Binding and Inhibitory Effect of the Dyes Amaranth and Tartrazine on

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Binding and Inhibitory Effect of Dyes Amaranth and Tartrazine on Amyloid Fibrillation in Lysozyme Anirban Basu, and Gopinatha Suresh Kumar J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b10465 • Publication Date (Web): 17 Jan 2017 Downloaded from http://pubs.acs.org on January 29, 2017

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The Journal of Physical Chemistry

Binding and Inhibitory Effect of Dyes Amaranth and Tartrazine on Amyloid Fibrillation in Lysozyme Anirban Basu* and Gopinatha Suresh Kumar* Biophysical Chemistry Laboratory Organic & Medicinal Chemistry Division CSIR-Indian Institute of Chemical Biology Kolkata 700 032, India

Corresponding Author’s Address Dr. Anirban Basu, Ph.D and Dr. G. Suresh Kumar, Ph.D Organic & Medicinal Chemistry Division CSIR-Indian Institute of Chemical Biology 4, Raja S. C. Mullick Road, Jadavpur Kolkata 700 032, INDIA Phone: +91 33 2499 5723 Fax: +91 33 2472 3967 e-mail: [email protected] (A. Basu) / [email protected] (G. Suresh Kumar)

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ABSTRACT: Interaction of two food colorant dyes, amaranth and tartrazine, with lysozyme was studied employing multiple biophysical techniques. The dyes exhibited hypochromic changes in the presence of lysozyme. The intrinsic fluorescence of lysozyme was quenched by both dyes; amaranth was a more efficient quencher than tartrazine. The equilibrium constant of amaranth was higher than that of tartarzine. From FRET analysis, the binding distances for amaranth and tartrazine were calculated to be 4.51 and 3.93 nms, respectively. The binding was found to be dominated by nonpolyelectrolytic forces. Both dyes induced alterations in the microenvironment surrounding the tryptophan and tyrosine residues of the protein; the alterations being comparatively higher for the tryptophans than the tyrosines. The interaction caused significant loss in the helicity of lysozyme, the change being higher with amaranth. The binding of both the dyes was exothermic. The binding of amaranth was enthalpy driven while that of tartrazine was pre-dominantly entropy driven. Amaranth delayed lysozyme fibrillation at 25 μM while tartrazine had no effect even at 100 μM. Nevertheless, both dyes had a significant inhibitory effect on fibrillogenesis. The present study explores the potential anti-amyloidogenic property of azo dyes.

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INTRODUCTION Food colorant azo dyes with aromatic rings in conjunction with N=N linkages can undergo reductive cleavage to form potentially carcinogenic aromatic amines.1,2 Cleavage of N=N linkages often yeild highly carcinogenic benzidine molecule.3,4 Amaranth (AMTH hereafter, Figure 1A) and tartrazine (TZ hereafter, Figure 1B) are two azo dyes widely used as food colorants. AMTH is a reddish or brownish dye used to color variety of foodstuff.1 It is toxic to human lymphocytes in vitro1 and causes allergic and asthmatic reactions in some sensitive individuals in the presence of drugs like aspirin.5,6 AMTH can also cause DNA damage in mice.7 Tartrazine is an orange-colored, mono azo pyrazolone dye present in soft drinks, cookies and cereals.1,8 It can invoke allergic reactions and hyperactivity in children.9 Besides, ingestion of TZ in children leads to restlessness and sleep disturbance.8 It can not only invoke oxidative stress by forming free radicals but also affect the hepatic and renal parameters.10 Since both AMTH and TZ are present in commonly consumed food items so they can easily enter into our body/system and interact with important biomolecules inside our body. Considering these potential adverse health effects there is an urgent need to study their effect in details at the functional biomacromolecular level to understand their transportation, distribution and toxicological actions in vivo.11-22 Lysozyme (LSZ) is an antimicrobial protein which is also referred to as muramidase.23,24 It is present in abundance in different tissues and secretions like milk, saliva, tears, and

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mucus. It is also found in the cytoplasmic granules of the polymorphonuclear

Figure 1. Molecular structure of (A) AMTH and (B) TZ.

neutrophils. It can provide protection against bacterial infections as it is endowed with the ability to cause damage to the bacterial cell wall by breaking the β-linkages between the N-acetyl-muramic acid and N-acetylglucosamine of the peptidoglycan. LSZ is also known for its antihistamine, antiviral, anti-inflammatory, antiseptic, and antineoplastic actions.25,26 It also serves as a food preservative.25-27 Since LSZ is small in size, naturally abundant, highly stable, can undergo amyloid aggregation, and bind drugs efficiently, it is employed as a model protein to understand protein folding, dynamics, and ligand interactions.28–30 LSZ is a monomeric globular protein with 129 amino acid residues.31,32 LSZ has six tryptophans (Trp), three tyrosines (Tyr), and four cross-linked disulfide bonds alongside many α-helices, β-sheets, turns, and loops in its secondary structure.33,34 The active site of LSZ contains a deep crevice which separates its two domains connected via an α-helix. One domain primarily comprises of β-sheet conformations while the second one is predominantly α-helical in nature.35 Elucidation of the crystal structure of 4


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LSZ has revealed that the three Trp residues (Trp-62, 63, and 108) are situated in the viscinity of the substrate binding site.36 As LSZ can reversibly bind to various endogenous and exogenous compounds37 it may modulate the distribution, metabolism and toxicity of many small molecules. Interaction of LSZ with potentially toxic compounds can result in the impairment of its aforementioned activities which are highly beneficial to humans. Furthermore, as LSZ can form amyloid fibrils under appropriate conditions studies on LSZ-small molecule interaction might enable us to develop potentially lead molecules as inhibitors for amyloid-related diseases.38 Amyloid fibrillation of proteins plays a critical role in many amyloid-related diseases such as Alzheimer’s disease, and type 2 diabetes.39–42 Amyloid fibrils generated by the wild-type of LSZ in vitro resemble the ultrastructures and biochemical properties of those obtained from pathological deposits in tissues. Screening of small molecules that can inhibit and disrupt fibrillogenesis of disease-related amyloid proteins is an interesting therapeutic strategy for the treatment of amyloid-related diseases. Here we probed the interaction of AMTH and TZ with LSZ by spectroscopy techniques, and calorimetry experiments. Since these two azo dyes are used to color many regularly consumed food items it is highly pertinent to monitor their influence on amyloid fibrillation. This study provides information on the various structural and conformational changes induced in LSZ by these two anionic food colorants and also enables us to understand their effects on LSZ fibrillation which may be useful for developing anti-amyloidogenic drugs. 5



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EXPERIMENTAL Materials. LSZ, AMTH and TZ were obtained from Sigma-Aldrich Corporation (St. Louis, MO, USA). LSZ was first purified according to the protocols reported in literature and was dialyzed into the experimental buffer.43 Concentration of LSZ was determined using a molar absorption coefficient value of 37,750 M-1 cm-1 at 280 nm.44 All experiments were performed in citrate-phosphate (CP) buffer containing 10 mM [Na+], of pH ~7.00 unless otherwise specified. Spectroscopic Studies. Absorbance titrations were performed on a Jasco V-660 spectrophotometer (Jasco International Co, Hachioji, Japan) following the protocols reported in the literature.12,25 Spectrofluorimetric studies were done on either a Shimadzu RF-5301 PC (Shimadzu Corporation, Kyoto, Japan) or a Quanta Master 400 unit (Horiba PTI, Canada) controlled with FelixGX spectroscopy software in quartz cuvettes following the procedures reported in details previously.21,25 FTIR spectra were acquired on a Bruker FTIR, TENSOR 27 spectrometer.21 Circular dichroism (CD) experiments were conducted on a Jasco J815 unit equipped with a temperature controller (PFD 425 L/15) in quartz cuvettes of 0.1 or 1 cm path length.25 The data was averaged from ten successive scans to improve the signal-to-noise ratio, smoothed within permissible limits and analyzed by Jasco software. Deconvolution of the far-UV CD spectrum of LSZ was also performed using OriginPro 8.5 software. Furthermore, far-UV CD was used to monitor the conformational changes in the LSZ samples upon fibrillation. Far-UV CD spectra, in the 190–260 nm range, of LSZ samples at varying 6


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time intervals were recorded after diluting 100 μl of the sample solution with 200 μl of glycine-HCl buffer in a 0.1 cm path length cuvette. All the spectra were recorded at 298.15 K at a scanning speed of 50 nm/min and smoothed within permissible limits. Differential Scanning Calorimetry (DSC). DSC studies were conducted on a MicroCal VP-DSC unit (MicroCal, Inc., Northampton, MA, USA, now Malvern Instruments ltd., Malvern, UK) following the protocols reported earlier.45 At first both the sample and reference cells were filled with degassed buffer and equilibrated for 15 min at the desired temperature. Thereafter, it was scanned within the required temperature range at a scan rate of 60 K . h-1 to obtain a stable reproducible base line. On the cooling cycle the sample chamber was loaded with LSZ and its complexes with AMTH and TZ and scanned. Finally, the DSC thermograms were analyzed using Origin 7.0 software provided with the unit. Isothermal Titration Calorimetry (ITC). ITC experiments were conducted on a MicroCal VP-ITC unit. AMTH and TZ solutions were injected from an auto-controlled rotating syringe into the sample chamber containing LSZ solution. Dilution experiments were also done to obtain the heat of dilution of AMTH and TZ by titrating the same volumes and concentrations of AMTH and TZ into the aqueous buffer. The dilution heats were then subtracted from the respective heats of AMTH/TZ–LSZ reaction to obtain the true heats of LSZ-AMTH/TZ complexation. These corrected heats were plotted as a function of the molar ratio and subsequently analyzed using a model for 'one set of binding sites' which afforded the equilibrium constant (Ka), and the enthalpy change (ΔH0) accompanying the 7



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complexation. The Gibbs energy change (ΔG0) and the entropic contribution (TΔS0) to the binding were subsequently deduced from the following standard thermodynamic relations G0 = -RT ln Ka = ΔH0 - TΔS0

