A biophysical investigation on the binding of proflavine with human hemoglobin: Insights from spectroscopy, thermodynamics and AFM studies

Journal of Photochemistry & Photobiology, B: Biology 165 (2016) 42–50

Contents lists available at ScienceDirect

Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol

A biophysical investigation on the binding of proflavine with human hemoglobin: Insights from spectroscopy, thermodynamics and AFM studies Anirban Basu, Gopinatha Suresh Kumar ⁎ Biophysical Chemistry Laboratory, Organic & Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Biology, Kolkata 700 032, India

a r t i c l e

i n f o

Article history: Received 18 July 2016 Received in revised form 26 September 2016 Accepted 11 October 2016 Available online 12 October 2016 Keywords: Proflavine Hemoglobin Spectroscopy Calorimetry

a b s t r a c t Interaction of proflavine with hemoglobin (Hgb) was studied employing spectroscopy, calorimetry, and atomic force microscopy. The equilibrium constant was found to be of the order 104 M−1. The quenching of Hgb fluorescence by proflavine was due to the complex formation. Calculation of the molecular distance (r) between the donor (β-Trp37 of Hgb) and acceptor (proflavine) suggested that energy can be efficiently transferred from the β-Trp37 residue at the α1β2 interface of the protein to the dye. Proflavine induced significant secondary structural changes in Hgb. Synchronous fluorescence studies showed that proflavine altered the microenvironment around the tryptophan residues to a greater extent than the tyrosine residues. Circular dichroism spectral studies showed that proflavine caused significant reduction in the α-helical content of Hgb. The esterase activity assay further complemented the circular dichroism data. The Soret band intensity of Hgb decreased upon complexation. Differential scanning calorimetry and circular dichroism melting results revealed that proflavine induced destabilization of Hgb. The binding was driven by both positive entropy and negative enthalpy. Atomic force microscopy studies revealed that the essential morphological features of hemoglobin were retained in the presence of proflavine. Overall, insights on the photophysical aspects and energetics of the binding of proflavine with Hgb are presented. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Proflavine is a novel acridine analog consisting of a planar polycyclic monocationic aromatic moiety with two amino groups exhibiting wide range of pharmacological activities [1–3]. Proflavine was used as an antiseptic as well as a disinfectant bacteriostatic [4,5]. It can cause frameshift mutations in viruses, bacteria and bacteriophages [6], and inhibit replication in cancer cells by intercalating between the DNA base pairs [7–13]. So, it has the potential to be developed as a chemotherapeutic agent [14–16]. Proteins perform a wide range of functions in living organisms including transportation of endogenous and exogenous molecules form one site to another and hence they are the prime choice for probing the pharmacological action of drugs [17]. Understanding the dynamics and biochemical consequences of drug–protein interactions is essential in pharmacology and drug development. Although the interaction of acridines with nucleic acids is well documented their interaction with proteins have not yet been properly characterized [18–27]. Hgb is an important protein whose structure and function are well established ⁎ Corresponding author at: CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Kolkata 700 032, India. E-mail address: [email protected] (G.S. Kumar).

http://dx.doi.org/10.1016/j.jphotobiol.2016.10.010 1011-1344/© 2016 Elsevier B.V. All rights reserved.

[28–31]. Hgb is present inside the red blood cells but it can become reactive and toxic on hemolysis, under diseased conditions or owing to the action of some drugs. This leads to its presence in the plasma getting exposed to various small molecules and drugs. Since Hgb can interact with a variety of small molecules the metabolism, distribution and efficacy of many drugs and biologically important compounds in vivo may be influenced by their affinity to Hgb [32–34]. The therapeutic effect of a drug is directly linked with its concentration in blood. The unbound drug can easily diffuse from blood to tissue/organ where the pharmacological activity occurs. Strong association between protein and drug molecules reduces the free drug concentration in blood and also decreases its pharmocodynamic effect. However, weak binding between protein and drug molecules results in shorter lifetime [35]. Thus, complexation between protein and drug molecules can influence the release of drugs from blood to receptors and also inhibit their rapid metabolism [36]. For this reason, it is pertinent to investigate the interaction of potential drugs like proflavine with Hgb to understand their metabolism and distribution in vivo. In this report, we investigated the effect of proflavine on Hgb studying the excited-state photophysics of proflavine within the microheterogeneous proteinous environment. Furthermore, we have evaluated the energetics of the interaction using sensitive calorimetric techniques to correlate the structural aspects with the energetics.

A. Basu, G.S. Kumar / Journal of Photochemistry & Photobiology, B: Biology 165 (2016) 42–50

43

2. Experimental

2.6. Atomic Force Microscopy Studies

2.1. Materials

Atomic force microscopy (AFM) studies were performed for Hgb and Hgb-PFV complexes. Hgb solution was diluted to a final concentration of 200 nM. Thereafter, it was centrifuged and filtered through 0.22 μm filter paper. 5 μL aliquot of Hgb was adsorbed onto a muscovite Ruby mica sheet (ASTM V1 grade Ruby Mica from MICAFAB, Chennai) and dried for 30 min in vacuum drier under inert atmosphere. For the Hgb-PFV complex, an equimolar mixture of Hgb and PFV was prepared, incubated for 10 min and adsorbed onto the mica. AAC mode AFM was done with a Pico plus 5500 ILM AFM (Agilent Technologies, USA) equipped with a piezo scanner. The images were processed using Picoview version 1.1 software (Agilent Technologies) while it was manipulated with the help of Pico Image Advanced version software.

Hgb and proflavine hydrochloride (PFV hereafter, Fig. 1) were obtained from Sigma–Aldrich Corporation (St. Louis, MO, USA). All the sample solutions were prepared in 10 mM citrate-phosphate buffer, pH 7.0 at (298.15 ± 0.01) K unless otherwise specified. The concentrations of PFV and Hgb were determined using molar absorption coefficient (ε) values of 42,000 M− 1 cm− 1 at 444 nm and 1,79,000 M− 1 cm−1 at 405 nm, respectively [37–42]. 2.2. Spectroscopic Studies Absorbance spectral studies were performed on a Jasco V660 double beam double monochromator spectrophotometer (Japan International Co., Hachioji, Japan). Steady state and time resolved fluorescence experiments were performed on either a Shimadzu RF-5301 PC (Shimadzu Corporation, Kyoto, Japan) or Quanta Master 400 unit (Horiba PTI, Canada) controlled with FelixGX spectroscopy software. Fourier transform infrared (FTIR) measurements were performed on a Bruker FTIR, TENSOR 27 spectrometer (Bruker Corp, USA) while circular dichroism (CD) studies were performed on a Jasco J815 spectropolarimeter (Japan International Co.). 2.3. Microcalorimetric Studies Differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) experiments were performed on MicroCal VP-DSC and MicroCal VP-ITC units, respectively, (MicroCal, Inc., Northampton, MA, USA, now Malvern Instruments, UK) following the procedures reported earlier [24,25,41]. In ITC, the heats of PFV-Hgb binding reaction, after appropriate correction for dilution, were plotted as a function of the corresponding molar ratio. The data were then analyzed to obtain the thermodynamic parameters and the equilibrium constant (Ka) [24,25]. 2.4. Dynamic Light Scattering Experiments Dynamic light scattering (DLS) measurements were performed on a HORIBA (SZ-100 OZ) dynamic light scattering particle size analyser [43]. The temperature was maintained at 298.15 K during the measurement.

