Article pubs.acs.org/JAFC
Targeting Proteins with Toxic Azo Dyes: A Microcalorimetric Characterization of the Interaction of the Food Colorant Amaranth with Serum Proteins Anirban Basu and Gopinatha Suresh Kumar* Biophysical Chemistry Laboratory, Chemistry Division, Council of Scientific and Industrial Research (CSIR)Indian Institute of Chemical Biology, Kolkata 700 032, India ABSTRACT: The interaction of amaranth with two homologous serum albumins from human and bovine (HSA and BSA) was studied by microcalorimetry. The binding stoichiometry for the complexation of amaranth to both BSA and HSA was around 1, and the equilibrium constants were (5.79 ± 0.07) × 105 and (1.76 ± 0.05) × 105 M−1, respectively. The binding reaction to HSA at 298.15 K was driven by a large negative enthalpic contribution and a small but positive entropic contribution, while to BSA, it was entirely enthalpy-driven and the entropic contribution was unfavorable. Parsing of the standard molar Gibbs energy revealed that the complexation was dominated by non-polyelectrolytic forces. Temperature-dependent isothermal titration calorimetry studies revealed that the enthalpic contribution increased and the entropic contribution decreased with the rise in the temperature but the Gibbs energy change remained almost unaltered. Differential scanning calorimetry results revealed that the binding reaction stabilized the serum albumins significantly against thermal unfolding. KEYWORDS: amaranth, serum albumins, toxicity, thermodynamics, thermal stability
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INTRODUCTION Azo dyes comprise of a class of synthetic compounds that are extensively used as food colorants. Azo dyes, although relatively less toxic, undergo biological or photochemical degradation in an aqueous environment, giving rise to degradation products that are potentially more toxic than the parent compound.1,2 Azo and nitro compounds can become reduced in sediments of the aqueous environment, resulting in the formation of carcinogenic amines.2,3 The carcinogenic effect of the synthetic azo dyes is also well-known.2,4,5 Their mechanism of toxicity has been reviewed.6 The cleavage of azo linkage can lead to the formation of a highly toxic carcinogenic molecule called benzidine, which can adversely affect different parts of our body, in particular, the urinary organs, stomach, kidney, brain, and liver.2,7 Amaranth (FD&C Red No. 2, E123) (Figure 1) is a synthetic azo dye that is used extensively as a food colorant in many
reactions inside the human body of sensitive people when it comes in contact with drugs such as aspirin.9,10 Synthetic azo dyes, such as amaranth, containing a NN functional group in conjunction with aromatic ring structures are also known to be reductively cleaved into aromatic amines, which are toxic, mutagenic, and carcinogenic.8,11 In light of the above evidence, a thorough safety assessment of amaranth is essential and the use of this food colorant needs to be strictly monitored by laws, regulations, and acceptable daily intake values. Serum albumins are the major multifunctional transport proteins present in the blood plasma. They perform myriad physiological functions, such as transportation and disposition of several endogenous and exogenous compounds, including fatty acids, proteins, metals, amino acids, and pharmaceutical compounds.12−15 The serum proteins also influence the transportation and distribution of many metabolites. Binding to serum proteins may be used as a vehicle to regulate and control the available free concentration of the azo dye for toxic action.16,17 Human serum albumin (HSA) and bovine serum albumin (BSA) are two homologous globular heart-shaped proteins having three structurally similar domains (I, II, and III), and each comprises of two subdomains (A and B).18−22 The two most important binding regions in these serum proteins are Sudlow’s site I (subdomain IIA) and site II (subdomain IIIA).23,24 Zhang et al. reported that amaranth binds in Sudlow’s site I (subdomain IIA) of HSA. 9 Spectroscopic, ultrafiltration, electrochemical, and kinetic studies on the interaction of amaranth and serum proteins have been already undertaken.9,25−27 Although the structural
Figure 1. Chemical structure of amaranth.
countries. Joint Food and Agriculture Organization (FAO)/ World Health Organization (WHO) expert committee on food additives (JECFA) recommended that the acceptable daily intake (ADI) level for amaranth is 0−1.5 mg/kg of body weight (bw).8 However, the toxic effects of amaranth are also welldocumented. It is known to invoke allergic and asthmatic © 2014 American Chemical Society
Received: Revised: Accepted: Published: 7955
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and sample cell was measured and converted to power. This data channel is referred to as the differential power (DP) between the reference and sample cell. Calibration of this signal was performed electrically by administering a known quantity of power through a resistive heater element located in the sample cell and then measuring the actual deflection of the DP data signal. The samples were incubated at 303.15 K for 10 min and scanned from 306.15 to 363.15 K at approximately 30 psi pressure at a scan rate of 50 K h−1. Prior to sample scans, the DSC unit was thermally stabilized by repeated buffer scans under an identical scan rate and in the same temperature range. Typically around 10−12 scans were needed to attain the baseline stability. A total of 40 μM of the serum protein solution was scanned in the aforementioned temperature range to obtain the DSC thermogram of the unbound protein. Thereafter, the serum proteins were incubated with the azo dye solution for 10 min to ensure complete complex formation and then scanned in the same temperature range to obtain the thermogram of the bound form. The thermograms were analyzed with the Origin software built-in with the unit. The non-2-state (cursor initiation) model of curve fitting was employed to fit the raw DSC thermograms. Each experiment was repeated 3 times with separate fillings. The Cp° of the protein solution was measured with respect to the Cp° of the buffer. The Cp° at a constant pressure is a temperature derivative of the enthalpy
aspects of the interaction have been studied, the calorimetric studies to elucidate the thermodynamics of the interaction have not yet been performed. Thus, the present work elucidates the thermodynamics of the interaction of amaranth with two homologous serum proteins using isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) techniques.