(1)

where, R is the gas constant and T is the temperature in kelvin (K). Atomic Force Microscopy (AFM) Imaging Studies. AFM imaging experiments were performed for LSZ and its complexes with AMTH and TZ. All the samples were prepared in autoclaved Milli-Q water which was filtered through 0.22 μm Millipore filters to remove any particles. The LSZ stock solution was diluted to a final concentration of 100 nM. Equimolar complexes of LSZ with AMTH/TZ were also prepared. For imaging of LSZ fibrils the solutions were diluted 100 fold with water. 5 μl of this sample solution was then adsorbed onto a freshly cleaved muscovite Ruby mica sheet (ASTM VI grade Ruby Mica from MICAFAB, Chennai, India). It was then dried for 30 min in vacuum drier under inert atmosphere. The complexes were incubated for 15 min prior to adsorption onto the mica sheet. AAC mode AFM was performed on a Pico plus 5500 ILM AFM (Agilent Technologies, USA) which was equipped with a piezo scanner of maximum range 9 μm. Micro fabricated silicon cantilevers of Nano Sensors (USA) were employed here. The resonance frequency of the cantilever oscillation was 146-236 kHz while the force constant was 21-98 N/m. The rate of the scan speed was 0.5 lines/s while taking the images (256 by 256 pixels). All the images were processed by flattening with the help of Picoview version 1.1 software (Agilent Technologies) while its

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manipulation was performed using Pico Image Advanced version software (Agilent Technologies). Preparation and Characterization of LSZ Fibrils. LSZ samples (10 mg/ml) were dissolved in freshly prepared glycine-HCl buffer, pH ~2.20, containing 100 mM sodium chloride and 1.54 mM NaN3.46-48 To prepare amyloid fibrils, 2 ml of the stock LSZ solution was transferred to a 25 ml glass tube, diluted to 20 ml with the glycine-HCl buffer and agitated with polytetrafluoroethylene coated micro stirring bars (220 rpm) at 333.15 K.48-50 At desired time intervals the glass tube was vortexed gently to homogenize the sample solution and then 1 ml aliquots of the sample were withdrawn for the following experiments. Thioflavin T (ThT) Fluorescence Experiments. ThT fluorescence experiments were performed on a Shimadzu RF-5301 PC spectrofluorimeter. A stock solution of ThT was prepared and its concentration was determined following the procedure reported previously.48,51 150 μl aliquots of LSZ solutions taken out at different time intervals with or without AMTH/TZ were diluted to 750 ml with the buffer. Thereafter, appropriate amounts of ThT solution were added so that the final concentration of ThT was 20 μM. Fluorescence intensities at 485 nm were recorded in slow speed by exciting the resultant mixtures at 416 nm. Congo Red (CR) Binding Assay. Freshly prepared 1 mM CR solution in DMSO was used for performing this assay. Aliquots of 50 μl LSZ solution were diluted with 1 ml of the buffer solution followed by addition of CR solution so that the final concentration of 9



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CR in the mixture was 10 μM. Thereafter, it was thoroughly mixed and incubated at room temperature for 30 min. The absorption spectra of the LSZ samples were taken in the 400–800 nm range. The absorbance at the respective absorption maxima of the dyes was noted and plotted against the corresponding time intervals. Nile Red (NR) Binding Assay. NR binding assay was performed following the protocols reported earlier with certain modifications.48 NR is a hydrophobic fluorescent dye often employed to prevent Raman and Rayleigh scattering interference.48 Prior to measurement, a stock solution of 1 mM NR was prepared in DMSO. Aliquots of 50 μl of LSZ samples taken at designated time points were diluted with 950 μl of buffer solution followed by addition of appropriate volumes of NR solution so that the final concentration of NR in the mixture was 1 μM. The mixture was incubated in dark for 30 min. Thereafter, the sample was excited at 550 nm and the NR fluorescence intensity was noted at its emission maximum. The variation in the position of the emission maximum as well as the fluorescence intensity at the emission maximum was plotted as a function of varying time intervals. Statistical Analysis. All data reported here are represented as mean ± standard deviation (S.D.) of four independent determinations unless otherwise mentioned. The combined standard uncertainty uc(x) linked with the data is provided by the following relationship uc(x) = {(u1(x))2 + (u2(x))2 + (u3(x))2 + ….………… + etc}1/2

(2)

where u1(x), u2(x), u3(x), etc. represent the individual uncertainties in the measurements.

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RESULTS AND DISCUSSION Spectrophotometric Studies. It is well known that the binding of ligands with proteins can be characterized classically through spectrophotometric studies20,25,26 where the former exhibits characteristic absorption spectra beyond 300 nm where LSZ does not absorb. The absorption spectral characteristics of AMTH and TZ have been described in details in earlier studies with nucleic acids.52,53 The visible absorption spectra of AMTH and TZ exhibited well-defined peaks at 521 and 427 nms, respectively, which provided a convenient tool to study the protein binding interaction. Figure 2 depicts the absorption spectral changes induced in AMTH and TZ upon interaction with LSZ. With increasing concentration of LSZ the absorption intensity of both the dyes weakened resulting in significant hypochromic effect. The absorption maxima of AMTH and TZ exhibited hypochromic shifts of 20% and 10%, respectively, in the presence of LSZ. This is essentially due to the coupling of the vacant π*-orbitals of the dyes with the π*orbitals of LSZ which results in an energy decrease and a reduction in the π–π* transition energy. The vacant π*-orbitals become partially occupied with electrons which causes reduction in the transition.54 However, there were no noticeable changes in the position of the absorption maxima for both AMTH and TZ, that is, neither any red nor any blue shift was observed.

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Figure 2. Absorption spectral changes of (A) AMTH (5.40 µM, curve1) treated with 5.40, 16.20, 37.80, 59.40, 81.00 and 102.60 µM of LSZ (curves 2–7) and (B) TZ (4.99 µM, curve1) treated with 15, 30, 50, 70, 90 and 110 µM of LSZ (curves 2–7).

Spectrofluorimetric Studies. Fluorescence is the most useful tool to study the binding interaction between ligands and proteins20,21,25,26 because the photophysical properties of the fluorophores of the proteins are highly responsive to changes in the polarity of their surroundings. Hence, changes in the intrinsic fluorescence of LSZ in the presence of ligands yield information on the nature and mode of the binding. LSZ was excited at 295 nm where Trp moieties are exclusively excited. A fluorescence emission maximum was obtained around 338 nm.25,44 LSZ contains six Trp residues positioned at 28, 62, 63, 108, 111, and 123 out of which 28, 108, 111, and 123 are positioned in the α domain while the others at 62, 63, and 108 are in the substrate binding cleft.25,44 Trp-62 and 63 are near the hinge region between α and β domains. Earlier study have revealed that Trp-62 and 12


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63 are most exposed to the solvent and hence they are highly susceptible to chemical modifications.55 The Trp-62 and 63 residues are highly exposed to light when LSZ is partly unfolded thereby causing an enhancement in its fluorescence intensity. Literature reports indicate that the Trp-62 and 108 moieties account for the intrinsic fluorescence of LSZ while the residues at 28, 63, 108, 111, and 123 contribute marginally.56,57 The Trp-63 residue is not buried in the hydrophobic core and lies near the active-site hinge region while the Trp-62 is wholly exposed to the solvent and Trp108 is buried away from the hydrophilic region.58 Hence, the Trp-62 and 63 moieties are the ones which are most likely to be influenced by the ligand and consequently the fluorescence studies yield information on their microenvironment. Effect of AMTH and TZ on the intrinsic fluorescence of LSZ is shown in Figure 3. The fluorescence of LSZ decreased gradually upon addition of AMTH and TZ; the quenching efficiency was markedly high for AMTH in comparison to TZ indicating AMTH can bind more efficiently

with

LSZ.

AMTH

induced

significantly

pronounced

changes

at

comparatively lower concentrations suggesting it has a remarkably higher affinity for LSZ. On the other hand, the quenching induced by TZ was modest indicating it has a moderate binding affinity for LSZ. There were also remarkable alterations in the position of the emission maximum of LSZ upon binding with AMTH and TZ. In the presence of AMTH there was a remarkable red shift of 10 nm while in presence of TZ there was a blue shift of 4 nm.

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Figure 3. Changes in the steady state fluorescence spectra of LSZ (5.60 µM, curve 1) on treatment with (A) 4, 12, 20, 28, 36 and 40 µM of AMTH (curves 2–7) and (B) 8, 24, 40, 52, 72 and 80 µM of TZ (curves 2–7).

Quenching can occur due to an inner-filter effect, collisional (dynamic) quenching, or ground-state complexation (static quenching).59 Corrections for the inner-filter effect of the two azo dyes on LSZ was performed. This correction was effected by measuring the absorbance values (Aex and Aem) at λex and λem for every concentration of AMTH and TZ followed by multiplying the apparent fluorescence intensity with a correction factor of e(Aex+Aem)/2 as reported earlier.12,60 Since the fluorescence intensity of LSZ diminished even after applying inner-filter effect corrections the mechanism of quenching may be either collision (dynamic) induced or due to complex formation (static) between LSZ and the ligand. It is well documented that the dynamic quenching constants increase with temperature while increase in temperature decreases the stability of the complexes in the case of static quenching mechanism causing a decrease in the magnitude of the quenching constant. Hence, temperature dependent fluorescence studies were 14


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performed at 288.15, 298.15 and 308.15 K, respectively, to decipher the quenching mechanism. The data were analyzed using the following Stern-Volmer equation22 Fo  1  K q o [Q]  1  KSV [Q] F

(3)

where, Fo = fluorescence of LSZ in the absence of the dye, F = fluorescence of LSZ in the presence of dye, KSV = quenching constant, Kq = quenching rate constant and [Q] = concentration of AMTH or TZ. The data obtained from temperature dependent fluorimetric experiments are depicted in Table 1. From the data it can be clearly seen that the KSV values decreased with rise in temperature which testified that the quenching mechanism was static in nature due to complex formation on binding of both dyes with LSZ. In other words specific ground state complexation was responsible for the quenching of the intrinsic fluorescence of LSZ in the presence of AMTH and TZ leaving dynamic collision effects if any to be negligible. The Kq values were also greater than 2.0 × 1010 M-1 s-1, which further testified for a static quenching mechanism.59,61 Fluorescence Lifetime Study. Lakowicz postulated that fluorescence lifetime measurements can conclusively distinguish between static and dynamic quenching processes.62 Static quenching is characterized by stable fluorescence life time values whereas significant alterations in the fluorescence life time values are the hallmark of dynamic quenching.62 Time resolved fluorescence decay of LSZ in the presence of AMTH and TZ was studied to decipher the dynamics of LSZ in presence of the food colorants. Fluorescence lifetime values along with their amplitudes for free and dye bound LSZ were deduced from their respective time-resolved fluorescence decay 15