3. Results and Discussion 3.1. Electronic Absorption Spectroscopy Hgb exhibits two major peaks in the 190–500 nm region. The first peak arises owing to π–π* transition of the carbonyl groups of the amino acid residues while the other one (~ 405 nm) is from the π–π* transition of porphyrin-Soret band [39,40]. The Soret band arises from the heme group embedded within the hydrophobic pocket formed through appropriate folding of Hgb's backbone [45–47]. Both these bands showed a decrease in absorbance on addition of PFV. The absorbance spectral changes in the Hgb spectrum upon addition of PFV are shown in Fig. 2A and the changes associated with the Soret band are highlighted in Fig. 2B. The Soret band's position remained virtually unaltered. The hypochromic change in the Soret band suggested the complexation between PFV and Hgb occured at the ground state. The changes in the absorbance of the Soret band were analyzed using the Benesi–Hildebrand equation [48]. 1 1 1 1  þ ¼ ΔA ΔAmax K BH ðΔAmax Þ ½PFV

ð1Þ

where ΔA = absorbance change at the Soret band, ΔA = maximum absorbance or absorbance of Hgb at the Soret band in absence of PFV, KBH = Benesi–Hildebrand binding constant and [PFV] = Concentration of PFV. The Benesi–Hildebrand association constant for PFV-Hgb complexation (KBH) was deduced to be 2.73 × 104 M−1 (Fig. S1A). 3.2. Effect of Proflavine on the Intrinsic Fluorescence of Hemoglobin

2.5. Esterase Activity Assay Esterase activity of Hgb was assayed by using p-nitro phenyl acetate (p-NPA) as the substrate according to the procedure reported previously [44]. The reactions were initiated by adding 1.5 mM alcoholic p-NPA to a reaction mixture containing 5.0 μM of Hgb and absorbance change at 400 nm due to the addition of p-NPA was recorded. The absorbance data were further corrected for p-NPA hydrolysis by the buffer. Thereafter, relative esterase activity was plotted as a function of the changing PFV concentration.

Intrinsic fluorescence of Hgb is due to the tryptophan (Trp) and tyrosine (Tyr) moieties in the polypeptide chain. The intrinsic fluorescence of Hgb is essentially due to the β-Trp37 residue at the α1β2 interface [49], and indicates transition from relaxed (R) to taut (T) form. Ligand bound (oxy) form is the R form while the deoxy form is the T form [50], and the relative fluorescence intensities of these two forms have significant differences. The fluorescence emission spectrum of Hgb was monitored by exciting at 295 nm which preferentially excited the Trp residues [51]. Hgb exhibited emission maximum at 329 nm revealing that the β-Trp37 residue is buried or is in a hydrophobic region [52]. The fluorescence intensity of Hgb steadily decreased in the presence of increasing PFV concentration. This suggested that PFV interacted with Hgb and the quenching was owing to specific complex formation. 3.3. Effect of Hemoglobin on the Fluorescence of Proflavine

Fig. 1. Molecular structure of PFV.

Proflavine is a highly fluorescent with emission maximum at 509 nm when excited at 444 nm. Since this peak is far away from the Hgb fluorescence maximum (329 nm) it provided a convenient handle to probe the effect of Hgb on the PFV fluorescence. Fluorescence intensity of PFV gradually decreased upon addition of increasing concentration of Hgb

44

A. Basu, G.S. Kumar / Journal of Photochemistry & Photobiology, B: Biology 165 (2016) 42–50

Fig. 2. (A) Representative absorption spectra of Hgb (curve 1) treated with increasing concentrations of PFV (curves 2–5). (B) 405 nm band of Hgb is highlighted here.

and the position of the emission maximum also blue shifted by 4 nm to 513 nm suggesting that the PFV molecules were buried in a more hydrophobic region inside the macromolecular proteinous environment (Fig. 3). The spectral titration data at the emission maximum was further utilized to determine the association constant using the aforementioned Benesi–Hildebrand protocol. The Benesi–Hildebrand equation afforded a linear plot, suggesting 1:1 interaction between PFV and Hgb. The KBH value for PFV–Hgb complexation was deduced to be 3.15 × 104 M−1 (Fig. S1B). 3.4. Temperature Dependent Fluorescence Studies and Elucidation of the Mode of Quenching Quenching of the intrinsic fluorescence at 329 nm of Hgb by PFV can be static or dynamic. Dynamic quenching constants enhance with temperature whereas in static quenching mechanism the equilibrium constant decreases with temperature due to weakening of the binding. Temperature-dependent fluorescence titrations were performed at 288.15, 298.15 and 308.15 K and the classical Stern-Volmer equation was used to analyze the data [53]. Fo ¼ 1 þ K q τ o ½Q  ¼ 1 þ K SV ½Q  F

ð2Þ

where Fo and F = fluorescence of Hgb in the absence and presence of PFV, respectively, KSV = Stern-Volmer quenching constant, [Q] = concentration of PFV, Kq = bimolecular quenching rate constant, τo =

average life time of Hgb and it is of the order of 10− 8 s [54]. All the data were corrected for the inner filter effect following the procedure described earlier [55,56]. The KSV and Kq values, decreased as the experimental temperature enhanced. KSV reduced from (3.02 ± 0.19) × 104 to (2.72 ± 0.14) × 104 to (2.24 ± 0.12) × 104 M−1 as the temperature increased from 288.15 to 298.15 to 308.15 K. This decreasing value of KSV with increasing temperature suggested weakening of the PFV-Hgb complexes at elevated temperatures. This result testified that the fluorescence quenching is because of specific ground state complexation that weakens with temperature. Besides, the Kq values were higher than 2.0 × 1010 M−1 s−1, suggesting that a static quenching mechanism occurred on complexation between PFV and Hgb [53]. 3.5. Fluorescence Lifetime Study According to Lakowicz's theory, fluorescence quenching through time-resolved fluorescence measurements can efficiently distinguish between static and dynamic processes [57]. Static quenching is manifested by identical fluorescence life time values while dynamic quenching is manifested by significant alterations in the fluorescence life time values [57]. From the time-resolved fluorescence decay curves of free and complexed Hgb their fluorescence lifetime values along with their corresponding amplitudes were determined. The sample was excited at 280 nm using LED radiation and the emission wavelength was fixed at 329 nm. Instrument response function (IRF) was determined based on the light signal scattered from Ludox and this was utilized for deconvolution of the fluorescence signal. The decay curves were fitted to a bi-exponential function. The fluorescence decay is given by the sum of the exponential functions as follows [57]. F ðt Þ ¼

X

  t α i exp − τi

ð3Þ

where F(t) = fluorescence intensity at time t and αi = pre-exponential factor corresponding to the ith decay time constant, τi. For multi exponential decay, the average lifetime τavg is given by [33]. τavg ¼

Fig. 3. Steady state fluorescence spectra of PFV (curve 1) treated with increasing concentrations of Hgb (curves 2–9).

