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Multispectroscopic and calorimetric studies on the binding of the food colorant tartrazine with human hemoglobin
Accepted Manuscript Title: Multispectroscopic and calorimetric studies on the binding of the food colorant tartrazine with human hemoglobin Author: Anirban Basu Ph.D Dr. Gopinatha Suresh Kumar Ph.D Dr. PII: DOI: Reference:
S0304-3894(16)30644-6 http://dx.doi.org/doi:10.1016/j.jhazmat.2016.07.023 HAZMAT 17879
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
11-4-2016 6-7-2016 7-7-2016
Please cite this article as: Anirban Basu, Gopinatha Suresh Kumar, Multispectroscopic and calorimetric studies on the binding of the food colorant tartrazine with human hemoglobin, Journal of Hazardous Materials http://dx.doi.org/10.1016/j.jhazmat.2016.07.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
REVISED MANUSCRIPT HAZMAT-D-16-01820R1
Multispectroscopic and calorimetric studies on the binding of the food colorant tartrazine with human hemoglobin
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.S. Kumar)
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Highlights Interaction of the food colorant tartrazine with human hemoglobin was studied. Tartrazine quenched the intrinsic fluorescence of hemoglobin efficiently. Tartrazine induced conformational changes in hemoglobin. Tartrazine altered the environment around the tryptophans more than the tyrosines. The binding was driven by positive entropy and negative enthalpy.
Abstract: Interaction of the food colorant tartrazine with human hemoglobin was studied using multispectroscopic and microcalorimetric techniques to gain insights into the binding mechanism and thereby the toxicity aspects. Hemoglobin spectrum showed hypochromic changes in the presence of tartrazine. Quenching of the fluorescence of hemoglobin occurred and the quenching mechanism was through a static mode as revealed from temperature dependent and time-resolved fluorescence studies. According to the FRET theory the distance between β-Trp37 of hemoglobin and bound tartrazine was evaluated to be 3.44 nm. Tartrazine binding led to alteration of the microenvironment around the tryptophans more in comparison to tyrosines of the protein from synchronous fluorescence results. 3D fluorescence and FTIR data provided evidence for conformational changes in the protein on binding which was confirmed as to lead to significant loss in the helicity of hemoglobin. The esterase activity assay 2
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 further complemented the circular dichroism data. Microcalorimetric study using isothermal titration calorimetry revealed the binding to be exothermic and driven largely by positive entropic contribution. Dissection of the Gibbs energy change proposed the protein-dye complexation to be dominated by non-polyelectrolytic forces. Negative heat capacity change also corroborated the involvement of hydrophobic forces in the binding process.
Keywords : Food colorant; tartrazine; hemoglobin; spectroscopy; calorimetry.
1. Introduction Food colorants, generally dyes or pigments obtained naturally as well as synthetically, are used to make the foodstuff more appealing and attractive. Synthetic colorants are of five classes; azo compounds (such as acid red 27, carmoisine and tartrazine), the triaryl methane group, chinophthalon analogs of quinoline yellow, xanthenes (such as erythrosine) and indigo colorants [1]. Tartrazine (TZ, Fig. 1), an orange-colored, mono azo pyrazolone dye (trisodium-5-hydroxy-1-(4-sulfonatophenyl)-4-(43
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 sulfonatophenylazo)-H-pyrazol-3-carboxylate), is used in soft drinks, juices, cookies, ice creams, snacks, drugs, shampoos, colognes, toothpastes and soaps [2,3]. The maximum acceptable daily intake of TZ recommended for human beings is around 7.5 mg/kg body weight [1,4]. Azo dyes containing N=N linkage and aromatic rings are potentially detrimental to human health. The azo dyes can be cleaved inside our body (small intestine) resulting in the release of highly toxic aromatic amines into the blood. The toxic degradation products can damage various body parts like the urinary organs, kidney, brain, stomach, and liver [5,6]. TZ can also trigger allergic reactions and hyperactivity in children [7]. TZ has been reported to alter the hepatic and renal parameters and induce oxidative stress by forming free radicals [8]. Apparently, its transportation and metabolism process in body could pose potential biological toxicity risk to humans. Hence, it is essential to study the effect of TZ at the functional biomacromolecular level to understand the transportation, distribution and toxicological actions in vivo [9-14]. Human hemoglobin (HHb) is an abundant blood protein carrying oxygen from lungs to different respiring tissues. It is also involved in dispersion of hydrogen peroxide and electron transfer to different body parts. HHb associated non-covalently with the erythrocytes as a tetramer. Each
subunits, and β-chain
contains 141 and 146 amino acid residues, respectively [15]. HHb lies in the red blood cells but it becomes reactive and toxic on hemolysis under diseased conditions and on usage of some drugs. Such conditions cause exposure of free HHb to small molecules in 4
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 the plasma. Thus, HHb, by interacting can influence the distribution, metabolism and toxicity of many small molecules, and therefore it is essential to understand its binding aspects with potentially toxic small molecules [10,13,14]. In this work, we have monitored the effect of TZ on the structural aspects of HHb using multispectroscopic experiments. Furthermore, microcalorimetric studies have been undertaken to elucidate the related energetics to correlate with the structural data. The present study provides useful insights into the effects of TZ on biological systems which may be interpreted in the light of its known toxicity. 2. Experimental 2.1. Materials HHb (H 7379) and TZ were obtained from Sigma-Aldrich Corporation (St. Louis, MO, USA). HHb was dialyzed into the buffer and its concentration was determined using a molar absorption coefficient of 1,79,000 M-1 cm-1 at 405 nm [16,17]. All the experiments were performed in 10 mM [Na+] citrate-phosphate (CP) buffer, at pH 7.0. 2.2. Spectroscopic studies Spectrophotometric experiments were performed on a Jasco V-660 spectrophotometer (Jasco International Co, Hachioji, Japan) in quartz cuvettes following the protocols described previously [16]. Fluorescence experiments were carried out on either a Shimadzu RF-5301 PC (Shimadzu Corporation, Kyoto, Japan) or a Hitachi F4010 (Hitachi Ltd., Tokyo, Japan) spectrofluorimeter in thermostated quartz cuvette following the protocols reported earlier [17]. Time resolved fluorescence experiments 5
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 were performed on Quanta Master 400 unit (Horiba PTI, Canada) controlled with FelixGX spectroscopy. Three dimensional-fluorescence studies were performed on a PerkinElmer LS55 spectrofluorimeter (PerkinElmer, Inc., USA). FTIR studies were done on a Bruker FTIR, TENSOR 27 spectrometer. Circular dichroism studies were performed on a Jasco J815 spectropolarimeter in 0.1 or 1 cm quartz cuvettes. 2.3. Isothermal titration calorimetry (ITC) ITC titrations were carried out on a MicroCal VP-ITC unit (MicroCal, Inc., Northampton, MA, USA). TZ solution was injected from a pre-programmed rotating syringe into the isothermal chamber containing the HHb solution. Immediately, a dilution experiment was performed to obtain the heat of dilution of TZ by titrating identical volumes and concentrations of TZ into the buffer alone. This dilution heat was then deducted from the corresponding heat of TZ–HHb reaction to obtain the real heat of HHb–TZ complexation reaction. The corrected heats were plotted against the molar ratio and analyzed with a model for one set of binding sites to obtain the equilibrium constant (Ka), the stoichiometry of binding (N) and the enthalpy change (ΔH0). The Gibbs energy change (ΔG0) and the entropic contribution (TΔS0) were then calculated from the thermodynamic relationships reported previously [18]. 2.4. Dynamic light scattering (DLS) experiments The DLS measurements were conducted on a Malvern Zetasizer Nano ZS DLS system (Malvern Instruments Ltd., UK) as reported previously [19]. The DLS unit was equipped