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profiles. Excitation and emission wavelengths were 280 and 338 nms, respectively. The light signal scattered from Ludox afforded the instrumental response function which was utilized for the deconvolution of the fluorescence signals. Decay curves of LSZ and its complexes with AMTH and TZ were fitted to a bi-exponential function and the quality of the fits were evaluated from both the χ2 values and the residuals of the function fitted to the data. Fluorescence decay is given by63

F (t )    i exp(

t ) τi

(4)

where F(t) = fluorescence intensity at time t and αi = pre-exponential factor with respect to the ith decay time constant, τi. For multi exponential decay, the average lifetime τavg is given by64

 avg   ai i

(5)

where, τi = fluorescence lifetime and ai = relative amplitude with i ranging between 1 to 2. For free LSZ the fluorescence lifetimes values were calculated to be τ1=0.91 ns and

τ2=2.23 ns. The fluorescence lifetime values were deduced to be τ1=0.77 ns and τ2=2.08 ns in presence of AMTH. In presence of TZ the fluorescence lifetime values were τ1=0.81 ns and τ2=2.21 ns. The Trp residues divulge multi exponential decays,16,26 therefore we have not assigned independent components but the average fluorescence lifetime values have been reported to obtain a qualitative analysis. Average fluorescence lifetime of LSZ was 1.83 ns while its complexes with AMTH and TZ had average fluorescence lifetime values of 1.79 and 1.85 ns, respectively. Thus, time dependent 16


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fluorescence studies suggested that the fluorescence lifetime of free and LSZ complexed with the dyes were not drastically altered. This firmly establishes that the quenching of LSZ fluorescence is mainly static in nature and due to ground state complexation. Förster Resonance Energy Transfer (FRET) Studies. Formation of dye complexes can also result in transfer of excited energy from LSZ to AMTH/TZ molecules. Energy transfer efficiency studies lead to the measurement of distances between the bound

Figure 4. Spectral overlap (shaded portion) of the emission spectrum of LSZ and absorption spectrum of (A) AMTH and (B) TZ.

AMTH/TZ and the interaction site on LSZ. This knowledge is essential to unravel the structural and conformational features associated with the binding process.64,65 The proximity of the AMTH/TZ molecules to the Trp residue of LSZ can be determined using FRET measurements. The donor-acceptor duo is regarded to be within the Förster distance when the emission spectrum of LSZ overlaps with the absorption spectrum of 17



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AMTH/TZ (Figure 4). FRET depends on the reciprocal of the sixth power of the distance between LSZ and AMTH/TZ (r) and also on the Förster radius (Ro) which is the critical distance at 50% energy transfer efficacy. Efficiency of energy transfer (E) can be determined using the equation

Ro 6 F E  1  Fo Ro 6  r 6

(6)

Ro can be calculated the relationship

Ro 6  8.8 1025 k 2n4 φJ

(7)

where k2 = spatial factor of orientation between the emission and absorption dipoles of LSZ and AMTH/TZ, respectively, n = refractive index of the medium and φ = fluorescence quantum yield of LSZ. Overlap integral (J) of the emission and absorption spectrum of LSZ and AMTH/TZ, respectively, is given by the equation 

 F( λ )ε( λ ) λ dλ 4

J

0



(8)

 F( λ )dλ 0

where F(λ) = fluorescence of LSZ at wavelength λ; ε(λ) = molar absorption coefficient of AMTH/TZ at the wavelength λ. For LSZ, k2 = 2/3, n = 1.336 and φ = 0.14.25,44 Using these values E, J, Ro and r were deduced to be 0.031, 1.05 × 10-14 cm3 L mol-1, 2.54 and 4.51 nms, respectively, for AMTH-LSZ binding reaction. For LSZ-TZ association the values of E, J, Ro and r were deduced to be 0.096, 1.50 × 10-14 cm3 L mol-1, 2.70 and 3.93 nms, respectively. Since the distance between the food colrants and the Trp residues of 18


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LSZ is substantially less than the upper limit of 8 nm value so there is a fair possibility of efficient energy transfer from LSZ to the AMTH/TZ molecules.25,65 Furthermore, all the criteria of energy transfer theory are obeyed in both the cases. Firstly, LSZ can produce fluorescence light, secondly there is sufficient overlap between the fluorescence spectrum of LSZ and the absorption spectra of the food colorants (Figure 4), and finally the value of r in either system is significantly lower than 8 nm. Additionally, the value of r was higher than Ro which suggested that both AMTH and TZ were capable of efficiently accepting energy from the Trp residues of LSZ. These observations firmly testify in favor of energy transfer between the two food colorants and LSZ, which results from ground-state complexation between the two binding species. Therefore, further analysis to estimate the binding constant and number of binding sites is completely justified. Estimation of the Binding Affinity and the Number of Binding Sites. From the above studies it can be clearly postulated that the fluorescence quenching resulted from strong interaction and subsequent complex formation between LSZ and AMTH and TZ. After establishing the proof of complex formation between LSZ and AMTH/TZ, the equilibrium binding constant (KA) and the number of binding sites (n) were subsequently calculated from the following equation25,66,67

log

(Fo  F)  log K A  n log[Q] F

(9)

The values of KA and n for LSZ-AMTH/TZ complexation at 288.15, 298.15 and 308.15 K were calculated by plotting log ((F0 − F)/F) against log [Q]. The KA and n values 19



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obtained from temperature dependent fluorescence studies are depicted in Table 1. The binding affinity values of AMTH and TZ to LSZ at 298.15 K were deduced to be (1.09 ± 0.10) × 106 and (4.43 ± 0.04) × 104 M-1, respectively. From the KA values it is clear that TZ has moderate affinity for LSZ while AMTH has remarkably strong affinity for LSZ. Such high affinity of small molecules for LSZ has seldom been observed. The numbers of binding sites (n) were deduced to be (1.25 ± 0.07) and (1.10 ± 0.03), respectively, at 298.15 K suggesting only one kind of binding site exist for both food colorants on LSZ. It is highly likely that the two food colorants bound close to a Trp residue of LSZ. It is worth mentioning here that the n values remained close to unity at all the temperatures studied (Table 1). Thus, 1 : 1 binding stoichiometry was envisaged for the complexation of AMTH and TZ with LSZ. Salt Dependent Fluorimetric Studies. Salt dependent fluorescence experiments were performed to understand the nature of the molecular forces driving the binding process. It was found that the equilibrium constant decreased for both food colorants with increasing ionic strength. The data obtained from salt dependent fluorimetric studies are presented in Table 2. Increase in [Na+] caused decrease in the KA values suggesting

destabilization

of

the

LSZ-AMTH/TZ

complexes

at

higher

salt

concentrations. Thus, the degree of interaction was dependent on the amount of salt present in the medium. However, the n values did not vary significantly and remained close to unity suggesting 1 : 1 complexation between the two binding species at all the salt conditions. The standard molar Gibbs energy change (ΔG0) also decreased in 20


Page 21 of 74

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magnitude with increasing [Na+] suggesting weakening of the binding interaction, that is, the spontaneity of the reaction decreased. Verification of the Debye-Hückel Limiting Law. Let us consider the equilibrium given below between the LSZ and AMTH: LSZ + AMTH ↔ LSZ-AMTH complex

K eq 

aLSZ AMTH c    LSZ AMTH  LSZ AMTH  K A  LSZ AMTH aLSZ aAMTH c LSZc AMTH  LSZ  AMTH  LSZ  AMTH

(10)

where a denotes the activity, c is the analytical concentration, and γ denotes the activity coefficient of the ions present in the solution. Taking logarithm of the above equation we get, or, ln Keq  ln KA  (ln  LSZAMTH  ln  LSZ  ln  AMTH )

(11)

or, RT ln Keq  RT ln KA  RT (ln  LSZAMTH  ln  LSZ  ln  AMTH ) 0 0 or, GI 0  G  RT (ln  LSZ AMTH  ln  LSZ  ln  AMTH )

(12)

(13)

In the above equations KA is the equilibrium constant while Keq is the equilibrium constant when the activity coefficients are equal to unity. ΔG0 is dependent on the activity coefficients, which deviate from unity when other ions are present in the system. ΔG0I→0 is the standard molar Gibbs energy change when activity coefficients are

21



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Page 22 of 74

Table 1 Binding Data Derived for AMTH and TZ Binding to LSZ from Spectrofluorimetric Studies at Different Temperatures. Temperature/K

Stern-Volmer quenching

Quenching rate

Apparent binding

constant (KSV/M-1)

constant (Kq/M-1s-1)

constant (KA/M-1)

288.15

(1.47±0.06)×105

(1.47±0.06)×1013

(1.58±0.12)×106

1.27±0.08

298.15

(9.60±0.06)×104

(9.60±0.06)×1012

(1.09±0.10)×106

1.25±0.07

308.15

(7.67±0.05)×104

(7.67±0.05)×1012

(8.26±0.09)×105

1.18±0.05

288.15

(1.99±0.03)×104

(1.99±0.03)×1012

(7.89±0.06)×104

1.18±0.05

298.15

(1.77±0.02)×104

(1.77±0.02)×1012

(4.43±0.04)×104

1.10±0.03

308.15

(1.40±0.02)×104

(1.40±0.02)×1012

(2.64±0.03)×104

1.08±0.02

n

AMTH

TZ

(τ0 = ~10-8 s).