X

ai τi

ð4Þ

Here, τi = fluorescence lifetime and ai = relative amplitude. The fluorescence lifetime values were τ1 = 0.60 ns and τ2 = 2.96 ns for free Hgb. After the addition of PFV, the average fluorescence lifetime values were found to be τ1 = 0.58 ns and τ2 = 2.91 ns, respectively. Since the Trp residues divulge multi exponential decays [34] so independent components have not been assigned, instead the average fluorescence lifetime was exploited to obtain qualitative insights. Average fluorescence lifetime of free Hgb was 2.51 ns while in the presence of PFV it was marginally altered to 2.42 ns. Thus, the fluorescence lifetime of free Hgb was almost unperturbed in presence of PFV. This result

A. Basu, G.S. Kumar / Journal of Photochemistry & Photobiology, B: Biology 165 (2016) 42–50

reaffirmed that the quenching mechanism is mainly static and due to complexation between Hgb and PFV.

3.6. Measurement of Energy Transfer Förster resonance energy transfer (FRET) is regarded as a “spectroscopic ruler” for determining the molecular distances in macromolecular systems [58]. Besides divulging more details on the binding aspects, energy transfer efficiency measurements can also yield information on the distance between the bound PFV and its interaction site on Hgb, which is crucial for gaining insights into the structural and conformational aspects of the dye-protein complex [59,60]. The Förster theory proposes that the efficiency of energy transfer (E) may be dependent on the orientation of the transition dipoles of Hgb and the distance between Hgb and PFV should be in the range 2–8 nm [60,61]. The values of E, J (overlap integral of the fluorescence emission spectrum of Hgb and the absorption spectrum of PFV), Ro (critical distance at 50% energy transfer efficacy) and r (distance between the Hgb and PFV) were deduced to be 0.10, 1.88 × 10−14 cm3 L mol−1, 2.42 nm and 3.49 nm, respectively, using the equations reported earlier [62]. Thus, there is sufficient overlap between the fluorescence spectrum of Hgb and absorption spectrum of PFV (Fig. 4). The distance between Hgb and PFV is less than 8 nm so there is a fair possibility of energy transfer from the Trp residues of Hgb to PFV [61]. From these results we can unequivocally conclude that binding of PFV to Hgb originates from strong ground state complexation.

45

3.8. Salt Dependent Fluorescence Studies With increasing [Na+] the KA values decreased. Since the degree of interaction was found to be dependent on the amount of salt present in the medium, we have performed the experiments in buffer solutions containing 10 to 50 mM [Na+]. Increasing the [Na+] from 10 to 20 to 50 mM decreased the KA value from (3.32 ± 0.19) × 104 to (2.45 ± 0.13) × 104 to (1.33 ± 0.08) × 104 M−1. PFV is a monocationic ligand, and hence electrostatic forces may play a critical role in the complexation process. The slope of the plot of log KA versus log [Na+] was calculated to be (−0.573 ± 0.064) (Fig. 5A) using the relation reported earlier [65]. The ΔG0 values were determined to be (−6.17 ± 0.19), (−5.99 ± 0.13) and (−5.63 ± 0.08) kcal/mol, respectively, at 10, 20 and 50 mM [Na+]. This ΔG0 values were partitioned between polyelectrolytic (ΔG0pe) and non-polyelectrolytic (ΔG0t) components (Fig. 5B). The polyelectrolytic contribution was quantitatively estimated from the equation reported earlier [66]. At 10, 20 and 50 mM [Na+], the ΔG0pe contributions were determined to be (− 1.57 ± 0.19), (−1.33 ± 0.13) and (−1.02 ± 0.08) kcal/mol, respectively, which accounted for only 25, 22 and 18% of the total ΔG0. Thereafter, the non-polyelectrolytic component (ΔrG0t) was obtained from the difference between ΔG0 and ΔG0pe. The non-polyelectrolytic contributions were (−4.60 ± 0.19), (− 4.66 ± 0.13) and (− 4.61 ± 0.08) kcal/mol, respectively, at 10, 20 and 50 mM [Na+]. Hence, the binding was mainly driven by non-polyelectrolytic forces like π-π stacking, H-bonding, and van der Waals interactions. 3.9. Anisotropy Measurements

3.7. Elucidation of the Binding Parameters After establishing the complex formation between PFV and Hgb, the equilibrium constant (KA) and the number of binding sites (n) were calculated as follows [63,64]

log

ð Fo − FÞ ¼ logK A þ n log½Q  F

ð5Þ

The binding affinity of PFV for Hgb at 298.15 K was deduced to be (3.32 ± 0.19) × 104 M−1. Besides, the KA values decreased with increasing temperature suggesting destabilization of the PFV-Hgb complex at higher temperatures. The number of binding sites (n) was deduced to be (1.05 ± 0.05) at 298.15 K suggesting only one kind of binding site exists for PFV on Hgb. So, it is highly likely that PFV bound close to a Trp residue on Hgb.

Anisotropy results can be used as a guide to determine the most probable location of a ligand in the macromolecular environment of a protein. Anisotropy experiments were carried out as per Larsson and colleagues [67]. Enhancement in fluorescence polarization anisotropy (r) of PFV in the presence of Hgb testified for the complexation of PFV with Hgb. On association with Hgb the anisotropy of PFV enhanced from 0.008 to 0.040 at D/P (dye/protein molar ratio) 220. The binding of PFV to Hgb reduced its mobility thereby leading to an increase in the anisotropy of PFV. Furthermore, it also suggests that the fluorophore of PFV on complexation with Hgb is confined within a restricted environment. The enhancement in the fluorescence anisotropy of PFV upon complexation with Hgb permits the determination of the binding constant (Kr) independently using the equations given by Ingersoll and Strollo [68] described below, 1 1 ¼1þ fB K r ½L where; f B ¼

r−r F Rðr B −rÞ þ r−r F

ð6Þ

ð7Þ

Here, fF and fB = fractional fluorescence intensities of free PFV and PFV bound to Hgb, respectively, rF and rB = magnitude of the corresponding anisotropy values, and [L] = [Hgb]. R = correction factor obtained from the ratio of fB/fF. From the above relationship, plots of 1/fB versus 1/[L] were drawn and using the slope the binding constant for PFV-Hgb complexation was determined to be 3.33 × 104 M− 1. This value from the anisotropy result is consistent with those obtained from the spectroscopic studies (vide supra) and suggested the validity of this method. 3.10. Hydrophobic Probe Displacement Assay

Fig. 4. Spectral overlap (shaded portion) of the emission spectrum of Hgb and absorption spectrum of PFV.

