Journal of Photochemistry and Photobiology B: Biology 124 (2013) 50–62

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Journal of Photochemistry and Photobiology B: Biology

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pH-insensitive electrostatic interaction of carmoisine with two serum proteins:A possible caution on its uses in food and pharmaceutical industry

Shubhashis Datta, Niharendu Mahapatra, Mintu Halder ⇑Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur 721 302, India

a r t i c l e i n f o

Article history:Received 7 December 2012Received in revised form 5 April 2013Accepted 11 April 2013Available online 18 April 2013

Dedicated to Professor Mihir Chowdhury.

Keywords:CarmoisineSerum proteinsFluorescence quenchingSpecific bindingThermodynamic parameters

1011-1344/$ - see front matter � 2013 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.jphotobiol.2013.04.004

⇑ Corresponding author. Tel.: +91 3222 283314; faxE-mail address: mintu@chem.iitkgp.ernet.in (M. H

a b s t r a c t

Here we have investigated the binding of carmoisine, a water-soluble azo food colorant, with serum pro-teins (HSA and BSA) by fluorescence and UV–VIS spectroscopy, circular dichroism and molecular dockingstudies. Results indicate that fluorescence quenching of protein has been due to site-specific binding ofthe dye with biomacromolecules. Site marker competitive binding and molecular docking explorationsshow that interaction occurs in the sub-domain IIA of HSA and the sub-domains IIA and IB in the caseof BSA. Conformational investigation indicates that dye binding modifies the secondary structure of pro-teins and this also alters the microenvironment of the tryptophan(s). The interaction is found to be pH-insensitive which can have relevance to the toxicological profiles of the dye, and ionic strength depen-dence of binding can be exploited in protein purification mediated by such food colorants.

� 2013 Elsevier B.V. All rights reserved.

1. Introduction

Dyes constitute an important class of synthetic organic com-pounds with a wide range of applications in various industries likefood, fabric, drinks, medicine and cosmetics. Apart from their com-mercial applications they can also be used as important probe forunderstanding the mechanism of various photophysical processes[1]. Dyes have been detected and estimated by means of variousavailable analytical methods [2–4]. The exploration of their photo-physics is one of the interesting aspects to the researchers espe-cially when the excited state properties of these dyes areprofoundly altered by the change of microenvironment [5]. Fluo-rescence properties of some of these dyes are considerably affectedin constrained media [6] and also when these tend to interact withbiopolymers like protein [7,8]. Among these dyes, azo compoundsform a separate class, in which almost 60–70% of the whole dyevarieties are included. Their stability in heat and wide range ofpH leads to usage as coloring agents in food and cosmetics. Thesedyes show photoisomerization and have very low fluorescencequantum yield [9]. Carmoisine (Fig. 1a) is a well known anionicazo food dye. The presence of carmoisine has been detected by dif-ferent analytical techniques [10,11]. It is found to exhibit interest-ing fluorescence behavior in presence of viologens (dimethyl

ll rights reserved.

: +91 3222 282252.alder).

viologen and diheptyl viologen) quenchers in neat methanol [9].Likewise, it would be interesting to explore how carmoisine, inter-acts with serum proteins and other biopolymers.

The study of exogenous ligand binding of serum protein has al-ways been more interesting mainly due to the transport propertiesof these macromolecules [12,13]. Quite a good number of drugs aretransported in the circulatory systems in the form of complex withalbumin [14]. The affinity of albumin towards small molecules,drugs can be altered by the simultaneous binding of the endo-and exogenous ligands [13]. If the target ligand binds stronger inthe drug binding site, it will lead to a reduction of the drug binding.Thus this alteration of drug affinity in presence of a competitivelybinding small molecule can be utilized to tune the transportingfunction of proteins.

Acidic and basic side chains of proteins can vary their chargesby protonation/deprotonation due to change of local pH. At theprotein surface normally acidic residues (Glu, Asp, C-terminus)are deprotonated (i.e., becomes negatively charged) and the basicresidues (Lys, Arg, His and N-terminus) are protonated (i.e., be-comes positively charged). The importance of pH in the structureand function of proteins is well illustrated by the fact that about25% of its residues contain ionizable side chains [15]. The ioniza-tions that arise from changes in pH can generate strong electro-static interactions, which will inevitably have a direct influenceon molecular structure and binding affinity. It is well accepted thatpH-effect on proteins is complex in nature. This is mainly due tothe multiplicity of titrable sites, which are not only subjected to

SO3Na

OH

N

(a)

(b)

N

SO3Na

Fig. 1. Structure of (a) carmoisine and (b) crystal structure of HSA (PDB entry1AO6).

S. Datta et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 50–62 51

different environments but also coupled to one another in complexways. Therefore, considering the structural and functional aspectsof protein the importance of these interactions is immense. It gov-erns several aspects of biophysical processes and is therefore, ofgreat significance for both applied bioengineering and more funda-mental biochemical and medicinal research.

Human serum albumin (HSA) and bovine serum albumin (BSA)are two extensively studied proteins in literature [13,14]. The X-ray crystallographic structure of HSA (Fig. 1b) is known to be orga-nized into three homologous domains (I–III), that are composed oftwo sub-domains (A and B) [16]. It has only one tryptophan residue(Trp 214) which is located in the hydrophobic pocket in sub-do-main IIA [16]. BSA has almost 88% sequence homology with HSA[1] and there are two tryptophan residues (Trp 134 and Trp 213)of which Trp 134 is located on the surface of the molecule insub-domain IB and Trp 213 resides in the hydrophobic pocket/foldin sub-domain IIA [13]. The binding pockets for small exogenousligands of these two serum proteins are well identified. Azo dyesare found to have binding interactions with serum protein result-ing in secondary structural modification of the bio-molecule. Butthe effect of alteration of local charge and environment, due tothe change of pH of the solution, on the dye–protein interactionis not explored very well. For this, in the present work we attemptto explore the interaction of carmoisine, a negatively charged foodcolorant, with HSA and BSA as function of pH and ionic strength ofthe medium. In this study we have used general optical spectro-scopic techniques like UV–VIS, fluorescence, synchronous fluores-cence and circular dichroism (CD) spectroscopy.

2. Materials and methods

2.1. Materials

HSA (>98%) and BSA (�99%), hemin (>98%, HPLC), ibuprofen(>98%, GC), carmoisine (molecular weight 502.44, 98%) are pur-chased from Sigma–Aldrich and warfarin (analytical grade) is pur-chased from TCI Chemicals, Japan. All other chemicals are ofanalytical reagent grade. Ultra pure water is used throughout thestudy. All solutions are prepared in 5 mM sodium phosphate bufferof pH 4.8 (±0.1), 5.5 (±0.1), 6.3 (±0.1), and 7.4 (±0.1). The pH mea-surements are carried out with a pre-calibrated EUTECH pH 510ion pH-meter.