22


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Table 2 Binding Data Derived for AMTH and TZ Binding to LSZ from Spectrofluorimetric Studies at Different Salt Concentrations. [Na+]

Apparent binding

/mM

(KA/M-1)

ΔG0

ΔG0t

ΔG0pe

/(kcal/mol)

/(kcal/ mol)

/(kcal/ mol)

n

AMTH

TZ

constant

10

(1.09±0.10)×106

1.25±0.07

-8.24±0.10

-7.22±0.10

-1.02±0.10

20

(8.57±0.09)×105

1.26±0.08

-8.09±0.09

-7.22±0.09

-0.87±0.09

50

(5.97±0.07)×105

1.25±0.07

-7.88±0.07

-7.21±0.07

-0.67±0.07

10

(4.43±0.04)×104

1.10±0.03

-6.34±0.04

-5.29±0.04

-1.05±0.04

20

(3.58±0.03)×104

1.11±0.04

-6.21±0.03

-5.32±0.03

-0.89±0.03

50

(2.40±0.03)×104

1.04±0.02

-5.97±0.03

-5.29±0.03

-0.68±0.03

23



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Page 24 of 74

unity, that is, at zero ionic strength. The following equation can be written based on the Debye-Hückel limiting law for dilute solutions68-70 2 2 2 1/2 GI00  G0  2.303RTA(ZHb  PF  ZHb  ZPF ) I

(14)

For a fixed system like AMTH-LSZ, ξ is a constant. Magnitude and sign of ξ depends on the difference between the square of charges of AMTH and LSZ. For aqueous CP buffer, at T=298.15 K, A = 0.509. Therefore,

GI00  G0  2094.28ξI1/2 or, G0  GI00  2094.28ξI 1/2

(15) (16)

Positive slope of the plot of ΔG0 vs I1/2 indicates a negative value of ξ. Additionally, the intercept of the plot gives the value of ΔG0 at I=0. Linear plots of ΔG0 vs I1/2 for the complexation of LSZ with both AMTH and TZ established the validity of the DebyeHückel limiting law (Figure S1). Such behavior has also been reported earlier for ligandprotein systems.14 Plots of ΔG0 vs I1/2 for the complexation of AMTH and TZ with LSZ afforded positive slopes, which implied that ξ is negative. Since the equilibrium constant reduced with increasing salt concentration so it implies negatively charged food colorants bind in the positively charged pockets of LSZ. We know,

   ZLSZ  ZAMTH  – ZAMTH 2  2ZLSZZAMTH 2

(17)

Since ZAMTH = negative, so ZLSZ must be positive. Therefore, ξ is negative, which reinforces the above observation. Similarly, for TZ also ZLSZ is positive and ξ is negative since it is a negatively charged dye like AMTH. From the intercept, the values of ΔG0 at 24


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I=0 were estimated to be (-8.51 ± 0.19) and (-6.64 ± 0.19) kcal/mol, respectively. Thus, the KA values at I=0 were calculated to be 1.73 × 106 and 7.37 × 104 M-1, respectively, for AMTH and TZ. Parsing of the Standard Molar Gibbs Energy Between Polyelectrolytic and Nonpolyelectrolytic Components. Both AMTH and TZ are anionic in nature, so electrostatic forces are likely to play a crucical role in their complexation with LSZ. As per the polyelectrolytic theory of Manning the slope of the plot of log KA versus log [Na+] is associated with the number of counterions released related by the relation71,72

N(ion)  (

 log KA )T , P   zψ  log[Na  ]

(18)

where ΔN(ion) is the number of ions released upon binding of AMTH/TZ, z is the apparent charge of the bound dye while ψ is the fraction of counterion bound per LSZ. Slopes of the plot of log KA versus log [Na+] were deduced to be -(0.375 ± 0.027) and (0.384 ± 0.027), respectively, for AMTH and TZ (Figure S2). This provided an estimation of the extent of electrostatic forces involved in the binding reaction of AMTH and TZ with LSZ. The low slope values suggested that though anionic still the charges on AMTH and TZ are weakly involved in the interaction. The G0 was partitioned between electrostatic (G0pe) and non-electrostatic (G0t) components (Figure 5). The polyelectrolytic contribution was calculated from the relationship73 0 Gpe  zψRT ln([Na ])

(19)

25



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Page 26 of 74

At 10, 20 and 50 mM [Na+], the ΔG0pe contributions were determined to be (-1.02 ± 0.10), (0.87 ± 0.09) and (-0.67 ± 0.07) kcal/mol, respectively, for AMTH which accounted for only 12.38, 10.75 and 8.50% of the total values of G0. For TZ, the ΔG0pe contributions were (-1.05 ± 0.04), (-0.89 ± 0.03) and (-0.68 ± 0.03) kcal/mol, respectively, which constituted 16.56, 14.33 and 11.39% of the total G0. The non-polyelectrolytic contribution (rG0t) was calculated from the difference between ΔG0 and ΔG0pe. At 10, 20 and 50 mM [Na+], the nonpolyelectrolytic contributions (G0t) were (-7.22 ± 0.10), (-7.22 ± 0.09) and (-7.21 ± 0.07) kcal/mol, respectively, for AMTH while for TZ the G0t values were (-5.29 ± 0.04), (-5.32 ± 0.03)

and

(-5.29

±

0.03)

kcal/mol,

respectively.

Thus,

although

Figure 5. Partitioned polyelectrolytic (ΔG0pe) (shaded) and non-polyelectrolytic (ΔG0t) (black) contributions to the standard molar Gibbs energy at 10, 20 and 50 mM [Na +] for the complexation of (A) AMTH and (B) TZ with LSZ.

AMTH and TZ are anionic the electrostatic contributions are less significant for their complexation

with

LSZ.

Hence,

non-polyelectrolytic 26


forces

like

hydrophobic

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interactions, π-π stacking, H-bonding, van der Waals interactions etc. appear to dominate the binding process and their magnitudes remained almost unaltered at all the salt concentration studied. Anisotropy Measurements. Anisotropy is a useful probe to measure the extent of flexibility

and/or

tumbling

motion

of

small

molecules

upon

binding

to

biomacromolecules. Anisotropy can also yield information on the most probable location of a small molecule in the macromolecular proteineous environment. Anisotropy measurements were performed as suggested by Larsson and colleagues.74 Anisotropy was determined using the equation

A

( Ivv- I vh G ) ( Ivv+ 2 I vh G )

(20)

where I denotes the intensity and the subscripts correspond to the vertical or horizontal positioning of excitation and emission polarizers. The instrumental correction factor for correcting the polarizing effects in the emission monochromator and detector is given by G = Ihv/Ihh. The rapid increase in fluorescence polarization anisotropy values of AMTH on addition of increasing concentrations of LSZ testified for the strong complexation of AMTH with LSZ (Figure S3). The binding of AMTH to LSZ decreased its mobility and freedom of motion thereby causing an increment in the anisotropy of AMTH in the complexed form. Furthermore, it can be suggested that the fluorophores of AMTH molecules are trapped within a motionally restricted environment in the presence of LSZ. The anisotropy of TZ did not increase in presence of LSZ and

27



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Page 28 of 74

remained virtually unaffected. This further testifies that the interaction between TZ and LSZ is much weaker in comparison to that of AMTH in conformity with the results from the spectroscopic studies (vide supra). Synchronous Fluorescence Studies. Conformational changes in LSZ upon binding with AMTH and TZ were studied using synchronous fluorescence spectroscopy.75 The shape and intensity of the synchronous fluorescence spectra is dependent upon the difference between the excitation and emission wavelengths (Δλ). From Miller’s theory we know

Figure 6. Synchronous fluorescence spectra of LSZ (curve 1, 10 µM) in the presence of (A) 2, 10, 20, 30, 40, 50, 60, 70 and 80 µM (curves 2-10) of AMTH, (B) 5, 10, 20, 30, 40, 50, 60, 70 and 80 µM (curves 2-10) of AMTH, (C) 5, 20, 40, 60, 80, 100, 120, 150 and 200 µM (curves 2-10) of TZ, (D) 10, 20, 40, 60, 80, 100, 120, 150 and 200 µM (curves 2-10) of TZ. Panels A and C represent the synchronous fluorescence spectra when Δλ = 60 nm and panels B and D represent the synchronous fluorescence spectra Δλ = 15 nm.

28


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that the synchronous fluorescence spectra of LSZ yield information about the microenvironment around the Tyr residues when Δλ is 15 nm and about the microenvironment around the Trp residues when Δλ is 60 nm.76 Effect of AMTH and TZ on the synchronous fluorescence spectra of LSZ is shown in Figure 6. AMTH and TZ caused significant quenching of the fluorescence intensity with bathochromic shifts of 5 and 4 nms, respectively, when Δλ = 60 nm. Thus, in the presence of AMTH and TZ the Trp residues of LSZ were shifted to a more hydrophilic environment leading to a greater exposure to the solvent molecules. However, when Δλ = 15 nm AMTH and TZ caused quenching of the LSZ fluorescence with a marginal bathochromic shift of 2 nms. Such

a

small

bathochromic

shift

indicated

very

little

alterations

in

the

microenvironment around the Tyr residues. Therefore, AMTH and TZ effected more pronounced alterations in the polarity around the Trp-62 and Trp-63 residues in comparison to the Tyr residues. This observation further reiterates the involvement of Trp residues in the binding process corroborating the data obtained from fluorescence quenching and FRET studies (vide supra). ANS Displacement Assay. Hydrophobic probe displacement assay using 8-anilino-1naphthalenesulfonic acid (ANS) was performed to probe the preferred binding site of AMTH and TZ on LSZ. A hydrophobic probe like ANS is very sensitive to the changes in the microenvironment of LSZ and can therefore afford information about the hydrophobic binding regions on the surface of LSZ.77 In this assay, binding studies were performed in the presence of ANS under identical conditions, and the relative 29



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Page 30 of 74

fluorescence (F/F0) was plotted as a function of the ligand concentration (Figure S4). According to Stryer77,78 the interaction of ANS with the solvent exposed hydrophobic clusters of LSZ would cause noteworthy augmentation of the ANS fluorescence. From these plots it can be seen that AMTH and TZ can compete with ANS for the hydrophobic regions of LSZ. Thus, AMTH and TZ can expel the bound ANS molecules and bind at the hydrophobic regions of LSZ. FTIR Studies. Fourier transform infrared (FTIR) spectroscopy was performed to monitor the conformational changes in LSZ upon binding of AMTH and TZ.79 FTIR technique is an efficient tool to monitor the conformational changes in LSZ since it has conformation sensitive spectral signature in the infra-red region.79 LSZ is a monomeric protein of 129 residues31,32 containing two structural domains. The α-domain comprises of four α-helices and a 310 helix. The β-domain is made up of a triple helical antiparallel β-sheet, a 310 helix, along with a long loop.79 FTIR spectrum of LSZ and its complexes with AMTH and TZ are shown Figure S5. The most intense band at 1652 cm–1 is due to the amide I vibration which arises mainly due to the stretching of the amide C═O groups.80,81 The amide II band at 1544 cm–1 arises due to the coupling of the in-plane N–H bending vibration with the C–N stretching vibration of the peptide bond.81 As can be seen from Figure S5 the intensity and position of the amide bands were affected in the presence of AMTH and TZ. The amide I band was shifted from 1652 cm–1 to 1649 cm–1 in the presence of both AMTH and TZ. The similarity in the spectral changes indicated that the essential characteristics of the secondary structure LSZ were intact in the presence of AMTH and TZ. The shift towards lower 30


Page 31 of 74

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wavenumber is assigned to the interaction between LSZ and AMTH/TZ which is probably driven by hydrogen bonding, electrostatic, hydrophobic and hydrophilic interactions.21,82

Figure 7. Far-UV CD spectral changes of LSZ (curve 1, 10 µM) on treatment with (A) 5, 20, 40, 60, 70 and 80 µM of AMTH (curves 2-7) and (B) 5, 20, 40, 50, 70 and 80 µM of TZ (curves 2-7).