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 with a 633 nm He-Ne laser and the sample was maintained at 298.15 K and allowed to equilibrate for 120 s before measurement. 2.5. Esterase activity assay Esterase activity of HHb was assayed using p-nitro phenyl acetate (p-NPA) in accordance with the protocol reported in literature [20]. The reactions were initiated by the addition of 1.5 mM p-NPA from a stock solution containing 100 mM p-NPA in alcohol to a solution containing 5.0 µM of HHb in 10 mM CP buffer and the change in absorbance at 400 nm due to the addition of p-NPA was noted. The absorbance data was corrected for p-NPA hydrolysis in the buffer. 3. Results and discussion 3.1. Spectrophotometric studies HHb exhibited characteristic absorption peaks centered at 195 and 406 nm (Soret band) (Fig. 2A). The Soret band provided a tool to study the dye-protein binding reaction. In Fig. 2, the changes in the absorption spectra of HHb with increasing concentration of TZ are shown. The Soret band exhibited a hypochromic change of about 6% in the presence of TZ (Fig. 2B). Such spectral changes can be associated with a close contact between TZ and HHb. However, the position of the absorption maxima remained virtually unaltered in presence of HHb, that is, neither any red nor any blue shift was observed. The spectral titration data were analyzed using the relation [21] 1 1 1 1 A A max KBH (A max ) [M]
(1)
7
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 where ΔA is the absorbance change at 406 nm and [M] is the concentration of TZ. Subsequently, by plotting 1/ΔA against 1/[M], the Benesi–Hildebrand association constant (KBH) for TZ-HHb complexation was calculated from the intercept to slope ratio [21] and was deduced to be 1.07 × 105 M-1. 3.2. Spectrofluorimetric studies Intrinsic fluorescence of HHb arises from the β-Trp37 residue at the α1β2 interface [22], and acts as an indicator of the transition from the relaxed form (R) to the taut (tense) form (T) of HHb. The R form is regarded as the oxy (ligand bound) form while the T form is regarded as the deoxy form [23]. HHb has fluorescence emission maxima at 328 nm upon excitation at 295 nm revealing that the β-Trp37 residue lies in the hydrophobic region [24]. Effect of TZ on the HHb fluorescence is shown in Fig. 3. The fluorescence of HHb decreased gradually upon addition of TZ. This result indicates that TZ can bind with HHb efficiently and quench the intrinsic fluorescence of HHb. The mechanism of fluorescence quenching of HHb can be either static or dynamic. To investigate the mechanism of quenching, temperature dependent fluorescence studies were performed at 288.15, 298.15 and 308.15 K, respectively, and the data were analyzed using the Stern-Volmer equation [25]
Fo 1 Kq o [Q] 1 KSV [Q] F
(2)
where, Fo=fluorescence of HHb in the absence of TZ, F=fluorescence of HHb in the presence of TZ, KSV=quenching constant, Kq=quenching rate constant and 8
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 [Q]=concentration of TZ. The observed fluorescence intensities for HHb and HHb-TZ complexes were also corrected for inner filter effect according to the procedures reported in literature [10,26]. From these studies the KSV values were found to decrease with rising temperature suggesting a static quenching mechanism for HHb-TZ complexation. Hence, the intrinsic HHb fluorescence was quenched in the presence of TZ due to specific ground state complexation and dynamic collision effects were negligible. To calculate the binding affinity of TZ to HHb it was presumed that there are independent binding sites to a set of equivalent sites on HHb and the apparent binding constant (KA) along with the number of binding sites per protein (n) were determined using the relation [27,28]
log
(Fo F) log K A n log[Q] F
(3)
The binding affinity value (KA) was calculated to be 1.16×105 M-1 at 298.15 K and the number of binding sites per HHb molecule (n) was determined to be around unity. Furthermore, the KA values also decreased with increasing temperature suggesting destabilization of the HHb-TZ complex at higher temperatures. 3.3. Fluorescence lifetime study Lakowicz’s theory states that fluorescence quenching achieved through time-resolved fluorescence measurements can effectively distinguish between static and dynamic processes [29]. Static quenching mechanism is manifested by stable fluorescence life time values whereas dynamic quenching is manifested by significant changes in the 9
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 fluorescence life time values [29]. Fluorescence lifetime values and their corresponding amplitudes were calculated from the time-resolved fluorescence decay profiles of HHb and HHb-TZ complex. The sample was excited at 280 nm using LED radiation while the emission wavelength was fixed at 328 nm. Instrumental response function was evaluated based on the light signal scattered from Ludox and it was further utilized for the deconvolution of the fluorescence signals. The decay curves of HHb and HHb-TZ complex were fitted to a bi-exponential function and the quality of the fits were judged from the χ2 values and inspection of the residuals of the function fitted to the experimental data. Fluorescence decay is given by following equation [29]
F (t ) i exp(
t ) τi
(4)
where F(t) denotes the fluorescence intensity at time t and αi denotes the preexponential factor corresponding to the ith decay time constant, τi. For multi exponential decay, the average lifetime τavg is given by the following equation [30]
avg ai i
(5)
where, τi denotes the fluorescence lifetime and ai denotes the relative amplitude. For free HHb the average fluorescence lifetimes values were found to be τ1=0.42 ns and τ2=2.64 ns. On addition of TZ, the average fluorescence lifetime values slightly reduced to be τ1=0.33 ns and τ2=2.42 ns, respectively. The Trp residues divulge multi exponential decays [31], hence we have not assigned independent components but the average fluorescence lifetime value have been reported to obtain a qualitative analysis. Average fluorescence lifetime of native HHb was 1.95 ns while HHb complexed with TZ was 10
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 1.81 ns. Thus, time dependent fluorescence studies suggested that the fluorescence lifetime of native and bound HHb were not altered significantly. This observation firmly reaffirmed that the quenching of HHb-TZ emission is mainly static in nature and due to ground state complex formation between HHb and TZ. 3.4. Förster resonance energy transfer (FRET) studies The formation of TZ-protein complex may lead to transfer of excited energy from HHb to TZ molecules. Energy transfer efficiency measurements can afford information about the distance between the complexed dye and the interaction site on HHb, which is required to unravel the structural and conformational features of protein-dye complexation process [32,33]. If the emission spectrum of HHb overlaps with the absorption spectrum of TZ, the donor-acceptor pairs are considered to be within Förster distance and the energy transfer between the two can be calculated. FRET is dependent on the reciprocal of the sixth power of the distance between HHb and TZ (r) and on the critical distance at 50% energy transfer efficacy or the Förster radius (Ro). Efficiency of energy transfer (E) between HHb and TZ can be calculated using the equation
Ro 6 F E 1 Fo Ro 6 r 6
(6)
Ro can be estimated using the following relationship
Ro6 8.8 1025 k 2n4φJ
(7)
where k2 denotes the spatial factor of orientation between the emission and absorption dipoles of HHb and TZ, respectively, n and φ denote the refractive index of the medium 11
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 and the fluorescence quantum yield of HHb, respectively. The overlap integral (J) of the emission and absorption spectrum of HHb and TZ, respectively, is calculated from the equation
F ( λ )ε( λ ) λ dλ 4
J
0
(8)
F ( λ )dλ 0
where F(λ) and ε(λ) denotes the fluorescence of HHb and the molar absorption coefficient of TZ, respectively, at the corresponding wavelength λ. From k2=2/3, n=1.36 and φ=0.062 for HHb [34], the values of E, J, Ro and r were deduced to be 0.06, 1.16 × 1014
cm3 L mol-1, 2.23 and 3.44 nms, respectively, for TZ-HHb complexation process. Since
