL. Fluorescence emission of CHL (I) is relatively weak compared to the CHL (A) form when excited at 338 nm and 318 nm respectively. In case of CHL (I) incremental addition of BSA to the constant concentration of CHL (1.8 µM) resulted in the progressive enhancement of the fluorescence intensity (Figure 4A). A strong peak generated around 430 nm and the intensity of this new peak enhanced rapidly with the addition of BSA. BSA is sensitive towards fluorescence because of the presence of three crucial amino acid residues Tryptophan (Trp), Tyrosine (Tyr) and Phenylalanine (Phe) but the intrinsic fluorescence sensitivity of BSA appears only because of Trp residue.36 BSA has two Trp residues: Trp 134 and Trp 212. Trp emission dominates BSA fluorescence spectra to appear in the UV region.37,38 When excited at 280 nm, both Trp and Tyr residues emit fluorescence whereas upon excitation at 295 nm, fluorescence emission



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occurs due to Trp residue alone.39 Trp 134 residing at the surface in the hydrophilic region appears with an emission maximum at a higher wavelength than Trp 212 which is identified by a shorter wavelength maximum at 340 nm when BSA is excited at 295 nm.40

Figure 4. Fluorescence titration of CHL with increasing concentration of BSA at 25 °C: (A) Spectra 1-12 denote the emission spectrum of CHL (I) (1.8 µM) treated with 0, 0.9, 1.8, 3.6, 5.4, 9.1, 12.7, 18.0, 25.1, 32.2, 39.2 and 49.6 µM of BSA in CP buffer of pH 6.0; (B) spectra 19 denote the emission spectrum of CHL (A) (1.8 µM) treated with 0, 1.0, 2.9, 4.8, 8.6, 12.4, 18.1, 23.7 and 29.3 µM of BSA in CB buffer of pH 10.2. λex=338 nm and 318 nm for CHL (I) and CHL (A) respectively.

According to Foster41 the protein structure is pH dependent and there are several conformations at different pH. Here we have performed the whole experiment with CP buffer of pH 6.0 and CB buffer of pH 10.2. With increasing pH a blue shift of ~3 nm as well as decrease in fluorescence intensity at 340 nm is observed. These changes in emission spectra may be ascribed to the internalization of tryptophan moiety due to polarity change and the quenching of Trp-212 might be due to the deprotonation of Lys-204, 211, 221, 242 moieties which reside very close to the Trp-212 residue in the crystal structure of BSA.42 Almost identical CD spectra of BSA in the two buffer conditions (shown later) indicate the 3-D structural integrity of BSA under our experimental conditions. 14 ACS Paragon Plus Environment

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Clearly, the peak near 430 nm is not for the protein. So there must be some other phenomenon which takes place when BSA interacts with the iminium form of CHL. Possibly, in the excited state, BSA converts the CHL (I) into CHL (A) form which has a strong emission in the same wavelength region. Whereas with CHL (A), there is a progressive decrease in the fluorescence intensity of the emission maximum leading to a saturation with a significant blue shift (~11 nm) (Figure 4B). The different behaviours exhibited by the two forms of CHL restricted us to determine the binding constant from the fluorescence studies. To obtain the binding constant for protein-ligand interaction, we have examined the complexation of BSA in presence of different concentration of CHL. The ligand interaction with BSA may alter Trp fluorescence depending on the nature of interaction. Fixed concentration of BSA (1 µM) was titrated with increasing concentration of both forms of CHL in respective buffer solution leading to the saturation. The results obtained are presented in Figure 5. Both the results show decrease in the fluorescence intensity with slight red shift

Figure 5. Steady-state fluorescence spectra of BSA (1 µM) treated with different concentrations of CHL (I) and CHL (A) at 25 °C. Panel (A): curves 1-9 denote 0, 0.6, 1.2, 1.8, 2.4, 4.8, 8.4, 11.9 and 17.8 µM of CHL (I) in CP buffer of pH 6.0 and panel (B): curves 1-9 denote 0, 0.3, 0.6, 1.2, 1.8, 2.4, 3.6, 4.8 and 6.6 µM of CHL (A) in CB buffer of pH 10.2. λex=295 nm for BSA.