CD Studies. Confirmatory proof in favor of the conformational changes in LSZ upon binding with AMTH and TZ was obtained by monitoring the changes in the CD spectra. The far-UV CD spectrum of uncomplexed LSZ comprised of two negative peaks at 208 and 222 nms (Figure 7). These two peaks are the features of a predominantly α-helical structure.26 The peak at 208 nm represents the π–π* transition of the α-helix while the minima at 222 nm represents the n–π* transition for the α-helix as well as the random coil.25,26 In the presence of increasing concentrations of AMTH and TZ, the spectrum of LSZ reduced in magnitude (Figure 7). This suggested a reduction in the helical content of LSZ and indicated that the complexation of AMTH and TZ induced secondary structural changes in LSZ. The deconvoluted far-UV CD spectrum of LSZ and its complexes with 31



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Page 32 of 74

AMTH and TZ are also shown in Figure S6. Upon deconvolution the far-UV CD spectrum of LSZ afforded minima at around 205, 217 and 231 nms. The deconvoluted far-UV CD spectrum of LSZ complexed with AMTH afforded minima at 206, 219 and 230 nms while upon complexation with TZ the minima were observed at 204, 212 and 226 nms. The helical content of the protein and its complexes with the dyes were calculated using the following equation83,84 MRE[] 

observed CD(m deg) Cnl  10

(21)

Here MRE = mean residue ellipticity (deg . cm2 . dmol-1), C = concentration of LSZ (M), n = total number of amino acid residues and l = path length of the cuvette (cm) where the titration was performed. The α-helical content of LSZ was calculated uisng the equation   helix(%)  [

[]222  3000 ] 100 36000  3000

(22)

The α-helical content of uncomplexed LSZ was deduced to be 33% whereas those of AMTH and TZ bound LSZ were calculated to be 14% and 22%, respectively. Hence, both AMTH and TZ induced significant loss of the helical stability of LSZ, the effect being much more pronounced in case of the former. The binding also affected unfolding of LSZ conformation with the extended polypeptide chains revealing the hydrophobic cavities with concomitant exposure of the aromatic amino acid residues. To investigate the changes in the tertiary structure of LSZ caused by the binding of AMTH and TZ the near-UV CD spectral experiments were performed. CD spectra of proteins in the region, 250–300 nm, originates owing to the presence of disulphide bonds and the aromatic 32


Page 33 of 74

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chromophores of the amino acids (Trp, Tyr, Phe).25,40 The CD maxima at 284, 289 and 294 nms are attributed to the transitions of the Trp residues.25,40 AMTH caused significant alterations in the near-UV CD spectra of LSZ while TZ had very little effect (Figure S7). Such alterations imply changes in the microenvironment of the aromatic amino acid side chains owing to complexation with the indole rings of the Trp residues, 62, 63 or 108, leading to moderate unfolding/enhanced flexibility of the tertiary structure of LSZ. The first two residues are present on the molecular surface while the third one is at the end of the cleft. Though the interpretation of the alterations in the tertiary structure of LSZ is not conclusive, but along with the far-UV spectral changes and other spectroscopic studies, it testifies for the strong interaction of AMTH with LSZ. DSC Studies. Ligands on binding with biomacromolecules can alter their thermal stability. This change is often manifested by an increase/decrease in the thermal denaturation temperature of the biomacromolecule. DSC is an efficient tool to monitor the thermal transitions in biomacromolecules in solution. It can also be employed to study the energetics associated with temperature dependent protein folding-unfolding process.85,86 LSZ denatured exhibiting a single endothermic peak at (339.39 ± 0.08) K (Figure 8, curve 1). Upon complexation with AMTH the thermal denaturation temperature of LSZ was reduced to (334.07 ± 0.20) K while in the presence of TZ the melting temperature remained almost unaffected (Figure 8, curves 2 and 3). So AMTH effected a decrease in the melting temperature of LSZ by 5.32 K suggesting that the protein-dye

33



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Page 34 of 74

Figure 8. DSC thermogram of LSZ (20 µM, curve 1), LSZ-AMTH (curve 2) and LSZ-TZ (curve 3) complex at D/P (dye : protein molar ratio) = 25.

complexation destabilizes LSZ. In this context, it is worth mentioning that there were no noticeable changes in the shape of the DSC thermograms in the presence of AMTH and TZ suggesting that the complexation did not alter the denaturation process. Additionally, the enthalpy of transition of LSZ (ΔHcal) enhanced from (143.80 ± 1.97) kcal/mol to (335.40 ± 12.10) kcal/mol in presence of AMTH while in presence of TZ ΔHcal was reduced to (40.17 ± 0.53) kcal/mol. Characterization of the Complexation by ITC. ITC was employed for the calorimetric characterization of the AMTH/TZ -LSZ binding reaction. Solutions of AMTH, TZ and LSZ were extensively degassed on the MicroCal’s Thermovac unit before the experiment to avoid bubble formation. Fixed aliquots of AMTH and TZ solutions were titrated from the preprogrammed rotating syringe into the LSZ solution at fixed time intervals. Upper panels of Figure 9A and B depicts the ITC 34


Page 35 of 74

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Figure 9. ITC profiles for the titration of (A) AMTH and (B) TZ into LSZ solution at 298.15 K. The upper panel represents the raw data for the titration of successive aliquots of the two azo dyes into LSZ solution (curve at the bottom), along with the dilution profiles (curves on the top offset for clarity). The bottom panel shows the integrated heat data after correction of heat of dilution. The symbols (■) in this panel represent the data points and the solid lines represent the best-fit data.

profiles for the complexation of AMTH and TZ with LSZ at 298.15 K. The binding reaction was exothermic in both cases as it afforded negative peaks in the plot of power versus time. Each of the spikes in the figure corresponds to a single injection and the actual heat of 35



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Page 36 of 74

interaction was obtained by subtracting the heat of dilution of AMTH and TZ. This heat of dilution was obtained from the titration of identical amounts of AMTH and TZ into the buffer alone. The bottom panel of Figure 9A and B presents the corrected heats as a function of molar ratio. The experimental data were fitted to a 'one set of site' binding model since the integrated heat data exhibited only one binding event. The Ka values obtained from ITC at 298.15 K for the binding of AMTH and TZ were (1.01 ± 0.10) × 106 and (4.24 ± 0.06) × 104 M-1, respectively. These values are closely matching with those obtained from the fluorescence studies (vide supra). AMTH-LSZ complexation was driven purely by negative standard molar enthalpy contribution of (-9.98 ± 0.08) kcal/mol while the entropic contribution was unfavorable (TΔS0 = -1.79 ± 0.02 kcal/mol). In sharp contrast, the binding of TZ to LSZ was highly entropy driven (TΔS0 = 5.91 ± 0.03 kcal/mol) with a marginal negative enthalpic contribution of (-0.40 ± 0.03). So, even though both AMTH and TZ are azo dyes but there were remarkable differences in the energetics of their interaction with LSZ. Such a difference can be attributed to their structural variations leading to difference in the balance of the forces governing their complexation with LSZ. The large negative ΔH0 primarily originates due to the restriction imposed on the mobility of the AMTH molecules within the low dielectric macromolecular core of LSZ. Besides, the large negative ΔH0 is also a consequence of the strengthening of the H-bonding interactions between the amino acid residues of LSZ and AMTH. van der Waals interactions, originating as a consequence of the hydrophobic effect, also contribute to the negative ΔH0.87 The large positive TΔS0 in the case of TZ originates from the perturbation and release of condensed counterions and alterations 36


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in the layers of hydration of LSZ. Specific electrostatic interaction between the two binding species in the aqueous medium may be also responsible for the positive ΔS0 and negative ΔH0 values.88 The ΔG0 value for the complexation of LSZ with AMTH and TZ were calculated to be (-8.19 ± 0.10) and (-6.31 ± 0.06) kcal/mol, respectively. These negative values of the Gibbs energy change testified for the spontaneity of AMTH/TZ-LSZ binding reaction. Temperature dependent ITC experiments lend crucial insights into the type, nature and magnitude of forces governing the binding reaction, and also permits the determination of standard molar heat capacity change values (ΔCp0). The ΔCp0 values for the interaction of AMTH and TZ with LSZ were determined using the relationship, ΔCp0=[∂(ΔH0)/∂T]P

(23)

The temperature dependent ITC experiments were performed at 288.15, 293.15, 298.15 and 308.15 K. The pH of the buffer remained almost unaltered in this temperature range. All the thermodynamic parameters deduced at these temperatures for the complexation are collated

in

Tables

3

37


and

4.