the distance between TZ and the Trp residues of HHb is much less than the 8 nm value so there is a reasonable chance of efficient energy transfer from HHb to the TZ molecules [35]. Besides, the fact that r was higher than Ro suggested that TZ was capable of efficiently accepting energy from the β-Trp37 residue of HHb [36]. 3.5. Synchronous fluorescence studies Conformational changes associated with protein-dye interaction can be monitored through synchronous fluorescence studies [37]. According Miller’s theory [38], when Δλ=15 or 60 nm, the synchronous fluorescence spectra of HHb molecule yields information about the environment surrounding the tyrosine (Tyr) and Trp residues, where Δλ is the difference between the excitation and emission wavelengths. Decrease of intrinsic HHb fluorescence by TZ is then suggestive of the alteration in the polarity 12
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 surrounding the Tyr and Trp residues. Addition of TZ upon HHb with Δλ=60 and 15 nm resulted in marked decrease in the fluorescence of HHb with red shifts in the emission maxima by 6 and 3 nms, respectively (Fig. 4). Such red shift is characteristic of the alterations in the microenvironment surrounding the Trp residues to a more hydrophilic environment and higher exposure to the solvent molecules. However, there was a lesser shift in the emission maximum when Δλ=15 nm suggesting that relatively lesser alterations take place in the microenvironment around the tyrosine residues of HHb. Thus, the polarity of β-Trp37 of HHb was changed in the presence of TZ to a greater degree compared to the Tyr residues. This observation in conjunction with the FRET experiments unequivocally implied the participation of Trp residues in the binding process. 3.6. Three-dimensional (3D) fluorescence spectroscopy 3D fluorescence testifies for conformational changes in HHb upon binding of TZ on concomitant change of excitation and emission wavelengths. 3D fluorescence and the contour maps of native HHb and TZ-HHb complex are presented in Fig. 5. Peaks a (λex=λem) and b (λem=2λex) are the first and second-order Rayleigh scattering peaks, respectively. Peak 1 (λex=280 nm) is characteristic of the intrinsic fluorescence of Trp and Tyr residues [39]. This is due to the fact that when the excitation is at 280 nm the fluorescence due to phenylalanine is negligible. Additionally, there is another peak 2 (λex=230 nm) which is characteristic of the polypeptide backbone. Intensities of both peaks 1 and 2 decreased in presence of TZ but to different extents. The change of Stokes 13
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 shift along with the decrease of fluorescence intensities clearly indicate that there is specific binding interaction between TZ and HHb which induces secondary structural changes in HHb. 3.7. FTIR studies Fourier transform infrared (FTIR) spectroscopy was carried out for characterizing the conformational changes in HHb upon binding to TZ [40,41]. FTIR technique was used to study the conformational changes in HHb because it has conformation sensitive spectral signature in the IR region [41]. The FTIR spectra of HHb and HHb-TZ complex are shown Fig. 6. The amide-I band at 1651 cm-1 was monitored to understand the changes induced upon complexation with TZ. This band was shifted towards lower wavenumber, viz. 1644 cm-1, in presence of TZ indicating conformational change of HHb from more helical structure to β-sheet like aggregated structures [41,42]. The similar features of the two spectra suggested that HHb retained the basic characteristics of its secondary structure even in presence of TZ. This shift towards lower wave number is a consequence of the interaction between HHb and TZ and possibly the interaction was because of hydrogen bonding, electrostatic, hydrophobic and hydrophilic interactions [40,43]. 3.8. Circular dichroism studies Circular dichroism (CD) being a conformation sensitive tool may lend valuable data on the conformational changes induced in HHb upon binding of TZ. The CD spectrum of HHb has two characteristic far UV negative bands at 210 and 222 nm (Fig. 7) which are characteristic of the α-helical structure of HHb [44,45]. The band at 210 nm is due to π-π* transition of the α14
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 helix and the band at 222 nm band is a result of the n-π* transition for both the α-helix and the random coil. The ellipticity of these negative CD bands decreased in magnitude progressively on addition of TZ. This accounted for the secondary structural changes in the conformation of HHb upon binding with TZ (Fig. 7). The helical content of native HHb and HHb complexed with TZ was calculated using the following equation [46,47] MRE[]
observed CD(m deg) Cnl 10
(9)
where MRE=mean residue ellipticity (deg . cm2 . dmol-1), C=concentration of HHb (M), n=number of amino acid residues and l=path length of the cuvette (cm) in which the experiment was performed. The α-helical content of HHb was obtained uisng the equation helix(%) [
[]208 4000 ] 100 33000 4000
(10)
The α-helical content of native HHb was deduced to be 74% whereas that of TZ bound HHb was calculated to be 27%. Thus, TZ caused significant loss of the helical stability of HHb and also induced unfolding of the protein conformation with the extended polypeptide chains exposing the hydrophobic cavities with simultaneous exposure of the aromatic amino acid residues. 3.9. Microcalorimetric characterization of the interaction Isothermal titration calorimetry was employed for thermodynamic analysis of the biomolecular binding reaction. The upper panel of Fig. 8 represents the raw isothermal titration calorimetric profile for the TZ-HHb binding reaction at 298.15 K. The complexation process was found to be exothermic. As the integrated heat data showed 15
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 only one binding event so they were fitted to a one set of site binding model. The equilibrium constant value obtained from ITC is (1.29±0.06) × 105 at 298.15 K which is closely matched with that obtained from the earlier studies (vide supra). The binding of TZ to the HHb was favored by high positive standard molar entropic and relatively low negative standard molar enthalpic contributions. At 298.15 K the value of TΔS0 was calculated to be (6.79±0.03) kcal . mol-1 and the value of ΔH0 was (-0.18±0.03) kcal . mol-1. Large positive entropy is suggestive of the disruption and release of HHb bound water molecules and condensed counterions on interaction with TZ. Specific electrostatic interaction between HHb and TZ in the aqueous solution also contributes to the positive ΔS0 value and negative ΔH0 value [48]. The N value obtained from ITC analysis was (1.05±0.03) at 298.15 K. This value of N close to unity suggests 1:1 complexation between TZ and HHb and is in excellent agreement with that obtained from spectroscopy. The standard molar Gibbs energy change value for the TZ-HHb complexation process was determined to be (-6.97±0.06) kcal.mol-1. This negative value of ΔG0 accounts for the spontaneity of HHb-TZ complexation process. Analysis of HHb-TZ interaction in the [Na+] range 10-50 mM along with van’t Hoff analysis revealed the contribution arising from polyelectrolytic and non-polyelectrolytic forces. The relation between Ka and [Na+] has been described earlier by Record and coworkers [49,50]
(
log K a )T , P zψ log[Na ]
(11)
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 where z=apparent charge of bound TZ and ψ=fraction of counter ions bound per anion. The Ka values decreased with increasing [Na+] in the range 10 to 50 mM. The N values varied only marginally suggesting 1:1 complexation between HHb and TZ. From the dependence of Ka on [Na+] the standard molar Gibbs energy can be divided into polyelectrolytic (ΔG0pe) and non-polyelectrolytic (ΔG0t) contributions. The polyelectrolytic contribution to the standard molar Gibbs energy can be calculated using the relationship, ΔG0pe=-zψRTln([Na+]). ΔG0pe is actually indicative of the contribution arising from polyelectrolytic sources like the release of condensed counterions upon binding of TZ. On increasing the [Na+] from 10 to 50 mM the ΔG0pe contribution decreased. At 10 mM [Na+] ΔG0pe was maximum and it gradually decreased thereafter. On the other hand, ΔG0t was dominant and virtually unchanged at all the salt concentrations. Thus, the complexation process was dominated by contributions from non-polyelectrolytic sources, viz. van der Waals interaction, Hbonding and hydrophobic transfer. The constant pressure heat capacity change (ΔCp0) for TZ-HHb binding interactions was determined employing the relationship, ΔCp0=[∂(ΔH0)/∂T]P
(12)