2.2. UV–vis absorption spectra

The UV-VIS absorption spectra are obtained by scanning thesolution on a Shimadzu UV-2450 absorption spectrophotometeragainst solvent reference in the wavelength range 200–650 nm.For the determination of association constant from UV-VIS absor-bance data, the concentration of carmoisine is kept at 15 lM andthat of proteins is varied up to 22.5 lM.

2.3. Fluorescence measurements

The steady state fluorescence spectra are recorded on a JobinYvon - Spex Fluorolog-3 spectrofluorimeter equipped with temper-ature controlled water cooled cuvette holder. Each sample is kept for8–10 min before each measurement to ensure thermal equilibrium.Quartz cuvette having 1-cm path length is used. The steady-statefluorescence measurements have been carried out at three temper-atures, namely, 288, 300 and 308 K. In order to monitor the intrinsicfluorescence of protein from Trp residues only, the samples are ex-cited at 295 nm and the emission spectra are recorded from 310 to470 nm. The excitation and emission slits are kept at 5 nm and2 nm, respectively, in steady state fluorescence measurements.The concentration of HSA and BSA has been kept at 2 lM and3 lM, respectively. Concentrations of carmoisine are 0, 0.2, 0.42,0.64, 0.84, 1.06, 1.27, 1.7, 2.11, 2.7, 3.18 and 4 lM with HSA, and 0,0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.4, 3, 3.75, 4.5, 5.25, 6, and 7.5 lM with BSA.

Fluorescence lifetimes are measured by means of a single pho-ton counting apparatus (Horiba, Jobin Yvon, IBH Ltd., Glasgow,Scotland) equipped with a LED excitation source (kexc = 295 nm)with a peak emission at 349 nm and the signals are collected atthe magic angle 54.7� using a Hamamatsu microchannel plate pho-tomultiplier tube (R3809U). Data have been analyzed as sum of theexponential components with pre-exponential factors (ai) normal-ized to unity, using iterative reconvolution with IBH DAS-6 soft-ware. The perfectness of fit is judged in terms of a v2 value andweighted residuals.

The synchronous fluorescence spectra (SFS) are obtained bysimultaneously scanning the excitation and emission monochroma-tors. Both the excitation and emission slits are kept at 5 nm for thismeasurement. When the Dk value between excitation and emissionwavelength is set at some value, the synchronous fluorescence spec-tra can provide characteristic information of the local environmentnear some specific chromophore. Any alteration in SFS will corre-spond to binding and consequent change of micro-environment ofthe concerned chromophore near the binding site. We have repro-duced our results by repeating the SFS experiments for five times.

2.4. Circular dichroism spectra

Circular dichroism (CD) spectra are recorded on a Jasco-810automatic recording spectropolarimeter at 300 K under constant

52 S. Datta et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 50–62

nitrogen flush over a wavelength range of 190–260 nm with a scanspeed 50 nm/min. A quartz cell having path length 0.1 cm is usedand each spectrum is average of three successive scans. Corre-sponding buffer solution running under the same condition is ta-ken as blank and subtracted from the experimental spectra. TheCD spectra of protein in presence of carmoisine are also correctedby subtracting the corresponding CD spectra of the dye only. Herethe concentration of protein is kept at 9 lM and the molar ratio ofprotein to carmoisine is varied as 1:0, 1:0.5, 1:1, 1:1.5 and 1:2.5.

2.5. Docking studies

The crystal structure of HSA (PDB ID: 1AO6) is available in pro-tein data bank [17]. The 3D structure of BSA is generated usinghomology modeling. The amino acid sequence corresponding toBSA is obtained from the website of protein structure [17]. The3D structure of HSA (PDB ID: 1AO6) with chain A is used as a tem-plate structure for homology modeling using alignment methodavailable in Easypred 3D [18]. The structure is validated using PRO-CHECK [19] which measures the stereochemical quality of the pro-tein structure. The N-terminal 24 amino acid residues in the BSAsequence, not having an equivalence in the HSA template crystalstructure (PDB ID: 1AO6), are not considered in the model. Polarhydrogen atoms are added and water molecules are removed fromthe protein PDB file. The structure of carmoisine is optimized byPM3 prescription using MOPAC 2002 [20]. Later, the di-negativecarmoisine has been docked using AutoDock 4.2 onto the 3D struc-ture of HSA and BSA. AutoDock 4.2 [21] uses the Lamarckian genet-ic algorithm to search for the optimum binding site of smallmolecules in the protein. AutoDock reports a docked energy thatincludes a solvation-free energy term and intermolecular interac-tion energy of the ligand. Uncomplexed proteins and their dockedconformations (poses) with carmoisine with lowest energy areused for the calculation of accessible surface area (ASA) using theDiscovery Studio Visualizer 2.5 from Accelrys Software Inc [22].The change in ASA for residue is calculated as DASA = ASAprotein -� ASAprotein–carmoisine.

3. Results and discussion

3.1. Absorption studies

UV-VIS absorption measurement is a simple method to explorethe structural changes and monitor complexation in the groundstate [13]. We have recorded the absorption spectra of carmoisine,both the proteins (data not shown) and protein–carmoisine com-plex at different pH. The absorption spectra of carmoisine with

0.0

0.1

0.2

0.3

6 nm

[HSA] increases

Abs

orba

nce

Wavelength (nm)

(a)

450 500 550 600

(

Fig. 2. Absorbance spectra of carmoisine at pH 7.4 with increasing concentration of HSAprotein concentration is varied from 0–2.25 � 10�5 moles dm�3.

increasing concentration of HSA (Fig. 2a)and BSA (Fig. 2b)at pH7.4 are shown below.

With the increase of protein concentration the absorption maximaof carmoisine (Fig. 2) shifts towards shorter wavelength and theabsorbance decreases which is most likely due to ground state com-plexation. The shift of the peak towards shorter wavelength indicatesthat carmoisine has been incorporated into the protein pocket.

3.1.1. Determination of binding stoichiometry by Job’s methodWe have determined the stoichiometry for ground state com-

plexation of carmoisine with both the serum proteins. To findout the analytical binding stoichiometry Job’s method has beenemployed. Job’s plot is a diagram of a certain physical propertyand is related to the concentration in an equilibrium two-compo-nent-complex against volume or mole fraction of one of the twoconstituents [23]. The use of UV–VIS absorbance spectra is suitablefor this method. At first various volume fractions of equimolar car-moisine and serum proteins are mixed in such a way that the totalmoles remain constant. The absorption spectra of these solutionsare then recorded. We have monitored absorbance at 515 nm, cor-responding to spectral maxima, for the calculation using a modi-fied version of Job’s plot [24].