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MATERIALS AND METHODS
Materials. HSA (0.99 mass fraction purity, essentially fatty acid and globulin free), BSA (0.99 mass fraction purity, essentially fatty acid and globulin free), and amaranth (dye content of 85−95%) were procured from Sigma-Aldrich Co. LLC (St. Louis, MO). All of the experiments were performed in 10 mM citrate−phosphate buffer at pH 7.0, unless otherwise mentioned, where the serum albumins are in the normal (or N) form. The concentrations of HSA and BSA samples were spectrophotometrically determined using molar absorption coefficient values (ε) of 3.66 × 104 and 4.38 × 104 M−1 cm−1, respectively, at 280 nm. 28 The concentration of the dye was also determined spectrophotometrically using the ε value of 2.56 × 104 M−1 cm−1 reported in the literature.25 All of the buffer salts were of analytical grade. The aforementioned citrate−phosphate buffer was prepared in triple-distilled and deionized water. All of the solutions were filtered through filters with a pore size of 0.22 μM (Millipore India Pvt. Ltd., Bangalore, India). ITC. Thermodynamic characterization of the amaranth−serum albumin interaction was performed on a MicroCal VP-ITC calorimeter (MicroCal LLC, Northampton, MA). The protein solutions were prepared by dialyzing against the buffer. The dye solution was prepared in the buffer. Both the dye and protein solutions were extensively degassed prior to the titrations. The titrant, amaranth solution, was loaded into the rotating injector syringe, and the serum protein solution was placed in the calorimeter cell. The programmable dye−protein titration was controlled by the Origin 7.0 software (MicroCal) of the unit. The autocontrolled rotating syringe (351 rpm) injected 10 μL aliquots of the dye solution into the protein solution (1.4235 mL) at intervals of 240 s. This time interval was sufficient enough for the heat signal to return to the baseline after each heat spike generation. The duration of each injection was fixed at 10 s, and the initial delay before the first injection was 60 s. To correct for the thermal effect because of mixing and dilution, appropriate control experiments were performed by injecting an identical volume of the same concentration dye solutions into the experimental buffer alone. The integrated heat change was analyzed by means of nonlinear regression using the Origin software of the unit. The resulting corrected injection heats were then plotted against the molar ratio (χ) of amaranth/serum protein and fitted to a model for single binding sites. The binding equilibrium constant (K), standard molar enthalpy change (ΔrH°), and stoichiometry value “N” were obtained from the analysis of the titration curve. The values of the standard molar Gibbs energy change (ΔrG°) and standard molar entropic contribution (TΔrS°) were then calculated using the relationships
Δr G° = − RT ln(K ) = T Δr S° − Δr H °
C p° =
⎛ ∂H ⎞ ⎜ ⎟ ⎝ ∂T ⎠P
(2)
and hence, the calorimetric enthalpy change (ΔHcal) can be calculated by integration of Cp°. The transition temperature (Tm) is the temperature at which the excess heat capacity reaches its maximum value. Calibration of the DSC unit was performed with built-in calibration heaters, whereas the temperature calibration was performed using the MicroCal standards.
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RESULTS AND DISCUSSION Elucidation of the Thermodynamics of the Complexation from ITC Studies. ITC is the most efficient tool to thermodynamically characterize the ligand−protein binding phenomenon.30,31 The thermodynamics of the binding reaction are characterized by the equilibrium constant, the stoichiometry of the reaction, the standard molar Gibbs energy change, the standard molar enthalpy change, the standard molar entropy change, and the standard molar heat capacity change (ΔrCp°). ITC directly measures the equilibrium constant for the binding reaction by measuring the heat evolved on complexation of a ligand with protein. Thus, the distinct advantage of this protocol is that, from a single experiment, we can obtain the values of the equilibrium constant, the stoichiometry of the reaction (N), and the standard molar enthalpy change of binding. The standard molar Gibbs energy change and the standard molar entropy change of binding may be subsequently calculated using the standard relationships described above. The temperature dependence of the values of ΔrH°, obtained by performing the titration at varying temperatures, permits the calculation of ΔrCp°. Figure 2 presents the ITC thermograms for the binding reaction of amaranth to BSA and HSA at 298.15 K. It can be seen that the ITC thermograms were characterized by negative peaks in the plot of power versus time, suggesting the binding reaction to be exothermic. Each heat spike in the thermogram corresponds to an injection of the dye into the respective serum protein solution. It was corrected for heat effects resulting from dilution and mixing by subtracting the corresponding control heats derived from the injection of an identical amount of the amaranth into the buffer alone. The resulting corrected heats for amaranth−protein complexation were plotted as a function