Standard molar heat capacity affords useful insights into the kind of forces involved in HHb-TZ complexation process. ITC studies were performed in the temperature range 288.15-308.15 K. As the temperature increased the Ka values decreased but there was only a marginal change in the N value suggesting that a 1:1 complexation occurs 17
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 between TZ and HHb. On the other hand, the binding enthalpies became more negative whereas the entropy contribution decreased with rise in temperature. The variation of ΔH0 with temperature yielded the ΔCp0 value to be (-4.00±0.06) cal.K-1.mol-1. Such negative ΔCp0 have been reported for a variety of ligand-protein binding reactions [1618,51,52]. This negative ΔCp0 value is generally linked with alterations in hydrophobic or polar group hydration and is assumed to be associated with dominant hydrophobic forces in the complexation process. Changes in the solvent accessible surface area also contribute to this ΔCp0 value [53-55]. From Record’s relationship [56] viz. ∆G0hyd=(80 10) × ΔCp0, the Gibbs energy contribution arising from the hydrophobic transfer step (∆G0hyd) was calculated to be -0.32 kcal.mol-1. 3.10. Dynamic light scattering Dynamic light scattering (DLS) is an useful tool for obtaining the dimensions of a large number of biomacromolecular assemblies. Using DLS the size distribution (hydrodynamic diameter, dh) profile of HHb and its complex with TZ was determined. The average hydrodynamic diameter of the native HHb, dh~(88±15) Å, enhanced to dh~(136±3) Å upon complexation with TZ. Such an enhancement in the value hydrodynamic diameter of Hb can be attributed to arise from the aggregation of HHb upon denaturation in presence of TZ [57]. 3.11. Esterase activity assay HHb is endowed with an interesting enzymatic property, usually known an esteraselike activity. Allura red, an artificial azo dye, and its degradation products are known to 18
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 inhibit the esterase activity of carbonic anhydrase II [58,59]. In light of the significant conformational changes induced by TZ on the native conformation of HHb, it is pertinent to monitor the influence of TZ on the esterase activity of HHb as knowledge of the influence of TZ on the activity of HHb is essential to assess the food safety of TZ. The esterase-like activity of HHb is obtained from the absorbance of p-nitrophenol produced by the hydrolysis of p-nitrophenyl acetate upon action of HHb. An unit activity refers to the amount of the enzyme needed to liberate 1.0 mM p-nitrophenol per minute at 310.15 K [57]. Changes in the relative esterase activity of HHb with increasing concentration of TZ are shown in Fig. 9. It can be clearly seen from the figure that HHbTZ association produced discernible decrease in the esterase activity of HHb, which reaffirms the earlier fact that TZ causes breakdown of the native structure of HHb. 3.12. Chaotrope induced denaturation studies Fluorescence studies provide a convenient tool to examine the changes in the tertiary structure of HHb which also enables us to unravel its environmental stability. The effect of urea induced denaturation of HHb on its binding efficacy and on the overall photophysics of TZ was studied. Urea produced alterations in the steady state emission spectra of TZ bound HHb. Fig. 10A represents the changes in the relative emission intensity of HHb complexed with TZ upon addition of increasing concentration of urea. Addition of the chaotrope effected an enhancement in the emission intensity of HHb at 328 nm. The intrinsic fluorescence of HHb for Trp residues enhanced on addition of the chaotrope due to a structural conformation whereby the Trp residues and the heme 19
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 group are far apart for energy transfer to be restricted [60-62]. This happens due to the chaotrope induced denaturation of HHb. Therefore, chaotrope effected protein denaturation leads to a large increase in the fluorescence intensity by inhibition of nonradiative energy transfer from the Trp residues to heme prosthetic group. From Fig. 10B it can be seen that with gradual addition of the chaotrope (urea) the intensity of the emission band of TZ enhanced. The addition of the chaotrope effected destabilization of the TZ–Hb complex thereby exposing greater amount of TZ to the bulk buffer solution compared to the native HHb bound condition. This exposure of the earlier bound TZ molecules caused an enhancement in the fluorescence of TZ. Chaotropes can displace water molecules adjacent to TZ in the HHb microenvironment with simultaneous denaturation of HHb [25,57,63]. Hence, chaotrope induced destabilization of the complex is associated with a greater exposure of TZ to the bulk aqueous buffer solution compared to its HHb bound state in the native conformation of HHb [57]. This increased exposure of TZ can account for the enhancement in the intensity of the emission maximum. 4. Conclusions Spectroscopic studies testified for a strong binding reaction between TZ and HHb. The binding phenomenon was static in nature. FRET studies revealed that the binding interaction involved close contact of TZ with β-Trp37 residue at the α1β2 interface. Synchronous, 3D fluorescence and FTIR studies revealed strong conformational changes in HHb upon complexation with TZ. Furthermore, circular dichroism studies 20
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 indicated conclusively that in presence of TZ there was a significant loss in the helical stability of HHb. The esterase activity assay also supported this data. ITC studies revealed that the binding was exothermic and favored by large positive entropic and small negative enthalpic contributions. Negative value of the Gibbs energy suggested spontaneity of the binding interaction. Salt and temperature dependent microcalorimetric studies established the critical role of non-polyelectrolytic forces such as hydrophobic interaction, van der Waals interaction, H-bonding etc. in the complexation process. This work clearly delineates the mode, mechanism, affinity and energetics of the binding of TZ with HHb. Toxicity of pollutants like azo dyes which are introduced into the blood stream as a consequence of environmental exposure is a serious health concern for the humans. Since it is revealed that TZ can bind strongly to HHb and induce significant conformational changes its transportation and metabolism in the human body, it can pose potential biological toxicity risk. It is likely that the absorption of TZ in the blood plasma will adversely affect HHb function and can even lead to impairment of its activity. Hence, the selection of TZ as a food colorant must take into consideration the aforementioned issues on health hazard grounds. 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 recipient of Research Associateship from GenCODE (BSC0123).
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Figure legends Fig. 1. Molecular structure of TZ.
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Fig. 2. (A) Absorption spectral titration of HHb (curve1) with increasing amounts of TZ (curves 2–5). (B) The Soret band of HHb is magnified here.
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Fig. 3. Changes in the steady state fluorescence emission spectra of Hb (10 µM, curve 1) on treatment with 16-120 µM of TZ (curves 2-9).
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Fig. 4. (A) Synchronous fluorescence spectra of HHb (10 µM, curve 1) in the presence of 5-100 µM of TZ (curve 2-10) when Δλ=60 nm. (B) Synchronous fluorescence spectra of HHb (10 µM, curve 1) in the presence of 5-100 µM of TZ (curve 2-10) when Δλ=15 nm, respectively.
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Fig. 5. Three-dimensional fluorescence and contour spectra of Hb (A, B) and Hb- TZ (C, D) complex.
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Fig. 6. FTIR spectra of Hb (curve 1) and Hb-TZ complex (curve 2).
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Fig. 7. Far UV CD spectra of HHb (0.80 µM, curve 1) in presence of 2, 10, 20, 30, 40, 60 and 70 µM of TZ (curves 2-8).
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Fig. 8. Representative ITC profile for the titration of TZ with HHb. The upper panel represents the ITC profile for the titration of successive aliquots of 10 μL TZ into HHb 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 line represents the best-fit data.
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Fig. 9. Plot of variation in the relative esterase activity of HHb with increasing TZ concentration.