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of the emission maxima which may be due to a number of probable interactions between the protein and CHL such as excited state reactions, molecular rearrangements, energy transfer, ground state complexation, collision quenching etc.43 This change in fluorescence intensity indicates that the microenvironment around Trp residues in BSA molecule is altered on interacting with both forms of the alkaloid.44 When CHL (A) was added to the BSA solution, a strong peak generated around 430 nm due to the strong emission of CHL (A) itself in that particular region. In case of CHL (I), an isoemissive point appeared at 412 nm while for CHL (A), it appeared at 357 nm. Appearance of isoemissive point is indicative of the equilibrium between free CHL and the bound forms of CHL. The results obtained from this titration were used to determine the binding parameters. Evaluation of Binding Parameters. The binding of CHL to BSA can be represented by the following equation considering 1:1 complex formation,  → [ PD ] [ P ] + [ D ] ← 

Here P represents the protein and D stands for the alkaloid and PD is for protein-alkaloid complex. The equilibrium concentrations are given by [P], [D] and [PD] respectively. The data obtained from spectrofluorimetric titrations of BSA in presence of CHL are further analyzed using Benesi-Hildebrand equation45 and the binding constants are evaluated. ∆Fmax 1 = 1+ ..................(3) ∆F K BH [M ]

where ∆F = |Fx-F0| and ∆Fmax= |F∞-F0|. F0, Fx and F∞ are the fluorescence intensities of BSA in absence of CHL, at an intermediate CHL concentration and at a concentration of complete saturation with CHL respectively. KBH represents the binding constant of the complexation between BSA and CHL and [M] is the concentration of the variant (herein CHL). Using the titration data, the Benesi-Hildebrand plot has been constructed (Figure S1) and the binding constant (KBH) values obtained are presented in Table 1. The results evidently indicate that when CHL exclusively exists in alkanolamine form, binds more strongly than


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the iminium form indicating the higher affinity for complexation of CHL (A) over CHL (I) with BSA. The data, that we have obtained, are consistent with the result obtained by Hossain

et al.8 Table 1. Binding parameters for the complexation of CHL (I) and CHL (A) with BSA in respective buffers.a environment

binding constant (M-1)

CHL (I) - BSA

1.62 x105

CHL (A) - BSA

2.45 x105

Steady state fluorescence

CHL (I) - BSA

1.51 x105

polarization anisotropy

CHL (A) - BSA

2.56 x105

method

Benesi-Hildebrand

a

Average of three determinations.

Fluorescence Polarization Anisotropy. Steady-state fluorescence anisotropy values provide important information about the nature of the environment of fluorescence active molecules (fluorophores). It also provides useful information regarding the restriction arises on the mobility of the probe when the probe is placed in a confined environment. Therefore using the anisotropy values we can predict the proper position of the probe within its biological environment.43 In case of a fluorophore, fluorescence anisotropy increases on increasing the rigidity of its environment. So here, we have monitored anisotropy of both iminium and alkanolamine forms of CHL in presence of incremental addition of BSA. Figure 6 shows the variation of fluorescence anisotropy of CHL with increasing concentration of BSA. Initially the anisotropy value increases rapidly which is corroborated due to the increase in motional restriction on CHL within BSA environment. On the other hand, in the later stage no significant change in anisotropy value reflects saturation of complexation between CHL and BSA. It has been found that the anisotropy value increases ~3.2-fold for CHL (I) while ~4.4-fold enhancement is observed for CHL (A) in presence of BSA upon saturation. Significant enhancement in anisotropy value clearly indicates that both form of CHL is trapped in motionally restricted region within BSA. We did not measure the excitation and emission dipole orientations of CHL(I) and CHL(A). But 17 ACS Paragon Plus Environment


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the lifetime of the CHL isomers are different both in free and bound state (given in supporting information). Furthermore, the two species are orientated differently at the binding sites (as observed in the molecular docking studies) and these two effects might be