(1)
where R is the gas constant (8.314 472 J K−1 mol−1) and T is the temperature in kelvin (298.15 K). The thermodynamic parameters presented are averages of triplicate measurements. Periodic calibration of the ITC unit for volume of the calorimetric chamber, heat exchange, and injected volumes of titrant were performed. Furthermore, to ensure the accuracy of the calorimetric results, water−water dilution experiments were performed as per criteria of the manufacturer that the mean energy per injection was less than 5.46 μJ and the standard deviation was less than 0.063 μJ. Chemical calibration of the unit was performed with propan-1-ol of three different concentrations, 2, 5, and 10%, as proposed by Adão et al.29 DSC. The change in excess heat capacity (ΔrCp°) as a function of the temperature “T” was measured in a VP-DSC microcalorimeter (MicroCal LLC). The temperature difference between the reference 7956
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the magnitude of ΔrH° was higher for BSA in comparison to HSA. The strengthening of the H bonds in the low dielectric macromolecular interior of the serum albumins in conjunction with van der Waals interactions introduced as a consequence of the hydrophobic effect account for the observed negative ΔrH° values.32 Using the aforementioned standard relationship, the values of ΔrG° for the complexation of amaranth with BSA and HSA were evaluated to be −32.89 ± 0.07 and −29.94 ± 0.05 kJ mol−1, respectively. Thus, the binding of amaranth to BSA over HSA was favored by an additional Gibbs energy change of 2.95 kJ mol−1 (in absolute values). The entropy contributions for the binding reactions were calculated to be −7.11 ± 0.03 and 7.35 ± 0.02 kJ mol−1, respectively. Thus, it can be observed that the binding reaction in the case of HSA was driven by both negative standard molar enthalpy change and positive standard molar entropy contributions. However, for BSA, the binding reaction was entirely driven by negative standard molar enthalpic contribution. The standard molar entropic contribution was unfavorable, but the magnitude of the standard molar enthalpic contribution was large enough to overcome this unfavorable entropy term and make the overall reaction feasible. It is also noteworthy that the binding reaction was enthalpy-dominated for both the serum proteins. Influence of the Ionic Strength of the Medium upon Binding and Parsing of the Standard Molar Gibbs Energy Change. Salt-dependent ITC studies provide further insights into the molecular forces driving the binding reaction. To have knowledge about the role of the polyelectrolytic forces governing the complexation phenomena, salt dependence of the binding was studied as a function of four different Na+ concentrations, viz., 10, 20, 30, and 50 mM, by ITC experiments. The thermodynamic parameters obtained from these salt-dependent ITC studies are collated in Table 1. Upon increasing the salt concentration, the K value progressively decreased for both the serum proteins (Table 1). The K value decreased from (5.79 ± 0.07) × 105 to (5.02 ± 0.07) × 105 M−1 upon increasing the salt concentration from 10 to 20 mM. Upon further increasing the salt concentration to 30 mM, the K value decreased to (4.51 ± 0.06) × 105 M−1, and finally, at 50 mM, the K value was (3.51 ± 0.05) × 105 M−1. Thus, the K value decreased 1.65 times upon increasing the salt concentration from 10 to 50 mM. For HSA, also the K value decreased upon increasing the ionic strength of the medium. The K value reduced from (1.76 ± 0.05) × 105 to (1.29 ± 0.04) × 105 M−1 upon increasing [Na+] from 10 to 20 mM. At 30 and
Figure 2. ITC thermograms for the titration of amaranth with (A) BSA and (B) HSA at 298.15 K. The top panels represent the raw data for the sequential injection of the amaranth solution into the serum proteins along with the corresponding dilution profiles (curves on the top offset for clarity). The bottom panels show the integrated heat data after correction of heat of dilution. The data points (■) reflect the experimental injection heats, while the solid lines represent the calculated fit of the data.
of the molar ratio of amaranth/protein. The data points in the bottom panels of Figure 2 represent the experimental injection heats, whereas the solid lines are the calculated best fits of the experimental data. The data points were fitted with a Levenberg−Marquardt nonlinear least-squares curve fitting algorithm to calculate the values of K, N, and ΔrH°. At T = 298.15 K, the equilibrium constant for the binding reaction of amaranth to BSA and HSA was calculated to be (5.79 ± 0.07) × 105 and (1.76 ± 0.05) × 105 M−1, respectively. This value is in good agreement with that reported in an earlier structural study.9 The magnitude of the equilibrium constant suggested that amaranth has a stronger binding affinity for BSA in comparison to HSA. The N values for the binding reaction were calculated to be 1.28 ± 0.07 and 0.91 ± 0.03, respectively, for BSA and HSA. Thus, a 1:1 complexation scenario may be envisaged for the association of amaranth to both the serum proteins. The analysis of the ITC thermograms yielded the value of ΔrH° to be −40.00 ± 0.04 and −22.59 ± 0.03 kJ mol−1, respectively, for BSA and HSA at T = 298.15 K. Thus,