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Fig. 10. Plot of variation of relative fluorescence intensity of HHb-TZ complex at the emission maxima of (A) HHb and (B) TZ with increasing urea concentration.
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S0304-3894(16)30644-6 http://dx.doi.org/doi:10.1016/j.jhazmat.2016.07.023 HAZMAT 17879
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
11-4-2016 6-7-2016 7-7-2016
Please cite this article as: Anirban Basu, Gopinatha Suresh Kumar, Multispectroscopic and calorimetric studies on the binding of the food colorant tartrazine with human hemoglobin, Journal of Hazardous Materials http://dx.doi.org/10.1016/j.jhazmat.2016.07.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
REVISED MANUSCRIPT HAZMAT-D-16-01820R1
Multispectroscopic and calorimetric studies on the binding of the food colorant tartrazine with human hemoglobin
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.S. Kumar)
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Highlights Interaction of the food colorant tartrazine with human hemoglobin was studied. Tartrazine quenched the intrinsic fluorescence of hemoglobin efficiently. Tartrazine induced conformational changes in hemoglobin. Tartrazine altered the environment around the tryptophans more than the tyrosines. The binding was driven by positive entropy and negative enthalpy.
Abstract: Interaction of the food colorant tartrazine with human hemoglobin was studied using multispectroscopic and microcalorimetric techniques to gain insights into the binding mechanism and thereby the toxicity aspects. Hemoglobin spectrum showed hypochromic changes in the presence of tartrazine. Quenching of the fluorescence of hemoglobin occurred and the quenching mechanism was through a static mode as revealed from temperature dependent and time-resolved fluorescence studies. According to the FRET theory the distance between β-Trp37 of hemoglobin and bound tartrazine was evaluated to be 3.44 nm. Tartrazine binding led to alteration of the microenvironment around the tryptophans more in comparison to tyrosines of the protein from synchronous fluorescence results. 3D fluorescence and FTIR data provided evidence for conformational changes in the protein on binding which was confirmed as to lead to significant loss in the helicity of hemoglobin. The esterase activity assay 2
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 further complemented the circular dichroism data. Microcalorimetric study using isothermal titration calorimetry revealed the binding to be exothermic and driven largely by positive entropic contribution. Dissection of the Gibbs energy change proposed the protein-dye complexation to be dominated by non-polyelectrolytic forces. Negative heat capacity change also corroborated the involvement of hydrophobic forces in the binding process.
Keywords : Food colorant; tartrazine; hemoglobin; spectroscopy; calorimetry.
1. Introduction Food colorants, generally dyes or pigments obtained naturally as well as synthetically, are used to make the foodstuff more appealing and attractive. Synthetic colorants are of five classes; azo compounds (such as acid red 27, carmoisine and tartrazine), the triaryl methane group, chinophthalon analogs of quinoline yellow, xanthenes (such as erythrosine) and indigo colorants [1]. Tartrazine (TZ, Fig. 1), an orange-colored, mono azo pyrazolone dye (trisodium-5-hydroxy-1-(4-sulfonatophenyl)-4-(43
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 sulfonatophenylazo)-H-pyrazol-3-carboxylate), is used in soft drinks, juices, cookies, ice creams, snacks, drugs, shampoos, colognes, toothpastes and soaps [2,3]. The maximum acceptable daily intake of TZ recommended for human beings is around 7.5 mg/kg body weight [1,4]. Azo dyes containing N=N linkage and aromatic rings are potentially detrimental to human health. The azo dyes can be cleaved inside our body (small intestine) resulting in the release of highly toxic aromatic amines into the blood. The toxic degradation products can damage various body parts like the urinary organs, kidney, brain, stomach, and liver [5,6]. TZ can also trigger allergic reactions and hyperactivity in children [7]. TZ has been reported to alter the hepatic and renal parameters and induce oxidative stress by forming free radicals [8]. Apparently, its transportation and metabolism process in body could pose potential biological toxicity risk to humans. Hence, it is essential to study the effect of TZ at the functional biomacromolecular level to understand the transportation, distribution and toxicological actions in vivo [9-14]. Human hemoglobin (HHb) is an abundant blood protein carrying oxygen from lungs to different respiring tissues. It is also involved in dispersion of hydrogen peroxide and electron transfer to different body parts. HHb associated non-covalently with the erythrocytes as a tetramer. Each
subunits, and β-chain
contains 141 and 146 amino acid residues, respectively [15]. HHb lies in the red blood cells but it becomes reactive and toxic on hemolysis under diseased conditions and on usage of some drugs. Such conditions cause exposure of free HHb to small molecules in 4
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 the plasma. Thus, HHb, by interacting can influence the distribution, metabolism and toxicity of many small molecules, and therefore it is essential to understand its binding aspects with potentially toxic small molecules [10,13,14]. In this work, we have monitored the effect of TZ on the structural aspects of HHb using multispectroscopic experiments. Furthermore, microcalorimetric studies have been undertaken to elucidate the related energetics to correlate with the structural data. The present study provides useful insights into the effects of TZ on biological systems which may be interpreted in the light of its known toxicity. 2. Experimental 2.1. Materials HHb (H 7379) and TZ were obtained from Sigma-Aldrich Corporation (St. Louis, MO, USA). HHb was dialyzed into the buffer and its concentration was determined using a molar absorption coefficient of 1,79,000 M-1 cm-1 at 405 nm [16,17]. All the experiments were performed in 10 mM [Na+] citrate-phosphate (CP) buffer, at pH 7.0. 2.2. Spectroscopic studies Spectrophotometric experiments were performed on a Jasco V-660 spectrophotometer (Jasco International Co, Hachioji, Japan) in quartz cuvettes following the protocols described previously [16]. Fluorescence experiments were carried out on either a Shimadzu RF-5301 PC (Shimadzu Corporation, Kyoto, Japan) or a Hitachi F4010 (Hitachi Ltd., Tokyo, Japan) spectrofluorimeter in thermostated quartz cuvette following the protocols reported earlier [17]. Time resolved fluorescence experiments 5
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 were performed on Quanta Master 400 unit (Horiba PTI, Canada) controlled with FelixGX spectroscopy. Three dimensional-fluorescence studies were performed on a PerkinElmer LS55 spectrofluorimeter (PerkinElmer, Inc., USA). FTIR studies were done on a Bruker FTIR, TENSOR 27 spectrometer. Circular dichroism studies were performed on a Jasco J815 spectropolarimeter in 0.1 or 1 cm quartz cuvettes. 2.3. Isothermal titration calorimetry (ITC) ITC titrations were carried out on a MicroCal VP-ITC unit (MicroCal, Inc., Northampton, MA, USA). TZ solution was injected from a pre-programmed rotating syringe into the isothermal chamber containing the HHb solution. Immediately, a dilution experiment was performed to obtain the heat of dilution of TZ by titrating identical volumes and concentrations of TZ into the buffer alone. This dilution heat was then deducted from the corresponding heat of TZ–HHb reaction to obtain the real heat of HHb–TZ complexation reaction. The corrected heats were plotted against the molar ratio and analyzed with a model for one set of binding sites to obtain the equilibrium constant (Ka), the stoichiometry of binding (N) and the enthalpy change (ΔH0). The Gibbs energy change (ΔG0) and the entropic contribution (TΔS0) were then calculated from the thermodynamic relationships reported previously [18]. 2.4. Dynamic light scattering (DLS) experiments The DLS measurements were conducted on a Malvern Zetasizer Nano ZS DLS system (Malvern Instruments Ltd., UK) as reported previously [19]. The DLS unit was equipped