Figure 6. Anisotropy variation of CHL (I) (red circles) and CHL (A) (blue circles) as a

function of concentration of BSA. λex = 338 and 318 nm, and λem = 564 and 420 nm for CHL (I) and CHL (A) respectively.

together contributing to the difference in the limiting anisotropies at high [BSA]/[CHL] ratios. Our data reveal that the restriction is more in case of CHL (A) due to the higher affinity of BSA towards the alkanolamine form of CHL over the iminium one. According to Ingersoll and Strollo,30 the alkaloid-protein binding constant can be determined from the steady state anisotropy measurement using the following equation: 1 1 = 1+ ...................(4) fB K a [ P]

where Ka is the apparent binding constant; [P] is the concentration of BSA and fB is the fractional fluorescence contribution from the BSA-bound CHL, and it is defined as follows: fB =

r − rF R ( rB − r ) + (r − rF )

......................(5)

where rB and rF are the anisotropy values corresponding to the BSA-bound and free CHL respectively. R is a correction factor (R = IB/IF), introduced to ensure that the alkaloid experiences emission intensity modulation upon interaction with BSA. The plot of 1/fB versus 18 ACS Paragon Plus Environment

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1/[P] results a straight line (not shown) with the intercept ‘1’ and slope 1/Ka. The calculated Ka values obtained from the reciprocal of slopes are presented in Table 1 and the data are in excellent agreement with those values obtained from the spectrofluorimetric experiments. Thus, this method establishes its practical applicability and reliability for estimation of the binding constant.46 Analysis of Fluorescence Quenching Induced by CHL. Emission of serum albumins generally comes from three amino acid residues namely Tryptophan (Trp), Tyrosine (Tyr) and Phenylalanine (Phe). Since Phe has a very low quantum yield and Tyr has almost completely quenched fluorescence, the intrinsic fluorescence of BSA is exclusively due to Trp residue.47 The fluorescence quenching mechanism is generally divided into either static or dynamic. The former involves the ground state fluorophore-quencher complex formation, while excited state fluorophore-quencher complex formation takes place in case of dynamic quenching.43 In order to find out the probable quenching mechanism of the BSA-CHL complexation, the fluorescence quenching data are analyzed using Stern-Volmer equation43: Fo = 1 + K SV [Q ] = 1 + K qτ [ Q ].................(6) 0 F

where Fo and F are the fluorescence intensities of BSA in absence and presence of the quencher (CHL) respectively. KSV represents the Stern-Volmer quenching constant and Kq is the bimolecular quenching constant. [Q] is the concentration of the quencher, and τ0 is the lifetime of the protein molecule BSA in the absence of quencher CHL (4.1 ns for BSA). Kq can be obtained from the following equation: Kq =

KSV

τ0

.............(7)

Plots of Fo/F against [CHL] at three temperatures viz. 293, 298 and 303 K are shown in Figure 7 and the corresponding values of KSV and Kq are listed in Table 2. Figure 7 shows that the KSV value decreases with increase in temperature suggesting that the quenching 19 ACS Paragon Plus Environment


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mechanism is due to complex formation in the ground state known as static quenching. The value of maximum scatter collision quenching constant of different quenchers is found to be 2.0 × 1010 L.mol-1.s-1.43 In case of static quenching that explains the formation of a ground state complex, Kq value exceeds 2.0 × 1010 L.mol-1.s-1.48 The value of Kq obtained from three different temperatures are presented in Table 2. The values are of two orders of magnitude larger than the maximum diffusion collision quenching rate constant in dynamic quenching. This indicates that the fluorescence quenching mechanism is static one; in other words there was ground state complex formation between BSA and CHL. Moreover from the Table 2, it has been observed that the KSV value for BSA, when quenched by CHL (A), is approximately four times larger than that for CHL (I). This again establishes the greater association of CHL (A) with BSA.