8-Anilino-1-naphthalenesulfonic acid (ANS), a hydrophobic probe sensitive to microenvironmental changes in Hgb, can be used to gather insights into the hydrophobic binding regions on Hb surface [37,69].

46

A. Basu, G.S. Kumar / Journal of Photochemistry & Photobiology, B: Biology 165 (2016) 42–50

Fig. 5. (A) Variation of log KA versus log [Na+] for PFV-Hgb complexation. (B) Partitioned polyelectrolytic (ΔG0pe) (shaded) and nonpolyelectrolytic (ΔG0t) (black) contributions to ΔG0.

Two types of binding sites exist for ANS on Hgb depending on the availability of water molecules [70]. First binding site for ANS is driven largely by the electrostatic interaction between its sulfonate group and cationic groups of Hgb. N-terminus of the β-subunit of Hgb forms an aggregate of eight positive charges surrounding the central cavity which permits intercalation of the ANS molecules into the central cavity and also allows it to be enveloped by a protein globule hydrophobic environment [71,72]. PFV was added to free Hgb solution and Hgb-ANS complexes at 1:10 and 1:20 ratios. Subsequently, the relative fluorescence intensity of Hgb versus PFV concentration was plotted. This displacement assay showed that the PFV molecules can expel the bound ANS molecules from the central cavity of Hgb and bind at the hydrophobic region. 3.11. Synchronous Fluorescence Spectroscopy Conformational changes in Hgb consequent to the binding of PFV were studied using synchronous fluorescence spectroscopy [73]. Synchronous fluorescence spectroscopy was introduced by Lloyd [73] and it is widely used to characterize ligand-protein interactions [39–41, 74]. Synchronous fluorescence of Hgb yield insights into the microenvironment around Tyr residues when Δλ = 15 nm and around Trp residues when Δλ = 60 nm. Quenching of synchronous fluorescence of Hgb by PFV suggests alteration of the polarity around these amino acid residues [75]. Synchronous fluorescence spectra of Hgb in the presence of PFV are depicted in Fig. S2. PFV induced systematic gradual quenching of the synchronous fluorescence of Hgb (Δλ = 60 nm) along with a significant bathochromic shift of 9 nm (Fig. S2A). Similarly, for Δλ = 15 nm the synchronous fluorescence of Hgb also decreased in the presence of PFV with a red shift of 7 nm (Fig. S2B). A larger red shift

in the emission maximum for Δλ = 60 nm indicates that the environment around the Trp residues is altered to greater extent in comparison to the Tyr residues. Thus, the Trp residues lie in a more hydrophilic environment than the Tyr residues. This result reiterates the observations from quenching and energy transfer experiments, further confirming the potential role of the Trp residues in the complexation phenomenon. 3.12. FTIR Studies FTIR spectroscopy is useful to qualitatively study the average secondary structure of Hgb [45,76]. The FTIR spectra of Hgb and Hgb-PFV complexes are shown Fig. S3. The most important vibrational band in Hgb is the amide I band (1700–1600 cm−1) which arises due to C_O and C\\N stretching vibrations and is associated with the backbone conformation of Hgb. The amide I band shifted from 1649 cm−1 in free Hgb to 1644 cm−1 in PFV-Hgb complex (Fig. S3). This shift to lower wave number is a result of the interaction between Hgb and PFV and the complexation phenomenon was possibly driven by hydrogen bonds, hydrophobic and hydrophilic interactions [77]. Furthermore, a conformational change in Hgb from the helical structure to β-sheet like aggregated structure may also account for the small shifting towards lower wavenumber [78]. 3.13. Circular Dichroism Spectroscopy Curve 1 in Fig. 6A shows that the CD spectrum of Hgb in the far UV region contains two negative bands at 209 and 222 nm which are characteristic of its α-helical structure [79,80]. The CD signal of Hgb decreased in magnitude on addition of PFV which suggested conformational changes in Hgb (Fig. 6A). The helical content of free

Fig. 6. (A) Circular dichroism (far UV CD) spectral changes of Hgb (curve 1) on treatment with 2, 6, 8, 10, 12, 14 and 16 μM of PFV (curves 2–8). (B) Soret band CD spectral changes of Hgb (curve 1) on addition of 2, 12 and 25 μM of PFV (curves 2–4).

A. Basu, G.S. Kumar / Journal of Photochemistry & Photobiology, B: Biology 165 (2016) 42–50

47

increase in PFV concentration which suggested that the planarity of the porphyrin ring was altered upon complexation with the PFV molecules. 3.14. CD Melting Studies Thermal stability of Hgb may be significantly affected upon binding of PFV. To gather information on this the CD changes at 222 nm with increasing temperature was monitored. The CD melting profiles of Hgb and Hgb-PFV complex are presented in Fig. S4. The CD denaturation temperature of uncomplexed Hgb was found to be 336.11 K while Hgb-PFV complex melted at 331.05 K. The CD melting study thus showed that the binding reduced the thermal stability of Hgb (ΔTm) by 5.06 K. So PFV destabilized Hgb upon complexation corroborating the results of far UV CD studies. 3.15. Differential Scanning Calorimetry DSC is useful to monitor the energetics of temperature dependent protein folding-unfolding process [84–87]. Hgb exhibited a single endothermic peak at (335.02 ± 0.04) K (Fig. S5, curve 1) upon thermal denaturation. In the presence of PFV the thermal denaturation temperature of Hgb was reduced to (330.68 ± 0.03) K (Fig. S5, curve 2). So the melting temperature of Hgb was decreased by 4.34 K in the presence of PFV indicating that the dye binding destabilizes Hgb. The apparent excess enthalpy of transition of free Hgb decreased from (77.37 ± 0.07) to (76.57 ± 0.06) kcal/mol in the complexed form. Hence, the DSC data further corroborated the results obtained from CD melting studies. 3.16. Isothermal Titration Calorimetry Fig. 7. The upper panel represents the ITC thermogram for PFV-Hgb complexation at 298.15 K (curve on the top), along with the dilution profile (curve at the bottom 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.