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 38 of 74

Table 3 Thermodynamic Parameters for the Association of AMTH with LSZ from ITC Experiments at Different Temperatures.a Temperature/K

ΔH0/

TΔS0/

ΔG0/

ΔCp0/

(kcal/ mol)

(kcal/ mol)

(kcal/ mol)

(cal /K/ mol)

10-6 Ka/M-1

aAll

288.15

1.53±0.12

-9.81±0.07

-1.66±0.05

-8.15±0.12

293.15

1.26±0.11

-9.91±0.07

-1.73±0.04

-8.18±0.11

298.15

1.01±0.10

-9.98±0.08

-1.79±0.02

-8.19±0.10

308.15

0.81±0.10

-10.21±0.09

-1.88±0.01

-8.33±0.10

the data in this table are the average of four determinations.

38


-19.83±1.24

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Table 4 Thermodynamic Parameters for the Association of TZ with LSZ from ITC Experiments at Different Temperatures.a Temperature/K

ΔH0/

TΔS0/

ΔG0/

ΔCp0/

(kcal/mol)

(kcal/mol)

(kcal/mol)

(cal/K/mol)

10-4 Ka/M-1

aAll

288.15

7.88±0.07

-0.33±0.02

6.12±0.05

-6.45±0.07

293.15

6.14±0.06

-0.37±0.02

6.05±0.04

-6.42±0.06

298.15

4.24±0.06

-0.40±0.03

5.91±0.03

-6.31±0.06

308.15

2.53±0.05

-0.47±0.04

5.74±0.01

-6.21±0.05

the data in this table are the average of four determinations.

39


-6.91±0.89


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All the thermograms for both AMTH and TZ were exothermic and showed single binding event at all the temperatures studied. The Ka values decreased with increase in temperature indicating destabilization of the LSZ-AMTH/TZ complexes at elevated temperatures. Besides, there were remarkable variations in the ΔH0 and TΔS0 values. The negative ΔH0 values showed an increase in magnitude whereas the positive TΔS0 values decreased with increase in temperature. Plots of variation of ΔH0 with T are depicted in Figure S8. Slope of these plots afforded the ΔCp0 values to be (-19.83 ± 1.24) and (-6.91 ± 0.89) cal/mol K, respectively, for AMTH and TZ. Negative ΔCp0 values are a salient feature of many biomolecular interactions18,19,22,89,90 and is indicative of particular/specific alterations in the hydrophobic or polar group hydration. Additionally, negative ΔCp0 values are also suggestive of the involvement of a strong hydrophobic component in the binding reaction. Shift in the solvent accessible surface area can also contribute to the observed ΔCp0 values.91,92 In this study, the values of ΔCp0 obtained were non-zero suggesting dependence of ΔH0 on T. The higher magnitude of ΔCp0 for AMTH suggests greater extent of hydrophobic desolvation effects and conformational changes in LSZ consequent to AMTH binding in conformity with the CD and DSC data. The Gibbs energy components (ΔG0hyd) for the hydrophobic transfer step of the interaction of LSZ with AMTH and TZ were deduced to be -1.59 and -0.55 kcal/mol, respectively, using the relationship, ΔG0hyd = (80 ± 10) × ΔCp0, proposed by Record.93 Chaotrope Induced Denaturation Studies

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Fluorescence studies enable us to examine the changes in the tertiary structure of LSZ which also provides insights into its environmental stability. LSZ is made up of aromatic amino acids like most of the globular proteins. These amino acid residues are fluorescent but the fluorescence of the Trp moiety dominates the fluorescence of the biomacromolecule. Influence of urea (chaotrope) induced denaturation of LSZ on its efficacy to form complexes and on the overall photophysics of AMTH and TZ was studied. Urea caused significant changes in the steady state fluorescence spectra of AMTH and TZ bound LSZ. Figure S9 highlights the changes in the emission spectra of LSZ complexed with AMTH and TZ upon addition of increasing concentration of urea. Addition of the chaotrope caused an enhancement in the emission intensity of LSZ. This enhancement is a consequence of the exposure of the Trp residues embedded in the hydrophobic core of LSZ.94 The enhancement in the relative fluorescence intensity can then be used as a tool to monitor the extent of unfolding/denaturation of LSZ. Besides, the fluorescence spectrum of LSZ undergoes shift to a somewhat longer wavelength with the increasing concentration of urea.94 In this context, it is pertinent to mention that the shift was much more pronounced at a lower urea concentration for AMTH than TZ suggesting AMTH aided the urea induced denaturation of LSZ to a greater extent. This is in conformity with the results of CD and DSC studies where AMTH was shown to induce unfolding of LSZ more effectively than TZ. Changes in the fluorescence spectrum of LSZ bound AMTH and TZ upon addition of the chaotrope was also studied. Figure S10 shows the changes in the relative emission intensity of LSZ bound AMTH and TZ with increasing urea concentration. It is evident that addition 41



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of the chaotrope to the LSZ-AMTH/TZ complex caused progressive enhancement of the emission intensity of AMTH and TZ. This can explained on the basis that chaotrope addition caused destabilization of the LSZ-AMTH/TZ complex thereby leading to the exposure of more AMTH and TZ molecules to the bulk aqueous buffer solution compared to the complexed state. This exposure of the earlier bound AMTH and TZ molecules lead to an enhancement in the overall fluorescence intensity. Chaotropes such as urea can also effectively

expel

water

molecules

present

adjacent

to

the

probe in the

LSZ

microenvironment with simultaneous denaturation of LSZ.59,95,96 Hence, the chaotrope effected destabilization of the LSZ-AMTH/TZ complex is associated with a considerably higher exposure of the probe to the bulk aqueous buffer solution in comparison to the bound state in the native conformation of LSZ,95 thereby leading to an enhancement in the emission intensity of the probe. AFM Imaging Study AFM has been used here to probe whether the binding of AMTH/TZ induces any significant morphological changes in LSZ. The concentration of LSZ was kept 100 nM and its complexes with AMTH and TZ were prepared having D/P (dye : protein molar ratio) value equal to 1. Figure 10 depicts the AFM images of free LSZ and its complexes with AMTH and TZ in terms of topography along with the corresponding three-dimensional representations. The AFM imaging studies clearly testified for the complexation between LSZ and AMTH/TZ. Although the binding reaction did not induce any abrupt topographical alterations but there were subtle changes which resulted in an enhancement 42


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in the size of LSZ. The smaller molecules may represent the unreacted species present in the medium. There was a marked enhancement in the diameters of the LSZ molecules in presence of AMTH/TZ. This enhancement may be interpreted in terms of aggregation of

Figure 10. AFM images of LSZ (A, B) and its complexes with AMTH (C, D) and TZ (E and F). The corresponding graphs represent the height of LSZ and its complexes with AMTH and TZ.

the macromolecule in presence of the two anionic dyes. The extent of enhancement was different for the two complexes showing that the extent of aggregation is dependent upon the binding affinity of AMTH and TZ for LSZ. The average diameter of uncomplexed LSZ varied in the range 15-20 nm. In presence of AMTH the average diameter of the complex varied in the range 80-100 nm while in presence of TZ it varied in the range 40-56 nm. Thus, AMTH effected a markedly higher enhancement in the average diameter of LSZ in comparison to TZ. This greater enhancement can be attributed to a greater degree of 43



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macromolecular aggregation in presence of AMTH due to its higher affinity for LSZ. Hence, AFM imaging revealed that binding of both AMTH and TZ to LSZ (1:1 complexation) lead to an enhancement in the macromolecular diameter with the former causing a greater enhancement due to stronger complexation with LSZ. Inhibitory Effect on LSZ Fibrillogenesis Monitored by ThT and CR Assay Incubation of LSZ at low pH and high temperature causes amyloid fibrillogenesis. Temperature, ionic strength as well as LSZ concentration significantly influences the lag time of fibril formation.42,97,98 Here, LSZ was dissolved (10 mg/ml) in glycine-HCl buffer of pH ~2.20 and heated at 333.15 K with continuous agitation in the presence and absence of 25 µM AMTH and 100 µM TZ. At higher AMTH concentration turbidity appeared immediately upon addition of the dye to LSZ; therefore the concentration of AMTH was kept 25 µM for monitoring its inhibitory effect on fibrillogenesis. ThT fluorescence and CR absorption spectroscopy was checked to monitor the formation and growth of amyloid fibrils. ThT is a histochemical fluorescent staining agent widely used for characterizing the genesis and development of amyloid fibrils.48,99 The fluorescence intensity of ThT

increased

significantly upon complexation with the highly ordered linear array of β-sheet structure of amyloid fibrils.42,48,100 Upon complexation with ordered array of β-strands, ThT emits fluorescence which can be used as a convenient handle for monitoring the fibrillogenesis kinetics. The ThT fluorescence intensity at 485 nm for LSZ sample enhanced rapidly from 5 to 7 hrs, thereafter attaining an equilibration plateau (Figure 11). To study the effects of

44


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AMTH/TZ on fibrillogenesis, the ThT fluorescence intensities of LSZ samples in the presence and absence of the azo dyes was compared.

Figure 11. Variation of the fluorescence intensity of ThT at 485 nm with time for LSZ samples in the absence (•) and presence of AMTH (■) and TZ (▲).