Figure 7. Representative Stern-Volmer plots for the quenching of BSA by CHL at

different temperatures: (A) represents the quenching of fluorescence of BSA by CHL (I) and (B) represents the quenching of fluorescence of BSA by CHL (A) at 293 (black circles), 298 (blue circles) and 303 K (red circles) respectively.


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Table 2. Stern-Volmer constants for the quenching of BSA by CHL at different temperatures.a protein

CHL

temperature (K) 293

CHL (I) BSA CHL (A) a

298 303 293 298 303

KSV (L. mol-1) 8.37 x 104 7.97 x 104 7.18 x 104 38.31 x 104 32.22 x 104 27.47 x 104

Kq (L. mol-1. s-1) 2.04 x 1013 1.94 x 1013 1.75 x 1013 9.34 x 1013 7.86 x 1013 6.70 x 1013


Site Selective Binding of CHL from Fluorescence Displacement Studies. BSA is made out of a single polypeptide chain that contains 583 amino acids. This albumin structure possesses three homologous domains (labeled as I, II and III), and each of which is further divided into two sub-domains (A and B).49 Site I and site II are two major drug binding sites of BSA which are located within sub-domains IIA and IIIA, respectively.50 Most of the ligands are found to bind at site I to form complexes with serum albumins and only a few are known to bind at site II.20,26 The range of affinity constant varies from 104 to 106 L mol-1.51 In order to find out the binding site of CHL in BSA, site marker competitive binding experiments were carried out. During competitive binding interaction, the drug and the site marker both have similar sort of affinity to associate at the same binding site on the protein molecule, and the extent of protein-drug binding interaction can be exposed by monitoring the intensity change in the fluorescence spectra of the system.50 Warfarin is used as a typical site marker fluorescence probe to monitor the binding at site I while ibuprofen is distinctively used for the same at site II of BSA.27-29 Warfarin binds exclusively at site I (located in the sub-domain IIA) through hydrophobic interaction while ibuprofen specifically binds to site II (in sub-domain IIIA) involving hydrophobic, hydrogen bonding and electrostatic interactions.8,50,53-55 Site selective binding experiment with site I and site II 21 ACS Paragon Plus Environment


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markers warfarin and ibuprofen provides the concrete information regarding the CHL binding site which are obtained by monitoring the spectral changes in the fluorescence of CHL bound BSA. In the comprehensive binding experiment, the alkaloid was gradually added to the solution of BSA and site markers held in equimolar concentrations (1 µM) in CP buffer of pH 6.0 and CB buffer of pH 10.2 to visualise the change with CHL (I) and CHL (A) separately. The changes induced by the site markers are presented in Figure 8. Addition of CHL (I) and CHL (A) to the BSA-warfarin complex quenched the fluorescence intensity accompanied by an increase of wavelength emission maximum λmax (red shift) in the albumin spectrum (Figure 8A and 8B). On the other hand, in contrast to warfarin, a negligible change in fluorescence intensity of BSA was observed when ibuprofen was added to it. Both CHL (I)

Figure 8. Effect of site marker on the CHL-BSA complex: [Warfarin] = [BSA] = 1 µM with

CHL (I) (panel A) and CHL (A) (panel B) respectively. [Ibuprofen] = [BSA] = 1 µM with CHL (I) (panel C) and CHL (A) (panel D) respectively. In panel (A) curves 3-10 denote 1.5, 2.3, 3.0, 3.8, 4.5, 5.3, 6.0 and 6.8 µM and panel (C) curves 3-10 denote 1.5, 2.3, 3.0, 4.5, 6.0, 7.5, 9.0 and 10.5 µM of CHL (I) in CP buffer of pH 6.0 at 25 °C. In panel (B) curves 3-9 denote 0.7, 1.5, 2.3, 3.0, 3.8, 4.5 and 5.3 µM and (D) curves 3-12 denote 0.4, 0.7, 1.5, 2.3, 3.0, 3.8, 4.5, 5.3, 6.1 and 7.5 µM of CHL (A) in CB buffer of pH 10.2 at 25 °C. λex=295 nm for BSA.