and PFV bound Hgb was calculated using the equation reported previously [62,81,82]. The α-helical content of free Hgb was deduced to be 75%. Upon complexation with PFV it was reduced to 27%. Therefore, binding to PFV resulted in remarkable loss of the helical stability of Hgb with the extended polypeptide chains exposing the hydrophobic cavities. Soret band of Hgb originates from an electron dipole movement that permits π–π* transition commonly found in porphyrins [40,83]. The Soret band allows to monitor the ligand induced changes in the nature of the porphyrin ring. Hgb exhibited CD spectrum in the Soret region with a positive maximum at 414 nm and a small negative trough at 398 nm (Fig. 6B). The intensity of the positive band decreased with

ITC is widely used to study the energetics of ligand-protein binding reactions [84,86–88]. Fig. 7 presents the ITC thermogram for PFV-Hgb association at 298.15 K. The Ka value for Hgb-PFV complexation was evaluated to be (3.27 ± 0.05) × 104 M−1. The enthalpy change (ΔH0) was deduced to be (−3.20 ± 0.03) kcal/mol. Strengthening of the Hbonds in the hydrophobic environment of Hgb along with van der Waals interactions may account for this ΔH0 value [89]. The entropy contribution (TΔS0), which probably originates from the release of protein bound water molecules and counterions, was (2.96 ± 0.02) kcal/ mol. The Gibbs energy change (ΔG0) for the complexation process was calculated to be (−6.16 ± 0.05) kcal/mol. 3.17. Chaotrope Induced Denaturation Studies After deciphering the structural aspects of PFV-Hgb binding reaction, we monitored the influence of denaturation of Hgb on its binding efficacy. Fig. 8A represents the changes in the fluorescence spectra of PFV

Fig. 8. (A) Changes in the fluorescence spectra of PFV (1 μM) complexed with Hgb (10 μM) in the presence of 0–7 M urea (curves 1–5). Inset: variation of anisotropy of PFV-Hgb complex with urea concentration. (B) Plot of variation of fluorescence intensity of Hgb at 329 nm with increasing urea concentration.

48

A. Basu, G.S. Kumar / Journal of Photochemistry & Photobiology, B: Biology 165 (2016) 42–50

Fig. 9. Variation in the relative esterase activity of Hgb with increasing PFV concentration.

bound to Hgb in the presence of urea. It is evident that addition of urea to the complex of PFV and Hgb results in enhancement of the intensity of the emission band of PFV. The addition of the chaotrope resulted in destabilization of the PFV–Hgb complex thereby exposing more PFV to the buffer solution. This exposure of the previously bound PFV molecules accounted for an enhancement in the fluorescence of PFV. Chaotropes can expel water molecules next to the probe in the proteinous microenvironment with concomitant denaturation of Hgb [53,90,91]. Therefore, the chaotrope induced destabilization of PFV-

Hgb complex is linked with a higher degree of exposure of the probe to the buffer solution compared to its complexed state in the native conformation of Hgb [91]. In order to understand whether PFV is totally free or in a comparatively lesser but still somewhat restricted environment in the presence of urea anisotropy studies were undertaken. The fluorescence anisotropy decreased with increasing urea concentration (inset of Fig. 8A). However, in spite of decrease in the anisotropy of Hgb bound PFV with increasing urea concentration the anisotropy value remained higher than the value observed for free PFV, enabling to lead to the conclusion that PFV was not totally released into the buffer. The influence of the chaotrope on the fluorescence of Hgb was also monitored. Addition of urea caused an increase in the fluorescence of Hgb at 329 nm. A plot of the change in the emission intensity at 329 nm versus urea concentration is depicted in Fig. 8B. The intrinsic fluorescence of Hgb for Trp residues increases on addition of urea owing to a structural conformation where the emitting Trp residues and the heme group are far apart for energy transfer to be restricted [92–94]. The same trend was again reflected when another well known chaotrope guanidine hydrochloride was used in place of urea (not shown).

3.18. Dynamic Light Scattering Studies DLS experiment revealed the size distribution (hydrodynamic diameter, dh) profile of Hgb in buffer and in the presence of PFV. The average hydrodynamic diameter of Hgb in the absence and presence of PFV were (77 ± 9) and (207 ± 70) nm, respectively (Fig. S6). This increase in the average hydrodynamic diameter can be attributed to an

Fig. 10. AFM images of Hgb (A, B and C) and its complex with PFV (D, E and F).

A. Basu, G.S. Kumar / Journal of Photochemistry & Photobiology, B: Biology 165 (2016) 42–50

enhancement in the aggregation propensity of Hgb molecules upon complexation.

3.19. Esterase Activity Assay Hgb is also endowed with an interesting enzymatic property, known in terms of esterase-like activity. The esterase-like activity of Hgb is determined by monitoring the absorbance of p-nitrophenol as produced by the hydrolysis of p-nitrophenyl acetate on action of Hgb. Fig. 9 depicts the changes in the relative esterase activity of Hgb in presence of PFV. It can be seen that Hgb-PFV complexation resulted in a decrease in the esterase activity of Hgb, which further reiterates the earlier observation that the native Hgb structure is altered upon association with PFV.

3.20. Atomic Force Microscopy Imaging We have used AFM to monitor whether PFV induces any significant morphological changes in Hgb due to its interaction. Fig. 10A clearly represents the AFM image of free Hgb in terms of topography. Fig. 10B is a representation of the image in terms of amplitude. Fig. 10C is the three-dimensional AFM image of Hgb. The images clearly reveal the morphology of the tetrameric configuration of Hgb. Fig. 10D,E represents the AFM images of Hgb-PFV complex in terms of topography and amplitude, respectively. Fig. 10F is the three-dimensional representation of the Hgb-PFV complex. The AFM images revealed that the essential morphological features of the tetrameric configuration of Hgb molecules were retained in the presence of PFV. So this observation validates that PFV is less likely to cause damage or alter the morphology of important biomolecules like Hgb and thereby pose lesser toxic threats.

4. Conclusions This study delineates the structural aspects and thermodynamics of the interaction of PFV with Hgb. Spectroscopic evidence revealed that PFV binds with Hgb through complex formation at the ground-state. Fluorescence spectral data suggested that the interaction involved close contact of PFV with β-Trp37 residue of Hgb at the α1β2 interface. Synchronous fluorescence, FTIR and circular dichroism results suggested significant conformational changes in Hgb upon binding to PFV. Thermodynamic parameters obtained from ITC revealed that the complexation was favored by negative enthalpy and positive entropy. AFM imaging suggested that the morphology of Hgb remained intact upon complexation with PFV. This work lends crucial biophysical insights on the binding of PFV with Hgb which is essential to understand the delivery of PFV to different physiological targets. It is worth mentioning here that the affinity of PFV for Hgb is reasonably weaker in comparison to those reported for other therapeutic molecules [37,39]. So, the potential use of PFV in therapeutics can be justified on the basis that bio-distribution of PFV would be uniform and it would pose less toxic threats.