Figure 11 clearly shows that the variation in the ThT fluorescence intensities as a function of time. Each plot is sigmoidal in nature comprising of a lag phase where there is no noticeable fluorescence variation, followed by a growth phase where there is a sudden remarkable enhancement in fluorescence and finally an equilibrium phase where ThT fluorescence reaches saturation. From Figure 11 it can be seen that in presence of AMTH there was a noticeable delay in fluorescence enhancement (from 7 to 8 hr) suggesting AMTH effectively delayed the genesis and growth of amyloid fibrils. However, in presence of TZ no such noticeable delay in fluorescence enhancement was observed which signified that TZ did not delay the process fibrillogenesis. For LSZ the lag phase was observed until 5 hrs while for LSZ samples treated with AMTH and TZ the lag phase lasted till 7 and 5.5 hrs, respectively. This lag phase was accompanied by a 45



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Page 46 of 74

growth phase in the range 5-7, 7-8 and 5.5-6.5 hrs, respectively, for LSZ and LSZ coincubated with AMTH and TZ. The growth phase was followed by an equilibration plateau in all the cases. Furthermore, it was also observed that co-incubation of LSZ with 25 and 100 µM of AMTH and TZ, respectively, resulted in reduction of ThT fluorescence suggesting both the dyes had an inhibitory effect on fibrillogenesis (Figure 11). The percentage of ThT fluorescence reductions at 9 hr incubation were calculated using the following equation

% reduction in ThT fluorescence=

Io -I ×100% Io

(24)

where, Io and I are the ThT fluorescence of LSZ fibrils after appropriate corrections for inner filter effect in the absence and presence of AMTH/TZ. AMTH and TZ reduced the ThT fluorescence of mature fibrils by 47% and 73%, respectively, at 9 hr incubation. The higher reduction of ThT fluorescence by TZ in comparison to AMTH may be attributed to its higher concentration used in the study. CR binding assay is a complementary tool to detect the amyloid fibril formation in LSZ samples. Hence, CR binding assay was further employed to confirm the fibril-like nature of the LSZ samples. Tissues having amyloid deposits when stained with CR exhibit apple green birefringence in the presence of cross polarized light.48,101,102 Furthermore, binding with amyloid fibrils present in LSZ resulted in hyperchromicity in absorbance along with a red-shift in the maximum of CR to ~540 nm. Figure 12 depicts the change in CR absorbance at the respective absorption maxima for the LSZ 46


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samples in the absence and presence of AMTH and TZ at varying time intervals. It can be seen there is a considerable enhancement in the absorption of LSZ samples stained with CR along with a concomitant red shift in the absorption maxima. Such enhancement and red shift signifies the occurrence of ordered fibrillar structure containing β-strands in

Figure 12. Variation of the absorbance of CR at the respective absorption maxima with time for LSZ samples in the absence (•) and presence of AMTH (■) and TZ (▲).

the LSZ samples. In the presence of 25 µM AMTH and 100 µM TZ there was a significant reduction in the maximum absorbance suggesting a decrease in the formation of amyloid fibrillar species associated with cross β-pleated sheet. Furthermore, in case of AMTH there was a time delay in the enhancement of CR absorbance suggesting AMTH delays fibril formation in LSZ. This further reiterates our ThT fluorescence assay data where the enhancement of ThT fluorescence was delayed in presence of AMTH. Hence, ThT fluorescence and Congo red absorbance binding 47



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Page 48 of 74

assay unequivocally establishes that both the azo dyes has a concentration-dependent mitigating effect on the aggregation/fibril formation of LSZ. Secondary Structural Changes in LSZ in the Absence and Presence of the Dyes. To examine the conformational changes associated with fibril formation in LSZ we monitored the far-UV CD spectra of the LSZ samples in the absence and presence of AMTH and TZ. Initially, the LSZ samples in the absence and presence of AMTH/TZ exhibited minima at 209 and 222 nms which are characteristic of a predominantly αhelical structure (Figure S11). However, upon prolonged heating and concomitant stirring, the far-UV CD spectrum of LSZ showed a remarkable structural transition to a highly amyloidogenic β-sheet rich conformation. The structural transformation is characterized by a minimum at 218 nm. This structural transition in LSZ occurred around 6 hr while in presence of AMTH the structural transition was delayed to 7.5 hr. However, in presence of TZ the time period required for this structural transition remained virtually unaffected and occurred around 6 hr. In the presence of azo dyes the minima varied in the range 216-220 nm depending upon the incubation time and the nature/concentration of the dye used (Figure S11). Overall these observations permit us to conclude that AMTH can delay the α-to-β transition of LSZ very potently which in turn signifies that it can arrest the amyloid fibril-forming propensity of LSZ. Furthermore, intensity of the minima in the range 216-220 nm was found to comparatively greater at 9 hr suggesting the formation of more mature fibrils with time.

48


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NR Binding Assay. Effect of AMTH/TZ on the tertiary structure/surface hydrophobicity of LSZ was monitored by following the evolution of NR fluorescence emission with increasing incubation time. NR being a nonionic lipophilic fluorescent dye is often used as a tool to measure the lipid content inside the cell, microenvironmental change of biomolecules, as well as the polarity of many organic solvents.48,103 Initially, the emission maxima (λmax) of NR fluorescence emission was centered around 650 nm but after 6 hr it shifted to 620 nm and thereafter it varied marginally (Figure S12). The shift was markedly delayed in presence of AMTH to 7.5 hr while TZ did not alter the time period required for this shift significantly (Figure S12). This blue shift in the emission maxima was also accompanied by a marked fluorescence enhancement. As depicted in Figure S13 the emission maxima of NR fluorescence of the LSZ sample showed no noticeable increase in intensity in the first 5 hr of incubation. Thereafter, there was a slight increase at 5.5 hr followed by a dramatic enhancement in the 6-7 hr range. Thereafter, it reached an equilibration plateau suggesting saturation of NR fluorescence emission which can be related to the formation of completely mature fibrils. In case of AMTH this dramatic increase was considerably delayed and required 7.5-8 hr of incubation while in presence of TZ this enhancement almost coincided with LSZ and occurred in the 5.5 to 6 hr range (Figure S13). The blue shift with concomitant enhancement of NR fluorescence emission is indicative of the exposure of hydrophobic clusters which can be interpreted in terms of α-to-β transition of LSZ due to amyloid fibril formation. The drop in fluorescence intensity of NR fluorescence emission in 49



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Page 50 of 74

presence of TZ can be attributed to the 4-folds higher concentration of TZ used in this study. AFM Imaging of Fibrils. In order to attain further insights into the inhibitory effect of the azo dyes on fibrillogenesis AFM was employed. AFM enable us to monitor the morphological features of LSZ samples in the absence and presence of azo dyes at varying time intervals of incubation. As depicted in Figure 13A the LSZ samples displayed large quantities of shorter and unbranched fibrils at 6 hr incubation. After 9 hr of incubation the fibrils were found to be longer, compact and more matured which are the salient features of amyloid fibrils (Figure 13B). Figure 13C clearly depicts that in presence of AMTH no fibrils were formed after 6 hr of incubation. Only aggregates of LSZ molecules are evident here indicating clearly that AMTH can delay fibrillogenesis effectively. In Figure 13D we can clearly see that after 9 hr of incubation AMTH had an inhibitory effect on amyloid fibril formation. The fibrils were shorter, less matured and sparsely populated. In presence of TZ also fibril formation was inhibited. After 6 hr of incubation comparatively less dense, shorter and broken fibrils are formed while 9 hr of incubation procured fibrils which were comparatively shorter, less dense and less matured than the ones produced after 9 hr incubation of LSZ alone (Figure 13E and F). Thus AFM imaging conclusively indicate that AMTH and TZ attenuates amyloid fibril formation efficiently. Overall, ThT and CR assay in conjunction with AFM suggest that exposure of LSZ to AMTH and TZ results in bona fide suppression/prevention of amyloid fibril formation. 50


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Figure 13. AFM images of LSZ samples in the absence (A, B) and presence of AMTH (C, D) and TZ (E, F). Panels A, C and E represent the images after 6 hr of incubation while panels B, D and F represent the images after 9 hr of incubation.

In this context it is also pertinent to mention that the several research groups have probed the inhibitory effect of small molecules on fibrillogenesis in LSZ under varying conditions.38,48,104-117 Inhibitory effect of many small molecules like aromatic polyphenols,104–108 benzofurans,109 acridines,110 as well as flavones have been investigated.111–113 Influence of several biocompatible dyes like the food additive brilliant blue FCF,114 fast green FCF,115 brilliant blue G,116 methylene blue,117,118 Congo red119,120 and its lipophilic analog Chrysamine-G121 on fibrillogenesis have also been studied. Most of these molecules exhibited a concentration/dose-dependent inhibitory action against fibrillogenesis in protein. For example, the effect of food additives 51



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brilliant blue FCF and fast green FCF on fibrillogenesis in LSZ was studied at varying molar ratios and both the dyes showed a concentration-dependent inhibitory influence on fibrillation.114,115 The inhibitory effect of the non-toxic dietary pigment curcumin on fibrillogenesis in LSZ has also been investigated in the concentration range 1 to 100 μM and it was observed from ThT fluorescence emission assay (percentage reduction in ThT fluorescence) that curcumin inhibited fibrillogenesis more effectively at higher concentrations.105 Based on the same ThT fluorescence assay the inhibitory potency of AMTH and TZ were found to comparatively lower than that of curcumin at 25 and 100 μM concentrations, respectively. However, carnosine, a naturally occurring mammalian dipeptide, has been reported to inhibit fibrillogenesis at much higher concentrations (10 to 50 mM) than AMTH and TZ.48 Such screening and identification of small molecules that are potent inhibitors of fibrillogenesis is essential as it might lead to the development of better anti-amyloid therapeutics. CONCLUSIONS Both AMTH and TZ showed good affinity towards LSZ. However, the binding affinity of AMTH was significantly higher in comparison to TZ. The binding involved intimate ground state complexation and close contact with the Trp-62 and 63 residues at the cleft region of LSZ dominated by non-polyelectrolytic forces, such as, hydrophobic interaction, van der Waals interaction, H-bonding etc. The binding led to conformational changes in the protein, the effect being more pronounced in the case of the AMTH. AMTH thermally destabilized LSZ while TZ did not have any noticeable 52


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effect. The binding involved stark differences in the energetics of interaction. The binding of AMTH was purely enthalpy driven while TZ complexation was driven by large positive entropy changes. The equilibrium constant for AMTH-LSZ complexation was remarkably high and of the order of 106 M-1. Such high affinity is scarcely observed for ligand-protein interactions suggesting AMTH could lead to impairment of LSZ activity and thereby pose biological toxicity to humans. Furthermore, AMTH delayed the fibrillation of LSZ potently at 25 μM concentration while TZ affected no such delay in fibrillogenesis even at 100 μM concentration. However, both azo dyes had a significant inhibitory influence on amyloid fibril formation as evidenced by ThT fluorescence assay and AFM imaging. The present study advances new insights into the anti-amyloidogenic role of azo dyes that may be useful for their rational design as novel anti-amyloidogenic agents. The quest for potential inhibitors of amyloid fibrillation, and protein aggregation is a practical and viable therapeutic approach. Hence, such antiamyloidogenic agents might prove highly useful for the remedial treatment of protein aggregation diseases like amyloidoses. ASSOCIATED CONTENT Supporting Information Figures S1-13 depicting the variation of ΔG0 versus I1/2, variation of log KA versus log [Na+], variation of anisotropy versus P/D (LSZ/AMTH molar ratio), ANS displacement assay for LSZ in the presence of AMTH and TZ, FTIR spectra of LSZ, LSZ-AMTH and LSZ-TZ complex, deconvoluted far-UV CD spectra of LSZ and its complexes with 53