Table 1. Thermodynamic Parameters for the Association of Amaranth with BSA and HSA from ITC at Different Salt Concentrationsa protein
c(NaCl) (mM)
BSA
10 20 30 50 10 20 30 50
HSA
K (×10−5, M−1) 5.79 5.02 4.51 3.51 1.76 1.29 1.12 0.94
± ± ± ± ± ± ± ±
0.07 0.07 0.06 0.05 0.05 0.04 0.04 0.03
ΔrH° (kJ mol−1)
N 1.28 1.14 0.98 0.89 0.91 0.94 0.99 1.09
± ± ± ± ± ± ± ±
0.07 0.06 0.04 0.03 0.03 0.03 0.04 0.05
−40.00 −38.72 −36.94 −33.77 −22.59 −22.18 −22.04 −21.99
± ± ± ± ± ± ± ±
0.04 0.04 0.04 0.04 0.03 0.03 0.03 0.02
TΔrS° (kJ mol−1) −7.11 −6.18 −4.67 −2.12 7.35 6.99 6.78 6.40
± ± ± ± ± ± ± ±
0.03 0.03 0.02 0.01 0.02 0.01 0.01 0.01
ΔrG° (kJ mol−1) −32.89 −32.54 −32.27 −31.65 −29.94 −29.17 −28.82 −28.39
± ± ± ± ± ± ± ±
0.07 0.07 0.06 0.05 0.05 0.04 0.04 0.03
ΔrG°t (kJ mol−1) −29.43 −29.60 −29.64 −29.40 −25.50 −25.40 −25.44 −25.50
± ± ± ± ± ± ± ±
0.07 0.07 0.06 0.05 0.05 0.04 0.04 0.03
ΔrG°pe (kJ mol−1) −3.46 −2.94 −2.63 −2.25 −4.44 −3.77 −3.38 −2.89
± ± ± ± ± ± ± ±
0.07 0.07 0.06 0.05 0.05 0.04 0.04 0.03
a
All of the data in this table are derived from ITC experiments conducted in citrate−phosphate buffer of different [Na+] at pH 7.0 and 298.15 K. The standard uncertainties associated with K, N, ΔrH°, TΔrS°, ΔrG°, ΔrG°t, and ΔrG°pe are 6, 5, 4, 2, 6, 6, and 6%, respectively, for BSA. Similarly, the standard uncertainties associated with K, N, ΔrH°, TΔrS°, ΔrG°, ΔrG°t, and ΔrG°pe are 4, 4, 3, 1, 4, 4, and 4%, respectively, for HSA. 7957
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50 mM [Na+], the K values were calculated to be (1.12 ± 0.04) × 105 and (0.94 ± 0.03) × 105 M−1, respectively. Thus, for HSA, the K value decreased around 1.87 times upon increasing the ionic strength of the medium from 10 to 50 mM. However, the stoichiometry values remained largely unchanged with increasing the ionic strength of the medium. The N values suggested that 1:1 complexation occurs between amaranth and the serum proteins, irrespective of the salt concentration of the medium. However, the magnitude of ΔrH° decreased with an increasing ionic strength of the medium for both BSA and HSA. This decrease is probably due to shielding of the macromolecule by the sodium ions, which prevented the dye molecules from coming in contact with the serum albumins. The magnitude of ΔrG° also decreased with an increase in the ionic strength of the medium. At 10 mM [Na+], ΔrG° was calculated to be −32.89 ± 0.07 and −29.94 ± 0.05 kJ mol−1, respectively, for BSA and HSA. However, at 50 mM, it was reduced to −31.65 ± 0.05 and −28.39 ± 0.03 kJ mol−1. Thus, the value of ΔrG° decreased by 1.24 and 1.55 kJ mol−1 (in absolute values) for BSA and HSA, respectively, upon increasing [Na+] from 10 to 50 mM. There is a standard relationship that correlates the variation of the equilibrium constant with the sodium ion concentration33,34 ⎛ ∂ log K ⎞ Δr N (ion) = ⎜ = − zϕ + ⎟ ⎝ ∂ log[Na ] ⎠T , P
(3)
where ΔrN(ion) represents the number of ions released upon binding of the azo dye, z is the apparent charge of the bound dye, and φ is the fraction of [Na+] bound per anion. The plot of the variation of log K as a function of log [Na+] yielded straight lines (Figure 3). The slopes of these plots were calculated to be
Figure 4. Partitioned polyelectrolytic (ΔrG°pe) (hatched) and nonpolyelectrolytic (ΔrG°t) (black) contributions to the standard molar Gibbs energy (ΔrG°) at different Na+ concentrations for the complexation of (A) BSA and (B) HSA with amaranth.
The ΔrG°pe at any given [Na+] can be calculated quantitatively using the relationship35,36 ΔG°pe = −zϕRT ln([Na +])
The ΔrG°pe term accounts for the contribution arising from coupled polyelectrolytic forces, such as the release of condensed counterions upon binding of the synthetic azo dye amaranth. The ΔrG°t term accounts for the contribution arising from other non-polyelectrolytic sources, such as van der Waals interaction, π−π stacking, H bonding, and hydrophobic transfer. For BSA, ΔrG°pe at 10, 20, 30, and 50 mM [Na+] was −3.46 ± 0.07, −2.94 ± 0.07, −2.63 ± 0.06, and −2.25 ± 0.05 kJ mol−1, respectively. Similarly, ΔrG°pe was −4.44 ± 0.05, −3.77 ± 0.04, −3.38 ± 0.04, and −2.89 ± 0.03 kJ mol−1 for HSA at 10, 20, 30, and 50 mM [Na+], respectively. ΔrG°t contribution was calculated from the difference between ΔrG° and ΔrG°pe. At 10, 20, 30, and 50 mM [Na+], ΔrG°t was calculated to be −29.43 ± 0.07, −29.60 ± 0.07, −29.64 ± 0.06, and −29.40 ± 0.05, kJ mol−1, respectively, for amaranth−BSA complexation. Similarly, for the amaranth−HSA binding reaction, the magnitude of ΔrG°t was calculated to be −25.50 ± 0.05, −25.40 ± 0.04, −25.44 ± 0.04, and −25.50 ± 0.03 kJ
Figure 3. Plot of log K against log [Na+] for the binding of amaranth to BSA (■) and HSA (●).