Acknowledgements This work received funding from GenCODE (BSC0123) of the Council of Scientific and Industrial Research (CSIR), Govt. of India. Dr. Anirban Basu was supported by Research Associateship from the GenCODE project. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jphotobiol.2016.10.010.

49

References [1] N. Pal Singh, R. Kumar, D.N. Prasad, S. Sharma, O. Silakari, Synthesis and antibacterial activity of benzotriazole substituted acridines, Int. J. Biol. Chem. 5 (2011) 193–199. [2] L. Ngadi, A. Galy, J. Galy, J. Barbe, A. Crémieux, J. Chevalier, D. Sharples, Some new 1nitro acridine derivatives as antimicrobial agents, Eur. J. Med. Chem. 25 (1990) 67–70. [3] P. Belmont, J. Bosson, T. Godet, M. Tiano, Acridine and acridone derivatives, anticancer properties and synthetic methods: where are we now? Anti Cancer Agents Med. Chem. 7 (2007) 139–169. [4] S.C. Nash, A.S. Ketcham, R.R. Smith, Effect of local irrigation with proflavine hemisulfate on wounds seeded with tumor cells: an experimental study, Ann. Surg. 155 (1962) 465–471. [5] P. Ascenzi, M. Colosanti, M. Fasano, A. Bertollini, Stabilization of the T-state of human hemoglobin by proflavine, an antiseptic drug, IUBMB Life 47 (2008) 991–995. [6] L.R. Ferguson, W.A. Denny, The genetic toxicology of acridines, Mutat. Res. 258 (1991) 123–160. [7] A.K. Todd, A. Adams, J.H. Thorpe, W.A. Denny, L.P.G. Wakelin, C.J. Cardin, Major groove binding and ‘DNA-induced’ fit in the intercalation of a derivative of the mixed topoisomerase i/ii poison N-(2-(Dimethylamino) ethyl) acridine-4carboxamide (DACA) into DNA: X-ray, J. Med. Chem. 42 (1999) 536–540. [8] G.W. Rewcastle, G.J. Atwell, D. Chambers, B.C. Baguley, W.A. Denny, Potential antitumor agents. 46. Structure-activity relationships for acridine monosubstituted derivatives of the antitumor agent N-[2-(dimethylamino)ethyl]-9-aminoacridine-4carboxamide, J. Med. Chem. 29 (1986) 472–477. [9] G.J. Atwell, G.W. Rewcastle, B.C. Baguley, W.A. Denny, Potential antitumor agents. 50. In vivo solid-tumor activity of derivatives of N[2-(dimethylamino)ethyl]-acridine 4-carboxamide, J. Med. Chem. 30 (1987) 664–669. [10] J.M. Crenshaw, D.E. Graves, W.A. Denny, Interactions of acridine antitumor agents with DNA: binding energies and groove preferences, Biochemistry 34 (1995) 13682–13687. [11] S. Neidle, L.H. Pearl, P. Herzyk, H.M. Berman, A molecular model for proflavine-DNA intercalation, Nucleic Acids Res. 16 (1988) 8999–9016. [12] P. Tang, C.L. Juang, G.S. Harbison, Intercalation complex of proflavine with DNA: structure and dynamics by solid-state NMR, Science 249 (1990) 70–72. [13] M. Aslanoglu, Electrochemical and spectroscopic studies of the interaction of proflavine with DNA, Anal. Sci. 22 (2006) 439–443. [14] M. Demeunynck, F. Chamantray, A. Martelli, Interest of acridine derivatives in the anticancer chemotherapy, Curr. Pharm. Des. 7 (2001) 1703–1724. [15] W.A. Denny, Acridine derivatives as chemotherapeutic agents, Curr. Med. Chem. 9 (2002) 1655–1665. [16] W.A. Denny, Chemotherapeutic effects of acridine derivatives, Med. Chem. Rev. 1 (2004) 257–266. [17] U. Kragh-Hansen, S.O. Brennan, M. Galliano, O. Sugita, Binding of warfarin, salicylate, and diazepam to genetic variants of human serum albumin with known mutations, Mol. Pharmacol. 37 (1990) 238–242. [18] F. Quadrifoglio, V. Crescenzi, V. Giancotti, Calorimetry of DNA-dye interactions in aqueous solution: I. Proflavine and ethidium bromide, Biophys. Chem. 1 (1974) 319–324. [19] T. Biver, F. Secco, M.R. Tiné, M. Venturini, Equilibria and kinetics of the intercalation of Pt-proflavine and proflavine into calf thymus DNA, Arch. Biochem. Biophys. 418 (2003) 63–70. [20] B. García, J.M. Leal, R. Ruiz, T. Biver, F. Secco, M. Venturini, Change of the binding mode of the DNA/proflavine system induced by ethanol, J. Phys. Chem. B 114 (2010) 8555–8564. [21] W.D. Sasikala, A. Mukherjee, Molecular mechanism of direct proflavine–DNA intercalation: evidence for drug-induced minimum base-stacking penalty pathway, J. Phys. Chem. B 116 (2012) 12208–12212. [22] M. Dourlent, C. Hélenè, A quantitative analysis of proflavine binding to polyadenylic acid, polyuridylic acid, and transfer RNA, Eur. J. Biochem. 23 (1971) 86–95. [23] V. Kumar, A. Sengupta, K. Gavvala, R.K. Koninti, P. Hazra, Spectroscopic and thermodynamic insights into the interaction between proflavine and human telomeric Gquadruplex DNA, J. Phys. Chem. B 118 (2014) 11090–11099. [24] A. Basu, G. Suresh Kumar, Thermodynamic characterization of proflavine–DNA binding through microcalorimetric studies, J. Chem. Thermodyn. 87 (2015) 1–7. [25] A. Basu, G. Suresh Kumar, Calorimetric investigation on the interaction of proflavine with human telomeric G-quadruplex DNA, J. Chem. Thermodyn. 98 (2016) 208–213. [26] S.M.H. Evans, I.G.C. Robertson, J.W. Paxton, Plasma protein binding of the experimental antitumour agent acridine-4-carboxamide in man, dog, rat and rabbit, J. Pharm. Pharmacol. 46 (1994) 63–67. [27] B. Chakraborty, S. Basu, Interaction of BSA with proflavin: a spectroscopic approach, J. Lumin. 129 (2009) 34–39. [28] M.M. Cox, G.N. Phillips Jr., Handbook of Proteins: Structure, Function and Methods, 2, Wiley, West Sussex, 2007. [29] M.F. Perutz, Haemoglobin: structure, function and synthesis, Br. Med. Bull. 32 (1976) 193–194. [30] M.F. Perutz, M.G. Rossmann, A.F. Cullis, H. Muirhead, G. Will, A.C.T. North, Structure of haemoglobin: a three-dimensional Fourier synthesis at 5.5-Å resolution, obtained by X-ray analysis, Nature 185 (1960) 416–422. [31] M.F. Perutz, X-ray analysis of hemoglobin, Science 140 (1963) 863–869. [32] R.W. Lucek, C.B. Coutinho, The role of substituents in the hydrophobic binding of the 1,4-benzodiazepines by human plasma proteins, Mol. Pharmacol. 12 (1976) 612–619. [33] N. Shahabadi, M. Maghsudi, Z. Kiani, M. Pourfoulad, Multispectroscopic studies on the interaction of 2-tert-butylhydroquinone (TBHQ), a food additive, with bovine serum albumin, Food Chem. 124 (2011) 1063–1068.