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and CHL (A) induced quenching of the fluorescence of BSA-ibuprofen complex as in the absence of the site marker (Figure 8C and 8D). Thus CHL binding was affected very little, if at all, in presence of ibuprofen. Therefore we can say that, both CHL (I) and CHL (A) forms undergo complexation with the protein molecule at the site I in the sub-domain IIA of BSA. Combining the results obtained from fluorescence quenching and site selective binding we have come to a conclusion that Trp 212 residue resides near or within the binding site of CHL. Several small molecules are found to bind preferably to this location in BSA making the site favorable for small molecule-protein interaction.8,48,55-61 Fluorescence Resonance Energy Transfer (FRET) between CHL and BSA. FRET is an important tool in the investigation process of the structural conformations of biologically active molecules, such as spatial distribution, assembly of the protein-ligand complex etc. It is a very reliable method used for monitoring protein-ligand interactions and evaluating the distance between the donor and the acceptor.62 According to Förster’s nonradiative energy transfer theory, three factors influence the rate of energy transfer: (i) the relative orientation of the donor and acceptor dipoles, (ii) the extent of overlap between the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor, and (iii) the distance between the donor and the acceptor which is generally < 8 nm.63,64 Here, the donor is Trp residue of the protein (BSA) and the acceptor is CHL. The overlap (shaded) of the absorbance spectra of CHL (I) and CHL (A) forms with the fluorescence emission spectrum of BSA are shown in Figure 9A & 9B. A spectrum ranging from 300 to 550 nm was chosen to calculate the overlap integral and average distance between BSA and CHL using the following equations:

E = 1−

R6 F = 6 0 6 ..................(8) F0 R0 + r



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where E is the energy transfer efficiency, F and F0 are the fluorescence intensities in presence and in absence of CHL. r is the measure of the distance between the ligand and Trp moiety of BSA. Ro is the critical distance at 50% efficiency of energy transfer. Ro can be obtained from the following equation: R06 = 8.79 × 10 −25 K 2 N −4ϕ J ..................(9)

where K2 is the spatial factor of orientation, N is the refractive index of the medium and φ is the quantum yield of the donor. J is the overlap integral of the emission spectrum of the donor and the absorption spectrum of the acceptor (Figure 9) which can be obtained using the following equation: J=

∑ F (λ )ε (λ )λ 4 ∆λ ................(10) ∑ F ( λ ) ∆λ

where F(λ) is the emission intensity of the donor in the wavelength range of λ to (λ + ∆λ) and ε(λ) is the absorption coefficient of the acceptor at λ.65 For the present case, K2 = 0.67, N = 1.336 and φ = 0.15.66,67 The other parameters obtained using equations (8-10) are presented in

Figure 9. Overlap (shaded portion) of absorption spectrum of CHL and fluorescence

spectrum of BSA at 25 °C. Panel (A) and (B) represent absorption spectra of CHL (I) and CHL (A) and the emission spectra of BSA. The ratio of the concentration of [BSA]:[CHL] = 1:1. The λex of BSA was 295 nm.


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Table 3. It was observed that the average distance between the protein donor and the alkaloid acceptor after the interaction was < 8 nm for both forms of the alkaloid and 0.5Ro < r < 1.5Ro, which in turn establishes the fact that the energy transfer from BSA to CHL occurred with high probability.48 This data are in well agreement with the state of affairs associated to the Förster’s non-radiative resonance energy transfer theory and one more time indicate the occurrence of static quenching between BSA and CHL.66 This result also suggests that both the forms of bound CHL are found to be present in sub domain II of BSA which is also the residing area of Trp 212 residue. Lower r value in case of CHL (A) form indicates closer distance between Trp 212 and CHL (A). Table 3. Parameters related to fluorescence resonance energy transfer obtained at 25

°C.a protein

CHL

J (cm3. L. mol-1)

Ro (nm)

E

r (nm)

CHL (I)

1.96 x 10-14

2.86

0.58

2.71

CHL (A)