In this case the binding stoichiometry can be obtained by linearfitting of two lines [24]. The corrected absorbance (DA) of protein–carmoisine solutions is plotted against the mole fraction of carmoi-sine (Xcarmoisine). Now if carmoisine and protein do not react witheach other then the total absorbance of the solution (Atheoretical)should be equal to the sum of absorbance of their individualsolutions.

Atheoretical ¼ ePc0PXP þ eDc0

Dð1� XPÞ ð1Þ

where eP and eD are the molar extinction coefficient of protein andcarmoisine at 515 nm, respectively. c0

P and c0D are, respectively, the

concentration of the stock solutions of protein and carmoisine andis equal to 1.5 � 10�5 M. When they form complex the equationcan be written as follows:Aexp ¼ ePcP þ eDcD þ eccc ð2Þ

where ec is the molar extinction coefficient of the complex, and cP,cD

and cc are the concentration of protein, carmoisine and complex insolution, respectively. From the calculated value Atheoretical and mea-sured Aexp, the value of absorbance correction for these solutionscan be obtained and plotted against mole fraction of carmoisine(Fig. 3).

DA ¼ Aexp � Atheoretical ð3Þ

From the minima of the above plot the binding stoichiometryare found to be 1:1 and 1:1.3 (at 300 K) for HSA–carmoisine andBSA–carmoisine complex, respectively.

450 500 550 600

0.1

0.2

0.3

6 nm

[BSA] increases

Abs

orba

nce

Wavelength (nm)

b)

(a) and BSA (b). The concentration of carmoisine is 1.5 � 10�5 moles dm�3 and the

0.0 0.5 1.0

-0.15

-0.10

-0.05

0.00

Xcarmoisine

HSA BSA

Δ A

Fig. 3. Modified form of the Job’s plot for HSA–carmoisine and BSA–carmoisinecomplex at pH 7.4 at 300 K.

1x105 2x105 3x105

2x10-4

2x10-4

3x10-4

3x10-4

4x10-4

[Car

moi

sine

]/ΔA

[Protein]-1(moles dm-3)-1

BSA

HSA

Fig. 4. Benesi–Hildebrand plot at pH 7.4 for protein–carmoisine complex.

320 360 400 4400

5x104

1x105

2x105

2x105

HSA

[carmoisine] increases

Fluo

resc

ence

Int

ensi

ty (

a.u.

)

Wavelength (nm)

(a)

330 360 390 420 450

5x104

1x105

2x105

2x105

[carmoisine] increases

Fluo

resc

ence

Int

ensi

ty (

a.u.

)

Wavelength (nm)

(b)

BSA

Fig. 5. Emission spectra of HSA (a) and BSA (b), in the presence of differentconcentrations of carmoisine in pH 7.4 at 300 K. The concentration of HSA is2 � 10�6 and BSA is 3 � 10�6 moles dm�3. The concentration of carmoisine is variedup to 4 � 10�6 moles dm�3 and 7.5 � 10�6 moles dm�3 for HSA and BSA,respectively.

S. Datta et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 50–62 53

We have calculated the association constant (KB–H) for protein–carmoisine complex using Benesi–Hildebrand plot at all the pHfrom the absorbance data using the following equation [25]:

1DA¼ 1

KB�HDe½protein�½dye� þ1

De½dye� ð4Þ

where DA and De are the change in absorbance and molar extinc-tion coefficient of ligand at 515 nm, respectively, due to additionof different concentrations of protein. The association constanthas been calculated from the ratio of the intercept to slope fromthe plot of 1/DA vs 1/[protein]. The Benesi–Hildebrand (B–H) plotat pH 7.4 with HSA and BSA is shown below (Fig. 4).

The plot is linear for HSA–carmoisine system which indicates a1:1 complex. While for BSA–carmoisine complex, at higher concen-tration, the plot deviates from linearity, i.e. not a case of 1:1 com-plexation. The calculated value of association constant (KB–H) ofHSA and BSA with carmoisine (from the linear segment of Fig. 4)at pH 7.4 (at 300 K) is found to be (5.50 ± .05) � 105 and(1.25 ± .08) � 106, respectively.

3.2. Fluorescence studies

We have monitored the intrinsic fluorescence of tryptophanresidue of both HSA and BSA, and the spectra shows emission max-ima at 349 nm. The fluorescence measurements have been carriedout at pH 4.8, 5.5, 6.3, 7.4, and also in different ionic strengths (inthe presence of added salt).

When a fixed concentration of HSA (2 lM) or BSA (3 lM) ismixed with increasing concentrations of carmoisine, a consider-able decrease in fluorescence intensity is observed for both theproteins. The fluorescence spectra of HSA and BSA at 300 K in pH

7.4, as a function of carmoisine concentration, are shown inFig. 5 which shows that the emission maximum has been blue-shifted from 349 to 346 nm for both the proteins. This is indicativeof switching of tryptophan residues towards less polar environ-ment [26].

The fluorescence spectra are corrected using correction factordue to absorption of the quencher at the excitation and emissionwavelength of the fluorophore as reported elsewhere [27,28].

In order to understand whether the interaction of carmoisinewith both the protein is due to specific or non-specific binding,we have analyzed the fluorescence data by Scatchard method[29] which considers specific ligand–protein interaction. Thismethod is widely used for the graphical representation of recep-tor–protein binding data. The Scatchard equation for a single classof n independent binding sites is represented as follows:

v ¼ n� Kb � Lf

1þ ðKb � Lf Þð5Þ

where v represents binding ratio, i.e. the ratio of the bound ligand Lb

to the total protein Pt. Lf is the concentration of the free ligand atequilibrium. Kb is the binding constant and n is the number of bind-ing sites. The plot of Lb/Pt vs [carmoisine] using fluorescence data atpH 7.4 is shown below (Fig. 6) and it shows saturation after certainconcentration of carmoisine, i.e. saturation of ligand binding site inboth the proteins. Similar saturation of binding site with other li-gand is also reported elsewhere [30]. Therefore the interaction ofcarmoisine with both the serum proteins is specific to the ligandbinding site(s).

Scatchard equation is also used to calculate binding constantand binding site number. In order to estimate the binding constant,in some cases the free ligand concentration Lf in Scatchard equa-tion is approximated by the total ligand concentration Lt[31].

0 1 2 3 40.00

0.25

0.50

0.75

1.00L

b/Pt

[Carmoisine] (μmoles dm-3)

HSA+ carmoisine

(a)

0.0 2.6 5.2 7.80.00

0.25

0.50

0.75

1.00

Lb/ P

t

[Carmoisine] (μmoles dm-3)

BSA + carmoisine

(b)

Fig. 6. Saturation plot for HSA–carmoisine (a) and BSA–carmoisine (b) complexes atpH 7.4 using fluorescence data.