50

A. Basu, G.S. Kumar / Journal of Photochemistry & Photobiology, B: Biology 165 (2016) 42–50

[34] W. Peng, F. Ding, Y.-K. Peng, Y. Sun, Molecular recognition of malachite green by hemoglobin and their specific interactions: insights from in silico docking and molecular spectroscopy, Mol. BioSyst. 10 (2014) 138–148. [35] S. Tunç, O. Duman, B.K. Bozoğlan, Studies on the interactions of chloroquine diphosphate and phenelzine sulfate drugs with human serum albumin and human hemoglobin proteins by spectroscopic techniques, J. Lumin. 140 (2013) 87–94. [36] O. Duman, S. Tunç, B.K. Bozoğlan, Characterization of the binding of metoprolol tartrate and guaifenesin drugs to human serum albumin and human hemoglobin proteins by fluorescence and circular dichroism spectroscopy, J. Fluoresc. 23 (2013) 659–669. [37] S. Hazra, G. Suresh Kumar, Structural and thermodynamic studies on the interaction of iminium and alkanolamine forms of sanguinarine with hemoglobin, J. Phys. Chem. B 118 (2014) 3771–3784. [38] E. Antonini, M. Brunori, Hemoglobin and Myoglobin in Their Reactions With Ligands, North-Holland publishing Co., Amsterdam, The Netherlands, 1971. [39] S. Hazra, G. Suresh Kumar, Binding of isoquinoline alkaloids berberine, palmatine and coralyne to hemoglobin: structural and thermodynamic characterization studies, Mol. BioSyst. 9 (2013) 143–153. [40] S. Chatterjee, G. Suresh Kumar, Targeting the heme proteins hemoglobin and myoglobin by janus green blue and study of the dye–protein association by spectroscopy and calorimetry, RSC Adv. 4 (2014) 42706–42715. [41] A. Basu, G. Suresh Kumar, Binding of carmoisine, a food colorant, with hemoglobin: spectroscopic and calorimetric studies, Food Res. Int. 72 (2015) 54–61. [42] R. Sinha, M. Hossain, G. Suresh Kumar, Interaction of small molecules with doublestranded RNA: spectroscopic, viscometric, and calorimetric study of hoechst and proflavine binding to polyCG structures, DNA Cell Biol. 28 (2009) 209–219. [43] S. Pramanik, A. Saha, P. Sujatha Devi, Water soluble blue-emitting AuAg alloy nanoparticles and fluorescent solid platforms for removal of dyes from water, RSC Adv. 5 (2015) 3946–33954. [44] S. Sen, T. Bose, A. Roy, A.S. Chakraborti, Effect of non-enzymatic glycation on esterase activities of hemoglobin and myoglobin, Mol. Cell. Biochem. 301 (2007) 251–257. [45] M. Matsui, A. Nakahara, A. Takatsu, K. Kato, N. Matsuda, In situ observation of the state and stability of hemoglobin adsorbed onto glass surface by slab optical waveguide (SOWG) spectroscopy, Int. J. Chem. Biomol. Eng. 1 (2008) 72–75. [46] A.F. Nassar, J.F. Rusling, N. Nakashima, Electron transfer between electrodes and heme proteins in protein–DNA films, J. Am. Chem. Soc. 118 (1996) 3043–3044. [47] M. Mahato, P. Pal, B. Tah, M. Ghosh, G.B. Talapatra, Study of silver nanoparticle–hemoglobin interaction and composite formation, Colloids Surf. B: Biointerfaces 88 (2011) 141–149. [48] H.A. Benesi, J.H. Hildebrand, A spectrophotometric investigation of the interaction of iodine with aromatic hydrocarbons, J. Am. Chem. Soc. 71 (1949) 2703–2707. [49] B. Alpert, D.M. Jameson, G. Weber, Tryptophan emission from human hemoglobin and its isolated subunits, J. Photochem. Photobiol. 31 (1980) 1–4. [50] M.F. Perutz, G. Fermi, D.J. Abraham, C. Poyart, E. Bursaux, Hemoglobin as a receptor of drugs and peptides: x-ray studies of the stereochemistry of binding, J. Am. Chem. Soc. 108 (1986) 1064–1078. [51] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press, New York, 1983. [52] E.A. Burstein, N.S. Vedenkina, M.N. Ivkova, Fluorescence and the location of tryptophan residues in protein molecules, Photochem. Photobiol. 18 (1973) 263–279. [53] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press, New York, 1999. [54] J.R. Lakowicz, G. Weber, Quenching of fluorescence by oxygen. Probe for structural fluctuations in macromolecules, Biochemistry 12 (1973) 4161–4170. [55] R.F. Steiner, L. Weinry, Excited States of Protein and Nucleic Acid, Plenum Press, New York, 1971. [56] Y.-Q. Wang, H.-M. Zhang, B.-P. Tang, The interaction of C.I. acid red 27 with human hemoglobin in solution, J. Photochem. Photobiol. B 100 (2010) 76–83. [57] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, third ed. Springer Science + Business Media, New York, 2006 63–606. [58] A.N. Kapanidis, T.A. Laurence, N.K. Lee, E. Margeat, X. Kong, S. Weiss, Alternatinglaser excitation of single molecules, Acc. Chem. Res. 38 (2005) 523–533. [59] A. Mallick, B. Haldar, N. Chattopadhyay, Spectroscopic investigation on the interaction of ICT probe 3-Acetyl-4-oxo-6,7-dihydro-12H Indolo-[2,3-a] quinolizine with serum albumins, J. Phys. Chem. B 109 (2005) 14683–14690. [60] C. Jash, G. Suresh Kumar, Binding of alkaloids berberine, palmatine and coralyne to lysozyme: a combined structural and thermodynamic study, RSC Adv. 4 (2014) 12514–12525. [61] B. Valeur, J.C. Brochon, New Trends in Fluorescence Spectroscopy, Springer-Verlag, Berlin, 2001. [62] A. Basu, G. Suresh Kumar, Multispectroscopic and calorimetric studies on the binding of the food colorant tartrazine with human hemoglobin, J. Hazard. Mater. (2016), http://dx.doi.org/10.1016/j.jhazmat.2016.07.023. [63] O.K. Abou-Zied, O.L.K. Al-Shihi, Characterization of subdomain IIA binding site of human serum albumin in its native, unfolded, and refolded states using small molecular probes, J. Am. Chem. Soc. 130 (2008) 10790–10801.