1.28 x 10-14

2.66

0.67

2.30

BSA

a


Spectropolarimetric Results. The conformational changes in BSA secondary and tertiary structures induced by CHL (I) and CHL (A) were investigated using circular dichroism (CD) spectral studies. At both pH conditions, BSA showed two negative bands at 209 and 222 nm in the CD spectra which was the evidence of an α-helix in the advanced structure of a protein.68 The negative bands appeared mainly due to the charge transfer of n→π* transition,69 the so called negative Cotton effect70 in the peptide bond of α-helical structure of BSA. On the other hand, the benzophenanthridine alkaloid, CHL is optically inactive and hence it is also CD inactive in the entire range of UV-Vis region.4 To elucidate the changes in the BSA conformation upon



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binding with CHL (I) and CHL (A), the CD spectra were recorded in the region of 195-260 nm under varying the concentration of CHL. The results obtained are presented in Figure 10. The interaction results a decrease in negative ellipticity value without any considerable shift of the peaks in the wavelength range of far-UV CD. This observation clearly indicates partial unfolding of the helical structure and a decrease of the α-helix content in the protein.

Figure 10. Effect of CHL on intrinsic circular dichroic (CD) spectrum of BSA. In (A)

and (B) curves 1-5 denote CD spectrum of 0.28 µM of BSA treated with 0, 3.6, 10.8, 18.0 and 28.7 µM of CHL (I) and CHL (A) respectively at 25 °C.

Variation of the Secondary Structure of BSA. The percentage content of α-helix, β-sheet and random coil in the secondary structure of BSA at the both pH conditions was calculated using CDNN 2.1 software and the data obtained are tabulated in Table 4. It was found that the secondary conformation of the protein contains ~59% α-helix, ~22% β-sheet and ~18% random coil in both pH in absence of CHL. From the table it is clear that reduction of α-helical structure is more in case of CHL (A) compared to 26 ACS Paragon Plus Environment

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that for CHL (I). The decrease of the α-helical content and concomitant increase of the βsheet and random coil structure indicate unfolding of native protein structure. In case of CHL (I) there was a decrease of α-helix content by about 5%. On the other hand for CHL (A) it

Table 4. Effect of CHL (I) and CHL (A) on the secondary structure of BSA at 25 °C.a,b CHL

CHL (I)

CHL (A)

random

[CHL] / [BSA]

α-helix (%)

β-sheet (%)

0

59.4

22.1

18.5

25.43

58.1

22.8

19.1

50.75

57.0

23.3

19.7

75.98

55.6

24.1

20.3

101.11

54.8

24.5

20.7

0

58.4

22.6

19.0

25.43

56.1

23.8

20.1

50.75

54.2

24.8

21.0

75.98

52.9

25.4

21.7

101.11

51.8

26.0

22.2

coil (%)

a b

Average of three determinations; Error is less than 1%

was higher (~7%). When compared with the binding affinity differences, this structural change is less significant. The CD results give only an estimate about the global structural change induced by the small molecules. Our data reveal that protein unfolding is more in case of alkanolamine form compared to its counterpart iminium form. Similar observations have been reported for the interaction of two different isoforms of sanguinarine with serum albumins.8 Isothermal Titration Calorimetry (ITC) of BSA-CHL Interaction. Thermodynamic characterization of interaction of small molecules with macromolecules can be efficiently probed by employing isothermal titration calorimetry (ITC). We have used this tool to explore the energetics of the interaction of both forms of CHL and BSA. In Figure 11, representative calorimetric profiles of the titration of iminium and alkanolamine forms of 27 ACS Paragon Plus Environment


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CHL to BSA are presented. Each of the heat burst curve in the Figure 11 represents a single injection. The corresponding area under each heat burst curve was integrated to calculate the associated heat change. The heat liberated in each injection was corrected for the heat of dilution that was determined in a separate but identical experiments injecting CHL into the buffer alone. The resulting corrected heat is plotted against the respective D/P (CHL/BSA molar ratio) values in the lower panels of Figure 11, Here each data point reflects the corresponding injection heat and the solid lines show calculated fits of data. In both the cases the binding is found to be exothermic in nature. Calorimetric data were fitted to a single set of identical sites and this resulted a good fitting of the experimental data. The calculated binding and thermodynamic parameters that include the binding constants (Kb), number of occluded sites (N), enthalpy (∆H°) of binding, entropy (∆S°) of binding and free energy (∆G°) of