−0.303 and −0.389 for BSA and HSA, respectively. Hence, a sharper decrease in the K values was observed for HSA in comparison to BSA. The dependence of K upon [Na+] enabled ΔrG° to be partitioned into two components, viz., the nonpolyelectrolytic component (ΔrG°t) and the polyelectrolytic component (ΔrG°pe) (Figure 4) Δr G° = Δr G°t + Δr G°pe
(5)
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Table 2. Thermodynamic Parameters for the Association of Amaranth with BSA and HSA from ITC at Different Temperaturesa protein
T (K)
BSA
283.15 293.15 298.15 303.15 283.15 293.15 298.15 303.15
HSA
K (×10−5, M−1) 7.23 6.12 5.79 2.67 3.03 2.28 1.76 1.29
± ± ± ± ± ± ± ±
0.08 0.08 0.07 0.05 0.05 0.05 0.05 0.04
ΔrH° (kJ mol−1)
N 0.91 0.97 1.28 0.89 0.96 0.98 0.91 1.24
± ± ± ± ± ± ± ±
0.03 0.04 0.07 0.03 0.04 0.04 0.03 0.07
−27.77 −35.24 −40.00 −41.84 −14.73 −19.82 −22.59 −27.19
± ± ± ± ± ± ± ±
0.03 0.04 0.04 0.04 0.02 0.02 0.03 0.03
TΔrS° (kJ mol−1) 3.99 −2.76 −7.11 −10.35 14.98 10.25 7.35 2.47
± ± ± ± ± ± ± ±
0.05 0.04 0.03 0.01 0.03 0.03 0.02 0.01
ΔrG° (kJ mol−1) −31.76 −32.48 −32.89 −31.49 −29.71 −30.07 −29.94 −29.66
± ± ± ± ± ± ± ±
0.08 0.08 0.07 0.05 0.05 0.05 0.05 0.04
ΔrCp° (kJ K−1 mol−1) −0.73 ± 0.06
−0.60 ± 0.06
The standard uncertainties associated with K, N, ΔrH°, TΔrS°, ΔrG°, and ΔrCp° are 7, 4, 4, 3, 7, and 6%, respectively, for BSA. Similarly, the standard uncertainties associated with K, N, ΔrH°, TΔrS°, ΔrG°, and ΔrCp° are 5, 4, 2, 2, 5, and 6%, respectively, for HSA. a
mol−1, respectively. Thus, although large in magnitude, the non-polyelectrolytic component remained largely invariant with changing salt concentrations. However, the polyelectrolytic component decreased in magnitude with the increase in [Na+]. At the highest salt concentration studied, viz., 50 mM [Na+], the ΔG°pe term accounted for only 7 and 10% of the total standard molar Gibbs energy change for BSA and HSA, respectively. Hence, for both serum proteins, the binding reaction was dominated by non-polyelectrolytic forces, which accounted for ∼90% of the total standard molar Gibbs energy change. Thus, the dissection of the standard molar Gibbs energy into polyelectrolytic and non-polyelectrolytic components unequivocally established the dominant role played by non-polyelectrolytic forces in the protein−dye complexation process. Temperature Dependence of the Enthalpy: Determination of Heat Capacity Changes. To gain additional insights into the forces controlling the binding reaction of amaranth with serum albumins, temperature-dependent ITC studies were performed at four different temperatures, viz., 283.15, 293.15, 298.15, and 303.15 K. The pH of the solutions remained unchanged in this temperature range studied. The thermograms for amaranth−protein complexation were found to be sigmoidal at all four temperatures studied. The binding reaction was exothermic at all of the temperatures, yielding negative peaks in the plot of power versus time. The thermodynamic data derived from temperature-dependent ITC studies are collated in Table 2. The K values decreased, ΔrH° values became more negative with their magnitudes increasing, and the TΔrS° values also decreased with the rise in the temperature. However, the magnitude of ΔrG° remained largely unaltered with the temperature. The variations in the thermodynamic parameters with increasing the temperature are graphically represented in Figure 5. From the N values, it was clearly established that 1:1 complexation occurs between amaranth and the serum proteins, irrespective of the temperature. The standard molar specific heat capacity changes were derived from a plot of ΔrH° versus T at a constant pressure, using the following relationship: Δr Cp0 = [∂(Δr H °)/∂T ]P
Figure 5. Variation of ΔrG° (white), TΔrS° (hatched), and ΔrH° (black) with temperature for the complexation of amaranth with (A) BSA and (B) HSA.