[64] G.W. Zhang, Q.M. Que, J.H. Pan, J.B. Guo, Study of the interaction between icariin and human serum albumin by fluorescence spectroscopy, J. Mol. Struct. 81 (2008) 132–138. [65] M.T. Record Jr., T.M. Lohman, P. De Haseth, Ion effects on ligand-nucleic acid interactions, J. Mol. Biol. 107 (1976) 145–158. [66] M.T. Record Jr., R.S. Spolar, in: A. Revzin (Ed.), The Biology of Nonspecific DNA-Protein Interactions, CRC Press, Boca Raton, FL 1990, pp. 33–69. [67] A. Larsson, C. Carlsson, M. Jonsson, B. Albinsson, Characterization of the binding of the fluorescent dyes YO and YOYO to DNA by polarized light spectroscopy, J. Am. Chem. Soc. 116 (1994) 8459–8465. [68] C.M. Ingersoll, C.M. Strollo, Steady-state fluorescence anisotropy to investigate flavonoids binding to proteins, J. Chem. Educ. 84 (2007) 1313–1315. [69] L. Stryer, The interaction of a naphthalene dye with apomyoglobin and apohemoglobin: a fluorescent probe of non-polar binding sites, J. Mol. Biol. 13 (1965) 482–495. [70] D.A. Parul, S.B. Bokut, A.A. Milyutin, E.P. Petrov, N.A. Nemkovich, A.N. Sobchuk, B.M. Dzhagarov, Time-resolved fluorescence reveals two binding sites of 1,8-ANS in intact human oxyhemoglobin, J. Photochem. Photobiol. B 58 (2000) 156–162. [71] D. Matulis, R. Lovrien, 1-Anilino-8-naphthalene sulfonate anion-protein binding depends primarily on ion pair formation, Biophys. J. 74 (1998) 422–429. [72] V.E. Syakhovich, D.A. Parul, E.Y. Ruta, B.A. Bushuk, S.B. Bokut, 1,8Anilinonaphthalene sulfonate binds to central cavity of human hemoglobin, Biochem. Biophys. Res. Commun. 317 (2004) 761–767. [73] J.B.F. Lloyd, Synchronized excitation of fluorescence emission spectra, Nat. Phys. Sci. 231 (1971) 64–65. [74] A. Basu, G. Suresh Kumar, Interaction of toxic azo dyes with heme protein: biophysical insights into the binding aspect of the food additive amaranth with human hemoglobin, J. Hazard. Mater. 289 (2015) 204–209. [75] J.N. Miller, Recent advances in molecular luminescence analysis, Proc. Anal. Div. Chem. Soc. 16 (1979) 203–208. [76] P. Mandal, T. Ganguly, Fluorescence spectroscopic characterization of the interaction of human adult hemoglobin and two isatins, 1-methylisatin and 1-phenylisatin: a comparative study, J. Phys. Chem. B 113 (2009) 14904–14913. [77] X. Lu, G. Zou, J. Li, Hemoglobin entrapped within a layered spongy Co3O4 based nanocomposite featuring direct electron transfer and peroxidase activity, J. Mater. Chem. 17 (2007) 1427–1432. [78] T. Kamilya, P. Pal, M. Mahato, G.B. Talapatra, Effect of salt on the formation of alcohol-dehydrogenease monolayer: a study by the Langmuir-Blodgett technique, J. Phys. Chem. B 113 (2009) 5128–5135. [79] R.W. Woody, Circular dichroism, Methods Enzymol. 246 (1995) 34–71. [80] R.W. Woody, Theory of Circular Dichroism of Proteins, in Circular Dichroism and the Conformational Analysis of Biomolecules, Plenum Press, New York, 1996 25–30. [81] Y.H. Chen, J.T. Yang, H.M. Martinez, Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion, Biochemistry 11 (1972) 4120–4131. [82] Z.X. Lu, T. Cui, Q.L. Shi, Molecular Biology: Applications of Circular Dichroism and Optical Rotatory Dispersion, 1st ed. Science Press, Beijing, 1987. [83] M. Nagai, Y. Sugita, Y. Yoneyama, Circular dichroism of hemoglobin and its subunits in the Soret region, J. Biol. Chem. 244 (1969) 1651–1653. [84] L. Bernazzani, C. Ferrari, P. Gianni, V. Mollica, E. Tombari, Calorimetric investigation on the interaction of sodium taurodeoxycholate with human serum albumin, Thermochim. Acta 555 (2013) 7–16. [85] G.I. Makhatadze, P.L. Privalov, Energetics of protein structure, Adv. Protein Chem. 47 (1995) 307–425. [86] A. Basu, G. Suresh Kumar, Study on the interaction of the toxic food additive carmoisine with serum albumins: a microcalorimetric investigation, J. Hazard. Mater. 273 (2014) 200–206. [87] A. Basu, G. Suresh Kumar, Interaction of the dietary pigment curcumin with hemoglobin: energetics of the complexation, Food Funct. 5 (2014) 1949–1955. [88] L. Damian, Isothermal titration calorimetry for studying protein-ligand interactions, Methods Mol. Biol. 1008 (2013) 103–118. [89] P.D. Ross, S. Subramanian, Thermodynamics of protein association reactions: forces contributing to stability, Biochemistry 20 (1981) 3096–3102. [90] R. Breslow, T. Guo, Surface tension measurements show that chaotropic salting-in denaturants are not just water-structure breakers, Proc. Natl. Acad. Sci. U. S. A. 87 (1990) 167–169. [91] A. Ganguly, B. Kumar Paul, S. Ghosh, S. Dalapati, N. Guchhait, Interaction of a potential chloride channel blocker with a model transport protein: a spectroscopic and molecular docking investigation, Phys. Chem. Chem. Phys. 16 (2014) 8465–8475. [92] E.D. Goddard, in: K. Ananthapadmanabhan (Ed.), Protein Surfactant Interactions, CRC Press, Boca Raton, NY, 1993 (ch. 8). [93] M.N. Jones, Biological Interfaces, Elsevier, Amsterdam, 1975 101 (ch. 5). [94] S. De, A. Girigoswami, A fluorimetric and circular dichroism study of haemoglobineffect of pH and anionic amphiphiles, J. Colloid Interface Sci. 296 (2006) 324–331.