3.00 3.25 3.50

1.0

1.5

2.0

2.5

(F∞ -F

0)/

(F∞ -F

)

t{(F∞-F0)/(F-F0)}L (μmoles dm-3)

t{(F∞-F0)/(F-F0)}L (μmoles dm-3)

pH 4.8

pH 5.5

pH 6.3

pH 7.4

HSA

(a)

4 5 60

6

12

18

(F∞ -F

0 )/(F

∞ -F)

pH 4.8

pH 5.5

pH 6.3

pH 7.4

BSA

(b)

Fig. 7. Scatchard plot for HSA–carmoisine (a) and BSA–carmoisine (b) systems atpH 4.8, 5.5, 6.3 and 7.4.

54 S. Datta et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 50–62

Avoiding any such approximation, we have expressed Lf in terms ofthe total ligand concentration, Lt and total protein concentration, Pt

as follows [32]:

Lf ¼ Lt � Lb ¼ Lt � ðnhPtÞ ð6Þ

where h is the fractional occupancy of the total binding sites by theligand, and this can be expressed in terms of fluorescence signals asfollows [31]:

h ¼ F � F0

F1 � F0 ð7Þ

where F0 and F are the fluorescence intensity of protein in absenceand presence of carmoisine, respectively. F1 is the fluorescenceintensity of protein at the point of saturation. Now using Eqs. (6)and (7), the Scatchard equation (Eq. (5)) can be expressed as follows[33] (see the Supplementary File for details):

F1 � F0

F1 � F

!¼ KbLt

F1 � F0

F � F0

!� nKbPt

The plots using Eq. (8) for HSA/BSA–carmoisine system in all thepH at 300 K are shown in Fig. 7. The calculated binding constantusing the same equation at different pH for HSA/BSA–carmoisinecomplex is tabulated in Table 1.

From the magnitudes of binding constant it is clear that HSAshows a weaker binding affinity for carmoisine than does BSA,and also for both the proteins Kb is found to be insensitive to pH.The table also shows that binding site number is greater than unityin BSA, which is consistent with stoichiometry determinations andBenesi–Hildebrand plot. The binding constants (Kb) and bindingstoichiometry (n) for the HSA/BSA–carmoisine system have alsobeen calculated using above Eq. (8) at three different temperatures,288 K, 300 K and 308 K at pH 7.4 and are shown in Table 2.

The value of Kb has been found to decrease with increase oftemperature. The binding site number (n) is almost unity for

HSA, but it is higher than unity for BSA which indicates involve-ment of additional binding site in the latter.

3.2.1. Effect of ionic strength (added salt) of the mediumAccording to the results presented in the preceding sections,

site-specific binding of carmoisine with HSA and BSA is indicated,which may originate from the electrostatic interaction or hydro-phobic forces (or non-electrostatic modes). To elucidate whetherthe interaction of carmoisine and HSA or BSA is electrostatic ornot, we have performed the fluorescence measurements in pres-ence of three different concentrations of added NaCl (0, 0.05 and0.2 M at pH 4.8 and 7.4), that is in media of varying ionic strengths.By altering the ionic strength of the medium one can distinguishthe binding modes. The high ionic strength does not alter thestrength of hydrophobic interaction (or non-electrostatic modes)but strongly affects electrostatic binding [34,35].

We have calculated the magnitude of binding constant at pH 7.4in presence of salt using Eq. (8) and these are tabulated in the sup-porting information (Table T1, Supplementary file) which showsthat the magnitude of binding constant decreases with increasingsalt concentration for both cases. This indicates that an originallystronger protein–dye complex weakens in presence of salt whichmeans that electrostatic interaction is primarily responsible forbinding of carmoisine with serum proteins. Also it is importantto note that the value of binding constant with HSA is affected toa smaller extent by salt (NaCl) compared to BSA (see Table T1, Sup-plementary file) which indicates larger contribution of electrostaticinteraction in the latter.

3.3. Analysis of tryptophan fluorescence lifetime data

The specific serum protein–ligand interaction can also beunderstood by analyzing tryptophan fluorescence lifetime data inboth the serum proteins. Fluorescence decay of both HSA andBSA, at pH 7.4 (Fig. 8), shows bi-exponential decay with nanosec-

Table 1Variation of binding constant (Kb) as a function of pH for HSA/BSA–carmoisine systems at 300 K.a

pH HSA BSA

Kba n R¢ Kb

b n R¢

4.8 (2.97 ± 0.08) � 106 (0.99 ± .03) .9898 (6.08 ± .08) � 106 (1.20 ± .03) .99925.5 (2.99 ± 0.06) � 106 (1.03 ± .05) .9997 (6.16 ± .09) � 106 (1.24 ± .01) .98976.3 (2.93 ± 0.11) � 106 (1.01 ± .09) .9895 (6.62 ± .14) � 106 (1.23 ± .02) .99157.4 (2.98 ± 0.15) � 106 (1.04 ± .06) .9865 (6.81 ± .2) � 106 (1.25 ± .04) .9886

a Data are expressed as mean ± SD of experiments performed in triplicate in 5 mmol dm�3 sodium phosphate buffer.b Concentrations of carmoisine and proteins are expressed as moles dm�3. R¢ is the correlation coefficient for Kb values.

Table 2Binding and thermodynamic parameters for the complexation of carmoisine with HSA and BSA in pH 7.4 at different temperatures.a

Temp. (K) Kbb R¢ n DG0 (kJ mol�1) DH0 (kJ mol�1) DS0 (J mol�1 K�1) R§

HSA288 (5.04 ± 0.12) � 106 .9899 (1.02 ± .01) �36.95 ± .15 (�30.95 ± .13) (+20.85 ± .21) .999300 (2.98 ± 0.15) � 106 .9865 (1.04 ± .06) �37.20 ± .11 (�30.95 ± .13) (+20.85 ± .21) .998308 (2.18 ± 0.02) � 106 .9967 (0.99 ± .02) �37.37 ± .16 (�30.95 ± .13) (+20.85 ± .21) .999

BSA288 (1.01 ± .03) � 107 .9878 (1.25 ± .04) �38.61 ± .14 (�22.45 ± .16) (+56.12 ± .23) .995300 (6.81 ± .2) � 106 .9886 (1.25 ± .04) �39.28 ± .13 (�22.45 ± .16) (+56.12 ± .23) .999308 (5.51 ± .09) � 106 .9925 (1.20 ± .02) �39.73 ± .18 (�22.45 ± .16) (+56.12 ± .23) .997

a Data are expressed as mean ± SD of experiments performed in triplicate.b Concentrations of carmoisine and proteins are expressed as moles dm�3. R¢ and R§ are the correlation coefficient for Kb values and van’t Hoff plots.