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 with a 633 nm He-Ne laser and the sample was maintained at 298.15 K and allowed to equilibrate for 120 s before measurement. 2.5. Esterase activity assay Esterase activity of HHb was assayed using p-nitro phenyl acetate (p-NPA) in accordance with the protocol reported in literature [20]. The reactions were initiated by the addition of 1.5 mM p-NPA from a stock solution containing 100 mM p-NPA in alcohol to a solution containing 5.0 µM of HHb in 10 mM CP buffer and the change in absorbance at 400 nm due to the addition of p-NPA was noted. The absorbance data was corrected for p-NPA hydrolysis in the buffer. 3. Results and discussion 3.1. Spectrophotometric studies HHb exhibited characteristic absorption peaks centered at 195 and 406 nm (Soret band) (Fig. 2A). The Soret band provided a tool to study the dye-protein binding reaction. In Fig. 2, the changes in the absorption spectra of HHb with increasing concentration of TZ are shown. The Soret band exhibited a hypochromic change of about 6% in the presence of TZ (Fig. 2B). Such spectral changes can be associated with a close contact between TZ and HHb. However, the position of the absorption maxima remained virtually unaltered in presence of HHb, that is, neither any red nor any blue shift was observed. The spectral titration data were analyzed using the relation [21] 1 1 1 1 A A max KBH (A max ) [M]
(1)
7
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 where ΔA is the absorbance change at 406 nm and [M] is the concentration of TZ. Subsequently, by plotting 1/ΔA against 1/[M], the Benesi–Hildebrand association constant (KBH) for TZ-HHb complexation was calculated from the intercept to slope ratio [21] and was deduced to be 1.07 × 105 M-1. 3.2. Spectrofluorimetric studies Intrinsic fluorescence of HHb arises from the β-Trp37 residue at the α1β2 interface [22], and acts as an indicator of the transition from the relaxed form (R) to the taut (tense) form (T) of HHb. The R form is regarded as the oxy (ligand bound) form while the T form is regarded as the deoxy form [23]. HHb has fluorescence emission maxima at 328 nm upon excitation at 295 nm revealing that the β-Trp37 residue lies in the hydrophobic region [24]. Effect of TZ on the HHb fluorescence is shown in Fig. 3. The fluorescence of HHb decreased gradually upon addition of TZ. This result indicates that TZ can bind with HHb efficiently and quench the intrinsic fluorescence of HHb. The mechanism of fluorescence quenching of HHb can be either static or dynamic. To investigate the mechanism of quenching, temperature dependent fluorescence studies were performed at 288.15, 298.15 and 308.15 K, respectively, and the data were analyzed using the Stern-Volmer equation [25]
Fo 1 Kq o [Q] 1 KSV [Q] F
(2)
where, Fo=fluorescence of HHb in the absence of TZ, F=fluorescence of HHb in the presence of TZ, KSV=quenching constant, Kq=quenching rate constant and 8
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 [Q]=concentration of TZ. The observed fluorescence intensities for HHb and HHb-TZ complexes were also corrected for inner filter effect according to the procedures reported in literature [10,26]. From these studies the KSV values were found to decrease with rising temperature suggesting a static quenching mechanism for HHb-TZ complexation. Hence, the intrinsic HHb fluorescence was quenched in the presence of TZ due to specific ground state complexation and dynamic collision effects were negligible. To calculate the binding affinity of TZ to HHb it was presumed that there are independent binding sites to a set of equivalent sites on HHb and the apparent binding constant (KA) along with the number of binding sites per protein (n) were determined using the relation [27,28]
log
(Fo F) log K A n log[Q] F
(3)
The binding affinity value (KA) was calculated to be 1.16×105 M-1 at 298.15 K and the number of binding sites per HHb molecule (n) was determined to be around unity. Furthermore, the KA values also decreased with increasing temperature suggesting destabilization of the HHb-TZ complex at higher temperatures. 3.3. Fluorescence lifetime study Lakowicz’s theory states that fluorescence quenching achieved through time-resolved fluorescence measurements can effectively distinguish between static and dynamic processes [29]. Static quenching mechanism is manifested by stable fluorescence life time values whereas dynamic quenching is manifested by significant changes in the 9
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 fluorescence life time values [29]. Fluorescence lifetime values and their corresponding amplitudes were calculated from the time-resolved fluorescence decay profiles of HHb and HHb-TZ complex. The sample was excited at 280 nm using LED radiation while the emission wavelength was fixed at 328 nm. Instrumental response function was evaluated based on the light signal scattered from Ludox and it was further utilized for the deconvolution of the fluorescence signals. The decay curves of HHb and HHb-TZ complex were fitted to a bi-exponential function and the quality of the fits were judged from the χ2 values and inspection of the residuals of the function fitted to the experimental data. Fluorescence decay is given by following equation [29]
F (t ) i exp(
t ) τi
(4)
where F(t) denotes the fluorescence intensity at time t and αi denotes the preexponential factor corresponding to the ith decay time constant, τi. For multi exponential decay, the average lifetime τavg is given by the following equation [30]
avg ai i
(5)
where, τi denotes the fluorescence lifetime and ai denotes the relative amplitude. For free HHb the average fluorescence lifetimes values were found to be τ1=0.42 ns and τ2=2.64 ns. On addition of TZ, the average fluorescence lifetime values slightly reduced to be τ1=0.33 ns and τ2=2.42 ns, respectively. The Trp residues divulge multi exponential decays [31], hence we have not assigned independent components but the average fluorescence lifetime value have been reported to obtain a qualitative analysis. Average fluorescence lifetime of native HHb was 1.95 ns while HHb complexed with TZ was 10
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 1.81 ns. Thus, time dependent fluorescence studies suggested that the fluorescence lifetime of native and bound HHb were not altered significantly. This observation firmly reaffirmed that the quenching of HHb-TZ emission is mainly static in nature and due to ground state complex formation between HHb and TZ. 3.4. Förster resonance energy transfer (FRET) studies The formation of TZ-protein complex may lead to transfer of excited energy from HHb to TZ molecules. Energy transfer efficiency measurements can afford information about the distance between the complexed dye and the interaction site on HHb, which is required to unravel the structural and conformational features of protein-dye complexation process [32,33]. If the emission spectrum of HHb overlaps with the absorption spectrum of TZ, the donor-acceptor pairs are considered to be within Förster distance and the energy transfer between the two can be calculated. FRET is dependent on the reciprocal of the sixth power of the distance between HHb and TZ (r) and on the critical distance at 50% energy transfer efficacy or the Förster radius (Ro). Efficiency of energy transfer (E) between HHb and TZ can be calculated using the equation
Ro 6 F E 1 Fo Ro 6 r 6
(6)
Ro can be estimated using the following relationship
Ro6 8.8 1025 k 2n4φJ
(7)
where k2 denotes the spatial factor of orientation between the emission and absorption dipoles of HHb and TZ, respectively, n and φ denote the refractive index of the medium 11
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 and the fluorescence quantum yield of HHb, respectively. The overlap integral (J) of the emission and absorption spectrum of HHb and TZ, respectively, is calculated from the equation
F ( λ )ε( λ ) λ dλ 4
J
0
(8)
F ( λ )dλ 0
where F(λ) and ε(λ) denotes the fluorescence of HHb and the molar absorption coefficient of TZ, respectively, at the corresponding wavelength λ. From k2=2/3, n=1.36 and φ=0.062 for HHb [34], the values of E, J, Ro and r were deduced to be 0.06, 1.16 × 1014
cm3 L mol-1, 2.23 and 3.44 nms, respectively, for TZ-HHb complexation process. Since
the distance between TZ and the Trp residues of HHb is much less than the 8 nm value so there is a reasonable chance of efficient energy transfer from HHb to the TZ molecules [35]. Besides, the fact that r was higher than Ro suggested that TZ was capable of efficiently accepting energy from the β-Trp37 residue of HHb [36]. 3.5. Synchronous fluorescence studies Conformational changes associated with protein-dye interaction can be monitored through synchronous fluorescence studies [37]. According Miller’s theory [38], when Δλ=15 or 60 nm, the synchronous fluorescence spectra of HHb molecule yields information about the environment surrounding the tyrosine (Tyr) and Trp residues, where Δλ is the difference between the excitation and emission wavelengths. Decrease of intrinsic HHb fluorescence by TZ is then suggestive of the alteration in the polarity 12