(6)
The variation of ΔrH° with T afforded straight lines, the slopes of which resulted in values of −0.73 ± 0.06 and −0.60 ± 0.06 kJ K−1 mol−1, respectively, for the complexation of amaranth with BSA and HSA (Figure 6). Many ligand−protein and ligand−nucleic acid interactions have been characterized by such negative ΔrCp° values.28,32,37 Besides, negative ΔrCp° values are often associated with alterations in hydrophobic or polar group hydration and suggest that the binding reaction is
Figure 6. Plot of variation of ΔrH° as a function of the temperature for the complexation of BSA (■) and HSA (●) with amaranth.
dominated by hydrophobic interactions. The ΔrCp° value is lower for HSA compared to BSA, indicating differences in the 7959
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release of structured water upon complexation with amaranth. It is also an indicator of the different extent of hydrophobic interactions in the two systems studied. First, it is pertinent to note that the non-zero negative values of ΔrCp° suggest temperature dependence of ΔrH° and establish the involvement of hydrophobic forces in the complexation process. Second, the calculated values of ΔrCp° fall well within the range that is frequently observed for biomolecular interactions.28,38 The magnitude of the ΔrCp° value for amaranth−BSA complexation is slightly higher than that for amaranth−HSA complexation, indicating slight conformational differences in the complexation process and also differences in the disruption of the protein-bound water molecules and counterions. Besides, the removal of water molecules from hydrophobic interfaces, owing to electrostatic interaction, may also be responsible for the negative ΔrCp° term. The negative ΔrCp° values calculated for amaranth−serum protein complexation appears to denote that the binding is specific and accompanied by burial of the nonpolar surface area.39,40 The standard molar Gibbs energy contribution (ΔrG°hyd) from the hydrophobic transfer step of binding was calculated using the Records relationship,41 Δ r G° hyd = (80 ± 10)Δ r C p °. The Δ r G° hyd value for amaranth−BSA complexation was calculated to be −58.40 kJ mol−1, which is slightly higher than that calculated for amaranth−HSA complexation, viz., −48.00 kJ mol−1. Enthalpy−Entropy Compensation (EEC). EEC is a characteristic phenomenon of many ligand−protein interactions.28,42 EEC has been regarded as a natural consequence of finite ΔrCp° values by Cooper et al.43 They suggested that EEC is a consequence of quantum confinement effects, multiple weak non-covalent interactions, and limited free energy windows, which give rise to thermodynamic homeostasis that may be of evolutionary and functional advantage because it enables adaptation to harsher environments.43 EEC is also linked with solvent reorganization, accompanying biomolecular interactions.44−46 The ΔrH° values increased in magnitude, whereas the TΔrS° term decreased with increasing the temperature. However, the ΔrG° values remained almost unaltered with changing the temperature. The ΔrH° and TΔrS°, both of which are strong functions of T, compensated one another to make the overall ΔrG° temperature invariant. Linearity of the relationship of ΔrH° with TΔrS° with slope close to unity suggests complete compensation, and this occurs in systems with ΔrCp° ≠ 0 and ΔrCp° > ΔrS°. ΔrH° and ΔrG° were plotted as a function of TΔrS°, and the slopes of the plot of ΔrH° versus TΔrS° were deduced to be 1.01 ± 0.05 and 0.99 ± 0.06, respectively, for BSA and HSA (Figure 7). Linear relationship between ΔrH° and TΔrS° with slope close to unity suggested complete EEC in the two protein−dye systems studied. DSC Studies. DSC is an effective and reliable tool for monitoring protein folding−unfolding.30,47 To monitor the effect of the azo dye amaranth on the thermal stability of the two homologous serum proteins, the DSC thermograms of excess heat capacity versus temperature were analyzed with Origin 7.0 software. It was found that both the serum proteins, BSA and HSA, unfolded cooperatively, exhibiting a single endothermic peak with melting temperatures of 327.61 and 328.85 K, respectively (Figure 8). The binding reaction with amaranth resulted in remarkable enhancement of the thermal stability of both the serum proteins. Upon binding to amaranth, the thermal denaturation temperature (Tm) of BSA and HSA was enhanced to 335.56 and 341.01 K, respectively (Figure 8).
Figure 7. Plot of variation of ΔrH° (closed symbols) and ΔrG° (open symbols) as a function of TΔrS° for the binding of amaranth to BSA (■ and □) and HSA (● and ○).
Figure 8. DSC thermograms of (A) BSA and (B) HSA. Curves 1 and 2 represent the free protein and amaranth−protein complexes, respectively.
Thus, the two homologous serum proteins, BSA and HSA, were stabilized (ΔTm) by 7.95 and 12.16 K, respectively. In addition to the Tm values, the DSC thermograms upon analysis also afforded the calorimetric transition enthalpy (ΔrHcal). The value of ΔrHcal is model-independent and has no relation with the nature of the transition. It is pertinent to mention here that there was no significant change in the shape of the DSC thermograms, which enabled us to conclude that the denaturation process was not altered but only shifted to higher temperatures in the presence of the dye. The ΔrHcal value increased from 296.52 ± 0.08 kJ mol−1 in the unbound form to 683.76 ± 0.15 kJ mol−1 in the bound form for BSA. For HSA, the magnitude of ΔrHcal enhanced from 321.30 ± 0.14 kJ mol−1 in the unbound form to 759.36 ± 0.16 kJ mol−1 in the bound form. Thus, ΔrHcal increased by 387.24 ± 0.07 and 438.06 ± 7960
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0.02 kJ mol−1, respectively, for BSA and HSA upon binding to amaranth. The present study presents a detailed description of the thermodynamics of the interaction of the toxic food colorant amaranth with two homologous serum proteins. These microcalorimetric investigations lend deeper insights into the thermodynamics of the interaction of the toxic food additives with the serum proteins, which may prove critical in realizing the toxic and hazardous effects of food colorants on biological systems.