0

1000

2000

3000

4000

5000 IRF

[Q]= 0 mole dm-3

[Q]= 2 ×10-6 moles dm-3

[Q]= 4 ×10-6 moles dm-3

Cou

nts

Time (ns)

(a)

HSA

30 40

20 25 30 35 40 450

1000

2000

3000

4000

5000 IRF

[Q]= 0 mole dm-3

[Q]= 2 ×10-6 moles dm-3

[Q]= 4 ×10-6 moles dm-3

Cou

nts

Time (ns)

(b)

BSA

Fig. 8. Fluorescence decay profiles of HSA (a) and BSA (b) in absence and presenceof carmoisine at pH 7.4, kex = 295 nm and kem = 349 nm,[HSA] = [BSA] = 2 lmoles dm�3 and carmoisine concentration is varied as 0, 2 and4 lmoles dm�3.

S. Datta et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 50–62 55

ond lifetime components in the absence of ligand. In case of HSAdifferent conformations of tryptophan residue may be ascribed totwo decay components [36]. It has been found that between twodecay components for native BSA, one centered at 6.5 ns is contrib-uting to 90% of the total fluorescence and the other centered at1.73 ns is contributing to the rest of the fluorescence. A similar

observation with BSA has been interpreted elsewhere in terms ofa larger contribution to the longer lifetime component arising fromone of the two Trp residues and a larger contribution to the shorterlifetimes from the other [37].

We have calculated lifetimes by fitting these decay curves andthe number averaged fluorescence lifetime (savg) has been calcu-lated using the following the equation [37,38] and the correspond-ing values are shown in Table 3.

hsavgi ¼ a1s1 þ a2s2 ð8Þ

Here s and a is the time constant and relative amplitude,respectively, obtained from the fitted fluorescence decay. Table 3shows that with the addition of carmoisine the average fluores-cence lifetime (savg) of HSA is reduced to a smaller extent thanBSA which indicates a stronger interaction in the second case. Alsothe amplitude of both the longer and shorter lifetime componentsof BSA has been found to be affected by the presence of carmoisine.It has been pointed out elsewhere that when ligand binds in sub-domain IB of BSA, where Trp 134 is present, the amplitude and life-time of only the longer component of the decay changes. Also forHSA, when ligand binding does not occur at sub-domain IIA, whereTrp 214 is situated, it results in insignificant change in tryptophanlifetime [37]. Thinking along similar lines and analyzing the trendof lifetime data, in our experiments, it can be inferred that carmoi-sine binds in sub-domains IIA and IB of BSA containing the twotryptophan residues while in case of HSA it is likely to bind insub-domain IIA. The results of competitive ligand binding, as dis-cussed in Section 3.5, clearly support the above propositions.

If we look into the surrounding atmosphere of the ligand bind-ing pockets in serum protein [1], it can be found that there are fewpositively charged side chains (His, Arg, Lys), some negativelycharged (Asp and Glu) and neutral side chains as well. Also thedye remains in di-negative state in the studied pH range (the –OH pKa of carmoisine is determined to be 8, data not shown). Ithas been mentioned elsewhere that negative sulfonate groups ofazo dyes can bind electrostatically with basic amino acid sidechains [39,40]. Therefore, it is expected that the amino acid resi-dues which would interact with the negative dye by electrostaticforces should be mainly Arg and Lys. It should also be noted that

Table 3Lifetimes of fluorescence decay of HSA and BSA in phosphate buffer of pH 7.4 at different concentrations of carmoisine at 300 K.

[Protein] (lmoles dm�3) [Carmoisine] (lmoles dm�3) Lifetime (ns) Amplitude savg (ns) v2

s1 (ns) s2 (ns) A1 A2

HSA 2.0 0 2.84 7.30 14.80 85.20 6.63 1.102 2.42 6.80 16.70 83.30 6.06 1.034 2.17 6.47 20.84 79.16 5.57 0.99

BSA 2.0 0 1.73 6.50 7.17 92.83 6.15 0.982 1.50 6.32 21.09 78.91 5.30 1.054 1.25 5.69 48.90 51.10 3.50 1.08

0.0033 0.0034 0.00356.30

6.45

6.60

6.75

6.90

7.05

log

Kb

1/T (K-1)

HSA

BSA

Fig. 9. Van’t Hoff plot for the binding of carmoisine with HSA and BSA at pH 7.4.

56 S. Datta et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 50–62

while pH is increased from 4.8 to 7.4, side chain of His residue mayno longer remain charged. But no alteration of binding constantwith change of pH implies that His side chain is unlikely to beimportant here. It is important to note that change in the pH ofthe solution modulates the surface charges of the proteins [15].Since carmoisine binds in the hydrophobic pocket of serum pro-teins, surface charges should not have pronounced effect on itsbinding as charges in the pocket are likely to impart more effecthere due to the electrostatic nature of interaction [35].

3.4. Analysis of thermodynamic parameters and nature of binding

The interaction between small organic molecules and biologicalmacromolecules include hydrophobic forces, hydrogen bonding,van der Waals force and electrostatic interactions. The signs andmagnitudes of the thermodynamic parameters (DH0 and DS0)can account for the main forces involved in the binding [41]. Wehave used following two equations for calculation of the thermo-dynamic parameters.

log Kb ¼ �DH0

2:303RTþ DS0

2:303Rð9Þ

DG0 ¼ DH0 � TDS0 ð10Þ

The slope and intercept of the plot of logKb vs 1/T (Fig. 9) pro-vide the magnitude of binding enthalpy and binding entropy,respectively, in the studied temperature range. Now by pluggingin these values for different temperatures in Eq. (11) we have ob-tained the values of free energy change associated with the inter-action of serum proteins and carmoisine.