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 surrounding the Tyr and Trp residues. Addition of TZ upon HHb with Δλ=60 and 15 nm resulted in marked decrease in the fluorescence of HHb with red shifts in the emission maxima by 6 and 3 nms, respectively (Fig. 4). Such red shift is characteristic of the alterations in the microenvironment surrounding the Trp residues to a more hydrophilic environment and higher exposure to the solvent molecules. However, there was a lesser shift in the emission maximum when Δλ=15 nm suggesting that relatively lesser alterations take place in the microenvironment around the tyrosine residues of HHb. Thus, the polarity of β-Trp37 of HHb was changed in the presence of TZ to a greater degree compared to the Tyr residues. This observation in conjunction with the FRET experiments unequivocally implied the participation of Trp residues in the binding process. 3.6. Three-dimensional (3D) fluorescence spectroscopy 3D fluorescence testifies for conformational changes in HHb upon binding of TZ on concomitant change of excitation and emission wavelengths. 3D fluorescence and the contour maps of native HHb and TZ-HHb complex are presented in Fig. 5. Peaks a (λex=λem) and b (λem=2λex) are the first and second-order Rayleigh scattering peaks, respectively. Peak 1 (λex=280 nm) is characteristic of the intrinsic fluorescence of Trp and Tyr residues [39]. This is due to the fact that when the excitation is at 280 nm the fluorescence due to phenylalanine is negligible. Additionally, there is another peak 2 (λex=230 nm) which is characteristic of the polypeptide backbone. Intensities of both peaks 1 and 2 decreased in presence of TZ but to different extents. The change of Stokes 13
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 shift along with the decrease of fluorescence intensities clearly indicate that there is specific binding interaction between TZ and HHb which induces secondary structural changes in HHb. 3.7. FTIR studies Fourier transform infrared (FTIR) spectroscopy was carried out for characterizing the conformational changes in HHb upon binding to TZ [40,41]. FTIR technique was used to study the conformational changes in HHb because it has conformation sensitive spectral signature in the IR region [41]. The FTIR spectra of HHb and HHb-TZ complex are shown Fig. 6. The amide-I band at 1651 cm-1 was monitored to understand the changes induced upon complexation with TZ. This band was shifted towards lower wavenumber, viz. 1644 cm-1, in presence of TZ indicating conformational change of HHb from more helical structure to β-sheet like aggregated structures [41,42]. The similar features of the two spectra suggested that HHb retained the basic characteristics of its secondary structure even in presence of TZ. This shift towards lower wave number is a consequence of the interaction between HHb and TZ and possibly the interaction was because of hydrogen bonding, electrostatic, hydrophobic and hydrophilic interactions [40,43]. 3.8. Circular dichroism studies Circular dichroism (CD) being a conformation sensitive tool may lend valuable data on the conformational changes induced in HHb upon binding of TZ. The CD spectrum of HHb has two characteristic far UV negative bands at 210 and 222 nm (Fig. 7) which are characteristic of the α-helical structure of HHb [44,45]. The band at 210 nm is due to π-π* transition of the α14
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 helix and the band at 222 nm band is a result of the n-π* transition for both the α-helix and the random coil. The ellipticity of these negative CD bands decreased in magnitude progressively on addition of TZ. This accounted for the secondary structural changes in the conformation of HHb upon binding with TZ (Fig. 7). The helical content of native HHb and HHb complexed with TZ was calculated using the following equation [46,47] MRE[]
observed CD(m deg) Cnl 10
(9)
where MRE=mean residue ellipticity (deg . cm2 . dmol-1), C=concentration of HHb (M), n=number of amino acid residues and l=path length of the cuvette (cm) in which the experiment was performed. The α-helical content of HHb was obtained uisng the equation helix(%) [
[]208 4000 ] 100 33000 4000
(10)
The α-helical content of native HHb was deduced to be 74% whereas that of TZ bound HHb was calculated to be 27%. Thus, TZ caused significant loss of the helical stability of HHb and also induced unfolding of the protein conformation with the extended polypeptide chains exposing the hydrophobic cavities with simultaneous exposure of the aromatic amino acid residues. 3.9. Microcalorimetric characterization of the interaction Isothermal titration calorimetry was employed for thermodynamic analysis of the biomolecular binding reaction. The upper panel of Fig. 8 represents the raw isothermal titration calorimetric profile for the TZ-HHb binding reaction at 298.15 K. The complexation process was found to be exothermic. As the integrated heat data showed 15
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 only one binding event so they were fitted to a one set of site binding model. The equilibrium constant value obtained from ITC is (1.29±0.06) × 105 at 298.15 K which is closely matched with that obtained from the earlier studies (vide supra). The binding of TZ to the HHb was favored by high positive standard molar entropic and relatively low negative standard molar enthalpic contributions. At 298.15 K the value of TΔS0 was calculated to be (6.79±0.03) kcal . mol-1 and the value of ΔH0 was (-0.18±0.03) kcal . mol-1. Large positive entropy is suggestive of the disruption and release of HHb bound water molecules and condensed counterions on interaction with TZ. Specific electrostatic interaction between HHb and TZ in the aqueous solution also contributes to the positive ΔS0 value and negative ΔH0 value [48]. The N value obtained from ITC analysis was (1.05±0.03) at 298.15 K. This value of N close to unity suggests 1:1 complexation between TZ and HHb and is in excellent agreement with that obtained from spectroscopy. The standard molar Gibbs energy change value for the TZ-HHb complexation process was determined to be (-6.97±0.06) kcal.mol-1. This negative value of ΔG0 accounts for the spontaneity of HHb-TZ complexation process. Analysis of HHb-TZ interaction in the [Na+] range 10-50 mM along with van’t Hoff analysis revealed the contribution arising from polyelectrolytic and non-polyelectrolytic forces. The relation between Ka and [Na+] has been described earlier by Record and coworkers [49,50]
(
log K a )T , P zψ log[Na ]
(11)
16
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 where z=apparent charge of bound TZ and ψ=fraction of counter ions bound per anion. The Ka values decreased with increasing [Na+] in the range 10 to 50 mM. The N values varied only marginally suggesting 1:1 complexation between HHb and TZ. From the dependence of Ka on [Na+] the standard molar Gibbs energy can be divided into polyelectrolytic (ΔG0pe) and non-polyelectrolytic (ΔG0t) contributions. The polyelectrolytic contribution to the standard molar Gibbs energy can be calculated using the relationship, ΔG0pe=-zψRTln([Na+]). ΔG0pe is actually indicative of the contribution arising from polyelectrolytic sources like the release of condensed counterions upon binding of TZ. On increasing the [Na+] from 10 to 50 mM the ΔG0pe contribution decreased. At 10 mM [Na+] ΔG0pe was maximum and it gradually decreased thereafter. On the other hand, ΔG0t was dominant and virtually unchanged at all the salt concentrations. Thus, the complexation process was dominated by contributions from non-polyelectrolytic sources, viz. van der Waals interaction, Hbonding and hydrophobic transfer. The constant pressure heat capacity change (ΔCp0) for TZ-HHb binding interactions was determined employing the relationship, ΔCp0=[∂(ΔH0)/∂T]P
(12)
Standard molar heat capacity affords useful insights into the kind of forces involved in HHb-TZ complexation process. ITC studies were performed in the temperature range 288.15-308.15 K. As the temperature increased the Ka values decreased but there was only a marginal change in the N value suggesting that a 1:1 complexation occurs 17