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(12) Ishidate, M., Jr.; Sofuni, T.; Yoshikawa, K.; Hayashi, M.; Nohmi, T.; Sawada, M.; Matsuoka, A. Primary mutagenicity screening of food additives currently used in Japan. Food Chem. Toxicol. 1984, 22, 623− 636. (13) Carter, D. C.; He, X.; Munson, S. H.; Twigg, P. D.; Gernert, K. M.; Broom, M. B.; Miller, T. Y. Three-dimensional structure of human serum albumin. Science 1989, 244, 1195−1198. (14) Kratochwil, N. A.; Huber, W.; Muller, F.; Kansy, M.; Gerber, P. R. Predicting plasma protein binding of drugs: A new approach. Biochem. Pharmacol. 2002, 64, 1355−1374. (15) Ghuman, J.; Zunszain, P. A.; Petitpas, I.; Bhattacharya, A. A.; Otagiri, M.; Curry, S. Structural basis of the drug-binding specificity of human serum albumin. J. Mol. Biol. 2005, 353, 38−52. (16) 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. (17) Pan, X. R.; Liu, R. T.; Qin, P. F.; Wang, L.; Zhao, X. C. Spectroscopic studies on the interaction of acid yellow with bovine serum albumin. J. Lumin. 2010, 130, 611−617. (18) Foster, J. F. Albumin Structure, Function and Uses; Pergamon Press: Oxford, U.K., 1977; p 53. (19) He, X. M.; Carter, D. C. Atomic structure and chemistry of human serum albumin. Nature 1992, 358, 209−215. (20) Peters, T., Jr. All about Albumins. Biochemistry, Genetics and Medical Applications; Academic Press: San Diego, CA, 1996; p 9. (21) Dockal, M.; Carter, D. C.; Rücker, F. The three recombinant domains of human serum albumin. Structural characterization and ligand binding properties. J. Biol. Chem. 1999, 274, 29303−29310. (22) Huang, B. X.; Kim, H. Y.; Dass, C. Probing three-dimensional structure of bovine serum albumin by chemical cross-linking and mass spectrometry. J. Am. Soc. Mass Spectrom. 2004, 15, 1237−1247. (23) Sudlow, G.; Birkett, D. J.; Wade, D. N. The characterization of two specific drug binding sites on human serum albumin. Mol. Pharmacol. 1975, 11, 824−832. (24) Sudlow, G.; Birkett, D. J.; Wade, D. N. Further characterization of specific drug binding sites on human serum albumin. Mol. Pharmacol. 1976, 12, 1052−1061. (25) Murakami, K. Equilibrium and kinetic studies of the binding of tri- and tetra-anionic ligands to bovine serum albumin. Biophys. Chem. 1991, 41, 253−261. (26) Yoon, J.-H.; McKenzie, D.; Prichard, F. E. The binding of food dyes with human serum albumin. Arch. Pharmacal Res. 1996, 19, 269− 274. (27) Sun, W.; Jiao, K.; Wang, X.; Lu, L. Electrochemical studies of the reaction between albumin and amaranth and its analytical application. J. Electroanal. Chem. 2005, 578, 37−43. (28) 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. (29) Adão, R.; Bai, G.; Loh, W.; Bastos, M. Chemical calibration of isothermal titration calorimeters: An evaluation of the dilution of propan-1-ol into water as a test reaction using different calorimeters, concentrations and temperatures. J. Chem. Thermodyn. 2012, 52, 57− 63. (30) Bernazzani, L.; Ferrari, C.; Gianni, P.; Mollica, V.; Tombari, E. Calorimetric investigation on the interaction of sodium taurodeoxycholate with human serum albumin. Thermochim. Acta 2013, 555, 7− 16. (31) Damian, L. Isothermal titration calorimetry for studying protein−ligand interactions. Methods Mol. Biol. 2013, 1008, 103−118. (32) Ross, P. D.; Subramanian, S. Thermodynamics of protein association reactions: Forces contributing to stability. Biochemistry 1981, 20, 3096−3102. (33) Manning, G. S. The molecular theory of polyelectrolyte solutions with applications to the electrostatic properties of polynucleotides. Q. Rev. Biophys. 1978, 11, 179−246. (34) Record, M. T., Jr.; Anderson, C. F.; Lohman, T. M. Thermodynamic analysis of ion effects on the binding and conformational equilibria of proteins and nucleic acids: The roles of
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*Telephone: +91-33-2499-5723/2472-4049. Fax: +91-33-24723967. E-mail:
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[email protected]. Funding
This work was supported by the network project BIOCERAM (ESC0103) of the Council of Scientific and Industrial Research (CSIR), Government of India. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors extend their thanks to all of the members of the Biophysical Chemistry Laboratory for their cooperation and help and to the anonymous reviewers for their critical and judicious evaluation of the manuscript and helpful suggestions.