Ross and Subramanian [42] have discussed that the sign andmagnitude of thermodynamic parameters can conclude on the nat-ure of interaction taking place in protein ligand association pro-cesses as: (1) DH > 0 and DS > 0 corresponds to hydrophobicforce; (2) DH < 0 and DS < 0 corresponds to van der-Waals interac-tion and hydrogen bonding; (3) DH < 0 and DS > 0 corresponds toelectrostatic interaction. The calculated values of enthalpy and en-tropy change listed in Table 2, show that the association of carmoi-sine with both the protein is driven by negative enthalpy change(DH < 0) and positive entropy change (DS > 0). Negative enthalpychange (DH < 0) could correspond to van der-Waals interaction,hydrogen bonding and electrostatic interaction [42]. But here, thesign of entropy change (DS > 0) is clearly indicative of electrostaticinteraction. When ligand binds with protein predominantly byelectrostatic interaction, it is likely to be locally mobile becausethe electrostatic interaction is basically charge-charge interactionmostly occurring through space and does not lead to real bond for-mation (local motion of the ligand in an ion-pair is not restricted)[35,43]. Therefore, electrostatic interaction should be the majormode of binding in HSA/BSA–carmoisine complex. Also the contri-bution of entropy is lower for HSA–carmoisine complex comparedto the BSA–carmoisine complex indicating larger contribution ofelectrostatic forces in the latter which is also consistent with the

results of salt variation study. The negative values of DG0 at alltemperatures at pH 7.4 for both the proteins indicate the spontane-ity of binding. As discussed in the earlier section that positivelycharged side chain of amino acid residues present in binding pock-et in both the proteins are mainly Arg and Lys. So, electrostaticinteraction between the negative sulfonate groups of carmoisineand those positive side chains is likely to be responsible for site-specific binding.

3.5. Site marker competitive binding experiments

The principal regions where ligands bind in HSA and BSA areusually located in hydrophobic cavities of sub-domains IIA and IIIA,and the binding cavities associated with these sub-domains arealso referred to as sites I and II, respectively. The Trp 214 of HSAand Trp 213 of BSA are in the sub-domain IIA. Also in some casesthe ligand binds in the sub-domain IB which is also referred to assite III. The second tryptophan residue (Trp 134) of BSA is a partof sub-domain IB. To locate the precise binding site of carmoisine,the site marker competitive binding experiments are carried outusing drug which is known to specifically bind to a site or regionof HSA or BSA. As described in the literature, warfarin has beendemonstrated to bind in the sub-domain IIA while ibuprofen isconsidered as binder of sub-domain IIIA [37]. Hemin is the binderof sub-domain IB [37]. By monitoring the changes in the fluores-cence intensity of drug-bound HSA or BSA by the dye, where thedrugs are either site I (warfarin) or, site II (ibuprofen) or, site III(hemin) binders, information about the specific binding site of dyescan be obtained.

The result shows that addition of warfarin in equimolar ratiowith protein at pH 7.4 changes the fluorescence spectra of bothHSA and BSA (Supplementary Fig. S1 and S2) with a red shift ofthe emission maximum (by �10–25 nm) and slightly lower fluo-rescence intensity as compared with no-warfarin case. This obser-vation is similar to one reported elsewhere [37] and, it indicates

Table 4Binding constants (Kb) from competitive binding experiments for HSA/BSA–carmoi-sine systems at pH 7.4 at 300 K.a

Site marker Kbb

HSA BSA

None (2.98 ± 0.15) � 106 (6.81 ± 0.2) � 106

Ibuprofen (2.96 ± .12) � 106 (6.62 ± .05) � 106

Warfarin (1.01 ± .08) � 106 (5.32 ± .04) � 106

Hemin (2.99 ± .06) � 106 (4.47 ± .06) � 106

a Data are expressed as mean ± SD of experiments performed in triplicate in5 mmoles dm�3 phosphate buffer.

b Concentrations of carmoisine and protein are expressed in moles dm�3.

3.00 3.25 3.50

1.0

1.5

2.0

2.5 HSA + carmoisine(HSA : ibuprofen =1:1) + carmoisine (HSA : warfarin = 1:1) + carmoisine

(a)

3.5 4.0 4.5 5.0 5.50

4

8

12

16BSA + carmoisine(BSA : hemin=1:1) + carmoisine

(b)

(F∞ -F

0)/

(F∞ -F

)(F

∞ -F0)/

(F∞ -F

)

t{(F∞-F0)/(F-F0)}L (μmoles dm-3)

t{(F∞-F0)/(F-F0)}L (μmoles dm-3)

Fig. 10. Scatchard plot of HSA–carmoisine system (a) and BSA–carmoisine system(b) in presence of selected site markers, [HSA] = 2 lmoles dm�3, [BSA] = 3lmoles dm�3 and protein: site marker = 1:1 at pH 7.4.

Table 5Binding constants (Kb) for HSA/BSA–carmoisine systems with increasing ethanolpercentage at pH 7.4 at 300 K.a

% of Ethanol (v/v) Kbb

HSA BSA

0 (2.98 ± 0.15) � 106 (6.81 ± 0.2) � 106

5 (1.33 ± .05) � 106 (5.97 ± .03) � 106

10 (8.42 ± .06) � 105 (4.58 ± .06) � 106

a Data are expressed as mean ± SD of experiments performed in triplicate in5 mmoles dm�3 phosphate buffer.

b Concentrations of carmoisine and protein are expressed in moles dm�3.

S. Datta et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 50–62 57

incorporation of warfarin in the hydrophobic cavity of the protein.Now to this drug-bound protein we have added an increasing con-centration of carmoisine. In case of HSA the strength of its com-plexation with carmoisine is affected in presence of warfarin andleads to lowering of the binding constant (Table 4 and Fig. 10) com-pared to that in the absence of the warfarin. This shows thereplacement of warfarin from HSA–warfarin complex by carmoi-sine. On the other hand, for BSA the value of binding constant withcarmoisine is not altered to a large extent in the presence of war-farin. But while monitoring the emission spectra of BSA–warfarincomplex by exciting at 308 nm (corresponding to warfarin absorp-tion maxima) the emission maxima shows a red shift (Supplemen-tary Fig.S3) with the addition of dye. This indicates displacement ofwarfarin from its complex with BSA by carmoisine [44]. This is onlypossible if carmoisine binds in the warfarin binding site of BSA.Now the question is why don’t we get much change in the magni-tude of binding constant in presence of warfarin? The binding sitenumber from fluorescence data and binding stoichiometry fromJob’s method indicate that two binding sites are available for

carmoisine in BSA. This means although carmoisine binds in thewarfarin binding site of BSA but it also binds to an additional site-this may be a possible reason for such small decrease in bindingconstant in presence of warfarin.

In presence of ibuprofen the values of binding constant of boththe protein with carmoisine is not affected at all when carmoisineis added to the equimolar mixture of ibuprofen–HSA or BSA com-plex at pH 7.4 (Table 4 and Supplementary Fig. S4 and Fig. S5). Thisindicates that carmoisine does not bind in the ibuprofen-bindingsite.

In presence of hemin the binding constant (Table 4) of HSA withcarmoisine at pH 7.4 is insignificantly affected. It means carmoi-sine does not bind to the hemin binding site of HSA i.e., in thesub-domain IB. On the other hand, the presence of hemin lowersthe association of BSA with carmoisine at pH 7.4 and the corre-sponding Scatchard plot at pH 7.4 is shown in Fig. 10. So, the IBsub-domain of BSA is the second binding site for the ligand.