Figure 11. ITC profile for the binding of (A) CHL (I) and (B) CHL (A) to BSA at 25 °C. The upper

panels represent the raw data for the sequential injection of the CHL into the BSA (panel A and B, curves on the bottom) solutions respectively and CHL dilution control (curves on the top off set for clarity). The lower panels show the corresponding corrected heat signals versus molar ratio. Data (closed circles) were fitted to a one site model and the solid lines represent the best fit results.


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binding are presented in Table 5. Our binding data reveal that alkanolamine form of CHL binds more strongly to BSA compared to the iminium form. Binding parameters obtained from ITC experiments are in good agreement with the spectrofluorimetric results (Table 1). Binding of both forms of CHL with BSA was favoured by negative enthalpy change and positive entropy change. Negative free energy (∆G°) value indicates that both binding processes are thermodynamically favourable. More negative ∆G° value in case of CHL (A) is attributed to its higher binding constant with BSA.8 Table 5. Thermodynamic parameters for the interaction of BSA with CHL (I) and CHL (A) at 25 °C.a

a

∆G°298K ∆H° T∆S° (kcal.mol-1) (kcal. mol-1) (kcal.mol-1)

CHL

Kb (M -1)

N

CHL (I)

1.55 x105

0.225

-11.29

-10.50

0.79

CHL (A)

2.59 x105

0.137

-13.00

-11.81

1.19


Modeling of CHL Binding Site in BSA: Blind Docking Study The molecular docking study was employed to identify the probable binding location of the alkaloid within the protein molecule. The crystalline structure of BSA has revealed that it has three homologous domains: domain I (residue 1-183), domain II (residue 184-376) and domain III (residue 377-583), each of them containing two sub-domains (A and B).25 Two Trp residues (Trp 134 and Trp 212) of BSA are in the sub-domain IB and IIA respectively.61 Among the nine different conformers, the lowest binding energy conformer was taken for each docking simulation and the resultant of which was used to proceed for further analysis. The docked poses displayed in Figure 12A and 12D reveal that the sub-domain IIA of BSA is the favorable binding site for both forms of the alkaloid molecule. The right panel of Figure 12 (C and F) shows the protein molecule in near vicinity (within ~0.39 Å) of the alkaloid molecule. From figure it is clear that the environments of bound CHL (I) and CHL (A) on



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BSA are different in terms of the nearby amino acid residues. In other words, it seems that there is slight difference in the orientation of the two isoforms of the alkaloid at the binding site. This may be the another reason, in addition to the difference in lifetime values, for the difference in anisotropy values for CHL (A) and CHL (I) at high [BSA]/[CHL] molar ratio. It is also seen from the figure that both forms of CHL reside near the Trp 212 residue of the protein molecule which is responsible for its emission. As a result, the quenching of the fluorescence of BSA occurs in presence of both forms of CHL that we have obtained from fluorescence titration (Figure 5A and 5B). Thus the results obtained from experiment, justified theoretically.

Figure 12. Stereo view of the docked conformation of (A) CHL (I) and (D) CHL (A) with BSA. The

site of the interaction of CHL is magnified on the right panels (B & C for CHL (I) and E & F for CHL (A)) in the near vicinity (within 0.39 Å) of the protein at the interaction site.