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AMTH and TZ, near-UV CD spectral changes of LSZ on treatment with AMTH and TZ, variation of ΔH0 versus T, changes in the fluorescence spectra of LSZ complexed with AMTH and TZ in the presence of urea, bar chart representing the variation of relative fluorescence intensity of AMTH and TZ complexed with LSZ against increasing urea concentration, variation in the far-UV CD spectra of LSZ in the presence of AMTH/TZ at varying incubation times, variation in the emission maxima of NR with time for LSZ samples in the absence and presence of AMTH/TZ and variation of the fluorescence intensity of NR at the emission maxima with time for LSZ samples in the absence and presence

of

AMTH/TZ

are

available

free

of

charge

via

the

Internet

at

http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors Phone: +91 33 2472 4049. Fax: +91 33 2473 0284/5197. E-mail: [email protected] (A. Basu) / [email protected] (G. Suresh Kumar). Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS Financial assistance from the network project GenCODE (BSC0123) of the Council of Scientific and Industrial Research, Govt. of India is gratefully acknowledged. AB is a

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recipient of Research Associateship from GenCODE (BSC0123) of Council of Scientific and Industrial Research, Govt. of India. REFERENCES (1) Mpountoukas, P.; Pantazaki, A.; Kostareli, E.; Christodoulou, P.; Karelia, K.; Poliliou,

S.; Mourelatos, C.; Lambropoulou, V.; Lialiaris, T. Cytogenetic

Evaluation and DNA Interaction Studies of the Food Colorants Amaranth, Erythrosine and Tartrazine. Food Chem. Toxicol. 2010, 48, 2934-2944. (2) King-Thom, C. Mutagenicity and Carcinogenicity of Aromatic Amines Metabolically Produced from Azo Dyes. J. Environ. Sci. Health C 2000, 18, 51–74. (3) Ratna; Padhi, B. S. Pollution due to Synthetic Dyes Toxicity & Carcinogenicity Studies and Remediation. Int. J. Environ. Sci. 2012, 3, 940-955. (4) Chowdhury, G. Human Health Perspectives on Environmental Exposure to Benzidine: A review. Chemosphere 1996, 32, 267-291. (5) Zhang, G.; Ma, Y. Mechanistic and Conformational Studies on the Interaction of Food Dye Amaranth with Human Serum Albumin by Multispectroscopic Methods. Food Chem. 2013, 136, 442-449. (6) Nevado, J. J. B.; Cabanillas, C. G.; Salcedo, A. M. C. Simultaneous Spectrophotometric Determinat n of Three Food Dyes by Using the First Derivative of Ratio Spectra. Talanta 1995, 42, 2043-2051.

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(7) Tsuda, S.; Murakami, M.; Matsusaka, N.; Kano, K.; Taniguchi, K.; Sasaki, Y. F. DNA Damage Induced by Red Food Dyes Orally Administered to Pregnant and Male Mice. Toxicol. Sci. 2001, 61, 92–99. (8) Kashanian, S.; Zeidali, S. H. DNA Binding Studies of Tartrazine Food Additive. DNA Cell Biol. 2011, 30, 499-505. (9) Rowe, K. S.; Rowe, K. J. Synthetic Food Coloring and Behavior: A Dose Response Effect in a Double-Blind, Placebo-Controlled, Repeated-Measures Study. J. Pediatr. 1994, 125, 691-698. (10) Amin, K. A.; Abdel Hameid, H.; Abd Elsttar, A. H. Effect of Food Azo Dyes Tartrazine and Carmoisine on Biochemical Parameters Related to Renal, Hepatic Function and Oxidative Stress Biomarkers in Young Male Rats. Food Chem. Toxicol. 2010, 48, 2994–2999. (11) Messori, L.; Gabbiani, C.; Casini, A.; Siragusa, M.; Vincieri, F. F.; Bilia, A. R. The Reaction of Artemisinins with Haemoglobin: A Unified Picture. Bioorg. Med. Chem. 2006, 14, 2972-2977. (12) Wang, Y. -Q.; Zhang, H. -M.; Tang, B. -P. The Interaction of C.I. Acid Red 27 with Human Hemoglobin in Solution. J. Photochem. Photobiol. B 2010, 100, 76-83. (13) Pan, X.; Qin, P.; Liu, R.; Wang, J. Characterizing the Interaction Between Tartrazine and Two Serum Albumins by a Hybrid Spectroscopic Approach. J. Agric. Food Chem. 2011, 59, 6650-6656.

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(14) Bolel, P.; Datta, S.; Mahapatra, N.; Halder, M. Spectroscopic Investigation of the Effect of Salt on Binding of Tartrazine with Two Homologous Serum Albumins: Quantification by use of the Debye-Hückel Limiting Law and Observation of Enthalpy-Entropy Compensation. J. Phys. Chem. B 2012, 116, 10195-10204. (15) Wang, Y. -Q.; Zhang, H. -M. Investigations on the Binding of Human Hemoglobin with Orange I and Orange II. J. Photochem. Photobiol. B 2012, 113, 1421. (16) Peng, W.; Ding, F.; Peng, Y. K.; Jiang, Y. T.; Zhang, L. Binding Patterns and Structure–Affinity Relationships of Food Azo Dyes with Lysozyme: A Multitechnique Approach. J. Agric. Food Chem. 2013, 61, 12415-12428. (17) Li, Y.; Wei, H.; Liu, R. A probe to study the toxic interaction of tartrazine with bovine hemoglobin at the molecular level, Luminescence 2014, 29, 195–200. (18) Basu, A.; Suresh Kumar, G. Study on the Interaction of the Toxic Food Additive Carmoisine with Serum Albumins: A Microcalorimetric Investigation. J. Hazard. Mater. 2014, 273, 200-206. (19) Basu, A.; Suresh Kumar, G. Targeting Proteins with Toxic Azo Dyes: A Microcalorimetric Characterization of the Interaction of the Food Colorant Amaranth with Serum Proteins. J. Agric. Food Chem. 2014, 62, 7955-7962. (20) Basu, A.; Suresh Kumar, G. Interaction of Toxic azo Dyes with Heme Protein: Biophysical Insights into the Binding Aspect of the Food Additive Amaranth with Human Hemoglobin. J. Hazard. Mater. 2015, 289, 204–209. 57



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Figure Legends Figure 1. Molecular structure of (A) AMTH and (B) TZ. Figure 2. Absorption spectral changed of (A) AMTH (5.40 µM, curve1) treated with 5.40, 16.20, 37.80, 59.40, 81.00 and 102.60 µM of LSZ (curves 2–7) and (B) TZ (4.99 µM, curve1) treated with 15, 30, 50, 70, 90 and 110 µM of LSZ (curves 2–7). Figure 3. Steady state fluorescence spectra of LSZ (5.60 µM, curve 1) treated with (A) 4, 12, 20, 28, 36 and 40 µM of AMTH (curves 2–7) and (B) 8, 24, 40, 52, 72 and 80 µM of TZ (curves 2–7). Figure 4. Spectral overlap (shaded portion) of the emission spectrum of LSZ and absorption spectrum of (A) AMTH and (B) TZ. Figure 5. Partitioned polyelectrolytic (ΔG0pe) (shaded) and nonpolyelectrolytic (ΔG0t) (black) contributions to the standard molar Gibbs energy at 10, 20 and 50 mM [Na+] for the complexation of LSZ with (A) AMTH and (B) TZ. Figure 6. Synchronous fluorescence spectra of LSZ (curve 1, 10 µM) in the presence of (A) 2, 10, 20, 30, 40, 50, 60, 70 and 80 µM (curves 2-10) of AMTH, (B) 5, 10, 20, 30, 40, 50, 60, 70 and 80 µM (curves 2-10) of AMTH, (C) 5, 20, 40, 60, 80, 100, 120, 150 and 200 µM (curves 2-10) of TZ, (D) 10, 20, 40, 60, 80, 100, 120, 150 and 200 µM (curves 2-10) of TZ Panel A and C represent the synchronous fluorescence spectra when Δλ = 60 and panels B and D represent Δλ = 15 nm, respectively.

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Figure 7. Far-UV CD spectral changes of LSZ (curve 1, 10 µM) on treatment with (A) 5, 20, 40, 60, 70 and 80 µM of AMTH (curves 2-7) and (B) 5, 20, 40, 50, 70 and 80 µM of TZ (curves 2-7). Figure 8. DSC thermogram of LSZ (20 µM, curve 1), LSZ-AMTH (curve 2) and LSZ-TZ (curve 3) complex at D/P (dye : protein molar ratio) = 25. Figure 9. ITC profiles for the titration for the titration of (A) AMTH and (B) TZ into LSZ solution at 298.15 K. The upper panel represents the ITC profile for the titration of successive aliquots of the two azo dyes into LSZ solution (curve at the bottom), along with the dilution profiles (curves on the top offset for clarity). The bottom panel shows the integrated heat data after correction of heat of dilution. The symbols (■) represent the data points and the solid lines represent the best-fit data. Figure 10. AFM images of LSZ (A, B) and its complexes with AMTH (C, D) and TZ (E and F). The corresponding graphs represent the height of LSZ and its complexes with AMTH and TZ. Figure 11. Variation of the fluorescence intensity of ThT at 485 nm with time for LSZ samples in the absence (•) and presence of AMTH (■) and TZ (▲). Figure 12. Variation of the absorbance of CR at the absorption maxima with time for LSZ samples in the absence (•) and presence of AMTH (■) and TZ (▲). Figure 13. AFM images of LSZ samples in the absence (A, B) and presence of AMTH (C, D) and TZ (E and F). Panels A, C and E represent the images after 6 hr of incubation while panels B, D and F represent the images after 9 hr of incubation. 73



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Binding and Inhibitory Effect of the Dyes Amaranth and Tartrazine on

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