Therefore it may be concluded that carmoisine binds to HSA insub-domain IIA, i.e., warfarin binding site and this is near to the Trp214. While the binding sites in BSA are in sub-domains IIA (warfa-rin binding site) and IB (hemin binding site). So both the trypto-phan residues (Trp 134 and Trp 213) are accessible to carmoisinein this case.

3.6. Effect of ethanol on protein–carmoisine complexation

The effect of alcohol on binding of warfarin with HSA has beenpreviously studied by equilibrium dialysis and fluorescence meth-ods at pH 7.4 [45]. It is reported elsewhere that in presence of eth-anol the fluorescence intensity of bound warfarin is decreasedsignificantly due to changes in the surrounding environment ofthe warfarin in the binding site, and also because of replacementof bound warfarin by the alcohol [46]. In order to get further infor-mation of the dye binding site we have performed fluorescencemeasurements medium with varying ethanol content.

With increasing ethanol percentage at pH 7.4 the binding con-stant (Table 5) for HSA–carmoisine complex is significantly de-creased, while for BSA–carmoisine complex it decreases onlyslightly. From our earlier observation it is evident that carmoisinebinds in sub-domain IIA i.e., closer to Trp 214 and Trp 213 of HSAand BSA, respectively. It has one additional binding site with BSAwhich is in sub-domain IB i.e., closer to Trp 134. Now ethanol in-duced changes in native protein structure may lead to the directbinding of ethanol to the specific hydrophobic binding sites (sub-domain IIA of HSA and BSA) and/or displacement of ligands fromthose sites [47]. In our experiments, with 10% v/v ethanol contentthe change in secondary structure of both the serum protein is al-most negligible (see Section 3.8.1 for CD spectroscopy). So the de-crease in binding constant should be due to the displacement ofcarmoisine from its binding site in the sub-domain IIA by ethanol.Thus ethanol-induced displacement of ligand from HSA results indecrease of binding constant. On the other hand, in case of BSA–

58 S. Datta et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 50–62

carmoisine complex, ethanol is able to displace carmoisine fromsub-domain IIA only, but not from sub-domain IB. As a result thebinding constant for BSA–carmoisine complex is not decreased tothe same extent as HSA, as expected.

3.7. Urea denaturation studies

The effectiveness of urea as a protein denaturant is well known.It acts by increasing the aqueous solubility around the hydrophobicdomains of protein and/or weakening the hydrogen bonding in theprotein structure [48]. Thus tryptophan residues are exposed towater and F0 also reduces. So due to water exposure of amino acidresidues, the dye seems to have considerably reduced interactionwith the protein. Hence binding should decrease for both theproteins.

Recently urea has been used to investigate the probable loca-tion of probe in HSA by Leggio et al. [49], where they have pro-posed two intermediates during the denaturation process. In thepresence of 3 M urea domain I begin to open up, while domain IIand III remain closed (intact). At 4.35 M urea, domain II becomesunfolded. Domain III opens up only at high urea concentration(8 M). We have carried out the fluorescence measurements ofboth the proteins in presence of carmoisine at pH 7.4 in threedifferent concentrations of urea (0 M, 4 M and 6 M). We haveconsidered the volume correction (due to addition of urea) andthe Scatchard plots at different urea concentrations are shownbelow.

From the plot (Fig. 11) it is observed that binding of carmoisinewith HSA is affected only by 6 M urea concentration, whereas forBSA it is strongly affected by 4 M as well as 6 M urea concentra-tions. Since domain I opens up at 4 M urea, therefore, the probablelocation of carmoisine is only in the domain II with HSA and in thedomains I and II with BSA.

2.8 3.0 3.2 3.4

1.2

1.6

2.0

2.4 0 mole dm-3 urea

4 moles dm-3 urea

6 moles dm-3 urea

(a)

HSA

3.5 4.0 4.5 5.0 5.5

3

6

9

12

150 mole dm-3 urea

4 moles dm-3 urea

6 moles dm-3 urea

(b)

BSA

(F∞ -F

0)/

(F∞ -F

)(F

∞ -F0)/

(F∞ -F

)

t{(F∞-F0)/(F-F0)}L (μmoles dm-3)

t{(F∞-F0)/(F-F0)}L (μmoles dm-3)

Fig. 11. Scatchard plot of protein–carmoisine systems [HSA-(a), BSA-(b)] in thepresence of 0, 4, and 6 mol dm�3 urea in 5 mmol dm�3 buffer of pH 7.4.

3.8. Conformational investigations and related studies

3.8.1. Circular dichroism (CD) spectroscopyCD spectroscopy is a sensitive technique to monitor the second-

ary structural changes of protein upon interaction with ligand. TheCD spectra of HSA and BSA exhibit two negative bands at 208 and222 nm in the ultraviolet region, which are characteristic of thetypical a-helix structure of protein [50]. The negative peaks at208 and 222 nm both contribute to the n ? p� transition for thepeptide bond of a-helix [50].

The CD data are expressed in terms of mean residue ellipticity(MRE) in deg cm2 dmol�1 according to the following equation [51]:

MRE ¼ hobsðm degÞ10� nr � l� cp

ð11Þ

where cp is the molar concentration of the protein, nr is the numberof amino acid residues of the protein and l is the path length. The a-helix contents of free and complexed HSA or BSA are calculatedfrom mean residue ellipticity (MRE) values at 208 nm using the fol-lowing equation [52].

a-helix ð%Þ ¼ ð�MRE208 � 4000Þ33;000� 4000

� �� 100 ð12Þ

where MRE208 is the experimental MRE value of protein at 208 nm,4000 is the MRE value of the b-form and random coil conformationat 208 nm, and 33,000 is the MRE value of a pure a-helix at 208 nm.We have recorded the CD spectra of both the protein in absence andpresence of carmoisine at all the pH and in presence of differentethanol content of the medium. The CD spectra of both the proteinat pH 7.4 in presence of 10% (v/v) ethanol content of the mediumshows that the conformation of both the protein is only slightly af-fected (see Fig. S6, Supplementary file). We have also compared CDspectra of both the proteins in the absence and presence of high salt(0.2 M) (Fig. S7, Supplementary file) which indicates that there isvirtually no change in protein conformation (secondary structure)due to salt addition.

The value of a-helix for free HSA and BSA at all the pH is shownin Table 6 which shows that with the variation of pH the conforma-tion of both the protein is slightly changed. With increase of car-moisine the CD spectra of both the proteins (Fig. 12) change andthe a-helix content decreases indicating that the binding of thedye leads to the alteration of the secondary structure. Secondarystructure is linked to the biological activity of proteins [15]. There-fore the decrease in a-helical content may results in a loss of bio-logical activity of serum proteins upon interaction withcarmoisine. The a-helical content of both the protein in absenceand presence of carmoisine (protein: carmoisine ratio = 1:2.5) atall the pH are shown in Table 6.