CONCLUSION A multispectroscopic investigation was employed to monitor the interaction between the benzophenanthridine alkaloid CHL and plasma protein BSA. Taken together we can conclude that the alkanolamine form of the alkaloid CHL has greater binding affinity compared to that of the iminium form. Fluorescence measurement revealed that the intrinsic fluorescence of


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BSA was quenched through static quenching process leading to the formation of a ground state complex between CHL and BSA. The static quenching mechanism was further confirmed by fluorescence lifetime measurement which indicated no significant changes in the fluorescence lifetime of BSA upon interaction with CHL. The negative enthalpy and positive entropy values revealed that the hydrophobic forces played a major role in stabilizing the BSA-CHL complex. FRET analysis indicates that the distance between CHL and BSA is close enough (2.71 and 2.30 nm for iminium and alkanolamine form respectively) to occur non radiative energy transfer from BSA to CHL. Site selective binding experiment using well-known site markers warfarin and ibuprofen establishes that both the iminium and alkanolamine forms of CHL preferably bind at site I (sub-domain IIA) of BSA. CHL makes some changes in the protein conformation by reducing the percentage of α-helix. The protein unfolding process is more pronounced in presence of the alkanolamine form. Thermodynamic parameters indicate the stronger association of the alkaloid with BSA and the binding is favoured by strong hydrophobic forces. The more hydrophobic nature of the alkanolamine form over the iminium form is responsible for its higher binding constant. The relatively large value of entropy in case of the alkanolamine form clearly establishes its hydrophobic nature. This work provides some constructive information regarding the interaction of BSA with CHL which may be a helpful guideline for further investigation of CHL as therapeutic agent in addition to storage and transportation of drug in blood. ASSOCIATED CONTENT Supporting Information Representative Benesi-Hildebrand plot for the association of BSA with CHL (I) and CHL (A); fluorescence lifetime measurements: time-resolved fluorescence decay curves of CHL in absence and in presence of increasing concentration of BSA; life time data for CHL in absence and in presence of BSA in respective buffers at 25 °C; time-resolved fluorescence



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decay profiles of BSA in absence and in presence of increasing concentration of CHL; life time data for BSA in absence and in presence of CHL (I) and CHL (A) in respective buffers at 25 °C. ACKNOWLEDGEMENTS We sincerely thank Professor Nikhil Guchhait , University of Calcutta, India, for his valuable discussion and for providing his laboratory facilities. SB thanks the University Grant Commission, Government of India, for the award of RGNF research Fellowship. LH and ABP thank to the University Grant Commission, Government of India, for the award of Junior and Senior Research Fellowship respectively.

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Structures on the Conversion of Sanguinarine Alkanolamine Form to Iminium Form. J. Biomol. Struct. Dyn. 2002, 20, 455-464. (2)

Kumar, G. S.; Hazra, S. Sanguinarine, A Promising Anticancer Therapeutic:

Photochemical and Nucleic Acid Binding Properties. RSC Adv. 2014, 4, 56518-56531. (3)

Basu, P.; Bhowmik D.; Kumar, G. S. The Benzophenanthridine Alkaloid

Chelerythrine Binds to DNA by Intercalation: Photophysical Aspects and Thermodynamic Results of Iminium versus Alkanolamine Interaction. J. Photochem. Photobiol. B: Biology 2013, 129, 57-68. (4)

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Polyriboadenylic Acid by the Benzophenanthridine Plant Alkaloid Chelerythrine: A Spectroscopic Approach. RSC Adv. 2014, 4, 52815-52824. (5)

Sen, A.; Maiti, M. Interaction of Sanguinarine Iminium and Alkanolamine Form with

Calf Thymus DNA. Biochem. Pharmacol. 1994, 48, 2097-2102.


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M.; Bianco, S.; Bilia, A. R.; Schwalbe, H. et. al. Spectroscopic, Molecular Modeling, and NMR-spectroscopic Investigation of the Binding Mode of the Natural Alkaloids Berberine and Sanguinarine to Human Telomeric G-quadruplex DNA. ACS Chem. Biol. 2012, 7, 11091119. (7)

Giri, P.; Kumar, G. S. Spectroscopic and Calorimetric Studies on the binding of the

Photoxic and Cytotoxic Plant Alkaloid Sanguinarine with Double Helical Poly(A). J. Photochem. Photobiol. A: Chemistry 2008, 194, 111-121. (8)

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TOC GRAPHIC


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