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REFERENCES
(1) Sanchez-Prado, L.; Lompart, M.; Lores, M.; Garcma-Jares, C.; Bayona, J. M.; Cela, R. Monitoring the photochemical degradation of triclosan in waste water by UV light and sunlight using solid-phase microextraction. Chemosphere 2006, 65, 1338−1347. (2) Ratna; Padhi, B. S. Pollution due to synthetic dyes toxicity & carcinogenicity studies and remediation. Int. J. Environ. Sci. 2012, 3, 940−955. (3) Chen, H. Recent advances in azo dye degrading enzyme research. Curr. Protein Pept. Sci. 2006, 7, 101−111. (4) Weisburger, J. H. Comments on the history and importance of aromatic and heterocyclic amines in public health. Mutat. Res. 2002, 506−507, 9−20. (5) Umbuzeiro, G. A.; Freeman, H. S.; Warren, S. H.; Oliveira, D. P.; Terao, Y.; Watanabe, T.; Claxton, D. L. The contribution of azo dyes to the mutagenic activity of Cristais river. Chemosphere 2005, 60, 55− 64. (6) Puvaneswari, N.; Muthukrishnan, J.; Gunasekaran, P. Toxicity assessment and microbial degradation of azo dyes. Indian J. Exp. Biol. 2006, 44, 618−626. (7) Chowdhury, G. Human health perspectives on environmental exposure to benzidine: A review. Chemosphere 1996, 32, 267−291. (8) Mpountoukas, P.; Pantazaki, A.; Kostareli, E.; Christodoulou, P.; Kareli, D.; 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. (9) 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. (10) Nevado, J. J. B.; Cabanillas, C. G.; Salcedo, A. M. C. Simultaneous spectrophotometric determination of three food dyes by using the first derivative of ratio spectra. Talanta 1995, 42, 2043− 2051. (11) King-Thom, C. Mutagenicity and carcinogenicity of aromatic amines metabolically produced from azo dyes. J. Environ. Sci. Health, Part C: Environ. Carcinog. Ecotoxicol. Rev. 2000, 18, 51−74. 7961
dx.doi.org/10.1021/jf5025278 | J. Agric. Food Chem. 2014, 62, 7955−7962
Journal of Agricultural and Food Chemistry
Article
ion association or release, screening, and ion effects on water activity. Q. Rev. Biophys. 1978, 11, 103−178. (35) Record, M. T., Jr.; Spolar, R. S. In The Biology of Nonspecific DNA−Protein Interactions; Revzin, A., Ed.; CRC Press: Boca Raton, FL, 1990; p 33. (36) Record, M. T., Jr.; Ha, H. H.; Fisher, M. A. Analysis of equilibrium and kinetic measurements to determine thermodynamic origins of stability and specificity and mechanism of formation of sitespecific complexes between proteins and helical DNA. Methods Enzymol. 1991, 208, 291−343. (37) Basu, A.; Suresh Kumar, G. Minor groove binding of the food colorant carmoisine to DNA: Spectroscopic and calorimetric characterization studies. J. Agric. Food Chem. 2014, 62, 317−326. (38) Liu, C. C.; Richard, A. J.; Datta, K.; LiCata, V. J. Prevalence of temperature-dependent heat capacity changes in protein−DNA interactions. Biophys. J. 2008, 94, 3258−3265. (39) Spolar, R. S.; Record, M. T., Jr. Coupling of local folding to sitespecific binding of proteins to DNA. Science 1994, 263, 777−784. (40) O’Brien, R.; DeDecker, B.; Fleming, K. G.; Sigler, P. B.; Ladbury, J. E. The effects of salt on the TATA binding protein−DNA interaction of a hyperthermophilic archaeon. J. Mol. Biol. 1998, 279, 117−125. (41) Ha, J. H.; Spolar, R. S.; Record, M. T., Jr. Role of the hydrophobic effect in stability of site-specific protein-DNA complexes. J. Mol. Biol. 1989, 209, 801−816. (42) Olsson, T. S.; Ladbury, J. E.; Pitt, W. R.; Williams, M. A. Extent of enthalpy−entropy compensation in protein−ligand interactions. Protein Sci. 2011, 20, 1607−1618. (43) Cooper, A.; Johnson, C. M.; Lakey, J. H.; Nöllmanna, M. Heat does not come in different colours: Entropy−enthalpy compensation, free energy windows, quantum confinement, pressure perturbation calorimetry, solvation and the multiple causes of heat capacity effects in biomolecular interactions. Biophys. Chem. 2001, 93, 215−230. (44) Sharma, S.; Bharadwaj, S.; Surolia, A.; Podder, S. K. Evaluation of the stoichiometry and energetics of carbohydrate binding to Ricinus communis agglutinin: A calorimetric study. Biochem. J. 1998, 333, 539− 542. (45) Williams, B. A.; Chervenak, M. C.; Toone, E. J. Energetics of lectin−carbohydrate binding. J. Biol. Chem. 1992, 267, 22907−22911. (46) Gupta, D.; Cho, M.; Cummings, R. D.; Brewer, C. F. Thermodynamics of carbohydrate binding to galectin-1 from Chinese hamster ovary cells and two mutants. A comparison with four galactose-specific plant lectins. Biochemistry 1996, 35, 15236−15243. (47) Waldron, T. T.; Murphy, K. P. Stabilization of proteins by ligand binding: Application to drug screening and determination of unfolding energetics. Biochemistry 2003, 42, 5058−5064.
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