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 between TZ and HHb. On the other hand, the binding enthalpies became more negative whereas the entropy contribution decreased with rise in temperature. The variation of ΔH0 with temperature yielded the ΔCp0 value to be (-4.00±0.06) cal.K-1.mol-1. Such negative ΔCp0 have been reported for a variety of ligand-protein binding reactions [1618,51,52]. This negative ΔCp0 value is generally linked with alterations in hydrophobic or polar group hydration and is assumed to be associated with dominant hydrophobic forces in the complexation process. Changes in the solvent accessible surface area also contribute to this ΔCp0 value [53-55]. From Record’s relationship [56] viz. ∆G0hyd=(80 10) × ΔCp0, the Gibbs energy contribution arising from the hydrophobic transfer step (∆G0hyd) was calculated to be -0.32 kcal.mol-1. 3.10. Dynamic light scattering Dynamic light scattering (DLS) is an useful tool for obtaining the dimensions of a large number of biomacromolecular assemblies. Using DLS the size distribution (hydrodynamic diameter, dh) profile of HHb and its complex with TZ was determined. The average hydrodynamic diameter of the native HHb, dh~(88±15) Å, enhanced to dh~(136±3) Å upon complexation with TZ. Such an enhancement in the value hydrodynamic diameter of Hb can be attributed to arise from the aggregation of HHb upon denaturation in presence of TZ [57]. 3.11. Esterase activity assay HHb is endowed with an interesting enzymatic property, usually known an esteraselike activity. Allura red, an artificial azo dye, and its degradation products are known to 18
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 inhibit the esterase activity of carbonic anhydrase II [58,59]. In light of the significant conformational changes induced by TZ on the native conformation of HHb, it is pertinent to monitor the influence of TZ on the esterase activity of HHb as knowledge of the influence of TZ on the activity of HHb is essential to assess the food safety of TZ. The esterase-like activity of HHb is obtained from the absorbance of p-nitrophenol produced by the hydrolysis of p-nitrophenyl acetate upon action of HHb. An unit activity refers to the amount of the enzyme needed to liberate 1.0 mM p-nitrophenol per minute at 310.15 K [57]. Changes in the relative esterase activity of HHb with increasing concentration of TZ are shown in Fig. 9. It can be clearly seen from the figure that HHbTZ association produced discernible decrease in the esterase activity of HHb, which reaffirms the earlier fact that TZ causes breakdown of the native structure of HHb. 3.12. Chaotrope induced denaturation studies Fluorescence studies provide a convenient tool to examine the changes in the tertiary structure of HHb which also enables us to unravel its environmental stability. The effect of urea induced denaturation of HHb on its binding efficacy and on the overall photophysics of TZ was studied. Urea produced alterations in the steady state emission spectra of TZ bound HHb. Fig. 10A represents the changes in the relative emission intensity of HHb complexed with TZ upon addition of increasing concentration of urea. Addition of the chaotrope effected an enhancement in the emission intensity of HHb at 328 nm. The intrinsic fluorescence of HHb for Trp residues enhanced on addition of the chaotrope due to a structural conformation whereby the Trp residues and the heme 19
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 group are far apart for energy transfer to be restricted [60-62]. This happens due to the chaotrope induced denaturation of HHb. Therefore, chaotrope effected protein denaturation leads to a large increase in the fluorescence intensity by inhibition of nonradiative energy transfer from the Trp residues to heme prosthetic group. From Fig. 10B it can be seen that with gradual addition of the chaotrope (urea) the intensity of the emission band of TZ enhanced. The addition of the chaotrope effected destabilization of the TZ–Hb complex thereby exposing greater amount of TZ to the bulk buffer solution compared to the native HHb bound condition. This exposure of the earlier bound TZ molecules caused an enhancement in the fluorescence of TZ. Chaotropes can displace water molecules adjacent to TZ in the HHb microenvironment with simultaneous denaturation of HHb [25,57,63]. Hence, chaotrope induced destabilization of the complex is associated with a greater exposure of TZ to the bulk aqueous buffer solution compared to its HHb bound state in the native conformation of HHb [57]. This increased exposure of TZ can account for the enhancement in the intensity of the emission maximum. 4. Conclusions Spectroscopic studies testified for a strong binding reaction between TZ and HHb. The binding phenomenon was static in nature. FRET studies revealed that the binding interaction involved close contact of TZ with β-Trp37 residue at the α1β2 interface. Synchronous, 3D fluorescence and FTIR studies revealed strong conformational changes in HHb upon complexation with TZ. Furthermore, circular dichroism studies 20
REVISED MANUSCRIPT HAZMAT-D-16-01820R1 indicated conclusively that in presence of TZ there was a significant loss in the helical stability of HHb. The esterase activity assay also supported this data. ITC studies revealed that the binding was exothermic and favored by large positive entropic and small negative enthalpic contributions. Negative value of the Gibbs energy suggested spontaneity of the binding interaction. Salt and temperature dependent microcalorimetric studies established the critical role of non-polyelectrolytic forces such as hydrophobic interaction, van der Waals interaction, H-bonding etc. in the complexation process. This work clearly delineates the mode, mechanism, affinity and energetics of the binding of TZ with HHb. Toxicity of pollutants like azo dyes which are introduced into the blood stream as a consequence of environmental exposure is a serious health concern for the humans. Since it is revealed that TZ can bind strongly to HHb and induce significant conformational changes its transportation and metabolism in the human body, it can pose potential biological toxicity risk. It is likely that the absorption of TZ in the blood plasma will adversely affect HHb function and can even lead to impairment of its activity. Hence, the selection of TZ as a food colorant must take into consideration the aforementioned issues on health hazard grounds. 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 recipient of Research Associateship from GenCODE (BSC0123).
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Figure legends Fig. 1. Molecular structure of TZ.
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Fig. 2. (A) Absorption spectral titration of HHb (curve1) with increasing amounts of TZ (curves 2–5). (B) The Soret band of HHb is magnified here.
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Fig. 3. Changes in the steady state fluorescence emission spectra of Hb (10 µM, curve 1) on treatment with 16-120 µM of TZ (curves 2-9).
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Fig. 4. (A) Synchronous fluorescence spectra of HHb (10 µM, curve 1) in the presence of 5-100 µM of TZ (curve 2-10) when Δλ=60 nm. (B) Synchronous fluorescence spectra of HHb (10 µM, curve 1) in the presence of 5-100 µM of TZ (curve 2-10) when Δλ=15 nm, respectively.
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Fig. 5. Three-dimensional fluorescence and contour spectra of Hb (A, B) and Hb- TZ (C, D) complex.
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Fig. 6. FTIR spectra of Hb (curve 1) and Hb-TZ complex (curve 2).
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Fig. 7. Far UV CD spectra of HHb (0.80 µM, curve 1) in presence of 2, 10, 20, 30, 40, 60 and 70 µM of TZ (curves 2-8).
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Fig. 8. Representative ITC profile for the titration of TZ with HHb. The upper panel represents the ITC profile for the titration of successive aliquots of 10 μL TZ into HHb 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 line represents the best-fit data.
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Fig. 9. Plot of variation in the relative esterase activity of HHb with increasing TZ concentration.
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REVISED MANUSCRIPT HAZMAT-D-16-01820R1 Fig. 10. Plot of variation of relative fluorescence intensity of HHb-TZ complex at the emission maxima of (A) HHb and (B) TZ with increasing urea concentration.
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