The change in CD spectra can also be applied for the determina-tion of binding constant of carmoisine with protein [53]. We havemeasured the CD spectra of both HSA and BSA at pH 7.4 in pres-ence of different concentration of carmoisine. We have used Scott

Table 6a-Helix contents of BSA and HSA at pH 4.8, 5.5, 6.3 and 7.4 (at 300 K) in absence andpresence of carmoisine.

% a-Helix of BSA % a-Helix of HSA

[dye] (lmoles dm�3) 0 22.5 0 22.5

% a-Helix of BSA % a-Helix of HSA

pH4.8 65.10 63.43 66.75 65.955.5 65.43 63.41 66.82 65.826.3 66.05 64.35 67.65 66.537.4 66.08 64.28 67.94 66.96

205 210 215 220 225

-120

-112

-104

θ(m

deg )

Wavelength (nm)

(Protein:dye) =1:0

(Protein:dye) =1:2.5

(a)

HSA

205 210 215 220 225

-120

-114

-108

-102

θ(m

deg)

Wavelength (nm)

(Protein:dye) =1:0

(Protein:dye) =1:2.5

(b)

BSA

Fig. 12. CD spectra of HSA (a), BSA (b), free protein ) solid lines and complexedwith carmoisine ) dotted lines. [Proteins]: [dye] are 1:0 and 1:2 for both proteinsin 5 mmoles dm�3 phosphate buffer of pH 7.4.

260 270 280 290 300

0.2

0.4

0.6

0.8

1.0

266 268 270

0.2

0.4

Fluo

resc

ence

Inte

nsity

(a.u

)

Wavelength (nm)

270 280 290 300

0.3

0.6

0.9

Flu

ores

cen

ce I

nte

nsi

ty (

a.u

.)

Wavelength (nm)

Fluo

resc

ence

Int

ensi

ty (

a.u)

Wavelength (nm)

FWHM

remains same

[carmoisine] increasesFWHM increases

Fig. 13. Normalized synchronous fluorescence spectra of HSA with addition ofcarmoisine in 5 mmoles dm�3 phosphate buffer of pH 7.4 at Dk = 60 nm. (Top insetshows the same spectra in presence of 0.2 mol dm�3 NaCl, bottom inset shows plotin expanded scale.)

S. Datta et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 50–62 59

model [54] for analyzing CD-data (Fig. S8, Supplementary file) andthe calculated value of binding constant at pH 7.4 (at 300 K) isfound to be (1.01 ± .03) � 105 and (1.26 ± .01) � 106 for HSA–car-moisine complex and BSA–carmoisine complex, respectively. Bind-ing data from CD analysis again shows weaker interaction ofcarmoisine with HSA compared to BSA.

Fig. 14. The docked pose of carmoisine with BSA.

3.8.2. Synchronous fluorescence spectroscopy (SFS)Synchronous fluorescence spectroscopy can provide informa-

tion about the change of molecular micro-environment in thevicinity of chromophores. It involves simultaneous scanning ofthe excitation and emission monochromators while maintaininga constant wavelength interval (Dk). It is a useful method to studythe environment of amino acid residues by measuring the shift inthe wavelength of emission maxima (kSFS

max). The shift of kSFSmax de-

pends on the change of the polarity around the chromophoremicroenvironment. The synchronous fluorescence spectra providethe characteristic information of tyrosine or tryptophan residuesif Dk are set at 15 or 60 nm, respectively [55]. With the additionof carmoisine to both HSA and BSA the maxima of synchronousfluorescence spectra at pH 7.4, corresponding to tryptophan resi-dues, shows a little blue shift (about 2 nm). In contrast the tyrosinefluorescence spectra of HSA and BSA at pH 7.4 show no significantshift of the emission maxima. The results suggest that the polarityaround the tryptophan residues is decreased and hydrophobicity isincreased, yet the microenvironment around the tyrosine residueshave no discernable change due to dye binding. Also with increas-ing concentration of carmoisine at Dk = 60 nm i.e., the synchronousfluorescence spectra due to tryptophan becomes little broader andthe blue side of the spectra is shifted to a little shorter wavelengthside (FWHM changes from 22 nm to 26.5 nm) which is also anotherindication of the change of microenvironment of tryptophan resi-due while binding with carmoisine. Importantly, in presence of0.2 M NaCl, the broadening of the SFS is not observed even at

Dk = 60 nm (Fig. 13, inset, for HSA). These results indicate thatbinding of carmoisine with both the protein pushes tryptophanresidue towards less polar environment and presence of salt disfa-vors binding. This clearly suggests that the dye binding is primarilyelectrostatic in nature. Normalized synchronous fluorescence spec-tra of HSA at Dk = 60 nm with increasing concentration of carmoi-sine at pH 7.4 are shown in Fig. 13 (spectra for BSA is not shown).

3.8.3. Analysis of docking simulationsThe interaction between carmoisine and proteins is further ex-

plored with docking studies, where the di-negative dye has beendocked to determine the preferred binding sites in the protein.The docking results are very much consistent with the site-com-petitive drug displacement experiments and also with experimen-tal results from time-resolved data and steady-state fluorescenceand UV–Vis measurements. Competitive sitemarker binding exper-iments indicate single binding site in HSA and more than one bind-ing sites of carmoisine in BSA. As discussed earlier, stoichiometryfor HSA: carmoisine and BSA: carmoisine are found to be 1:1 and1:3, respectively. Considering these we have docked one carmoi-sine with HSA, and two with BSA. For docking with HSA the grid

Fig. 15. Closer look of carmoisine docked to BSA, near Trp 213 (a) and Trp 134 (b).

60 S. Datta et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 50–62

center is kept at 24.36, 34.57 33.09 corresponding to the co-ordi-nates of indole-N of Trp 214. While performing double docking,the first dye has been docked with BSA keeping the grid center at25.02, 35.49 and 34.32 which is the co-ordinates of indole-N ofTrp 213. The second dye molecule is then docked onto the lowestenergy first-ligand-docked pose keeping grid center at 52.74,21.71 and 22.49 corresponding to the co-ordinates of indole-N ofTrp 134.The docked pose of carmoisine with BSA is shown inFig. 14.

Fig. 14 shows the docking of carmoisine to the sub-domains IIA(site-I) and IB (site-III). A closer view of the first docked dye mole-cule is shown in Fig. 15a and the docked second carmoisine isshown in Fig. 15b. These figures show that Trp 213 and Trp 134are in the close vicinity of the docked ligands. The docking studiesof HSA show that carmoisine binds in the sub-domain IIA i.e. site –Inear the Trp 214 (Supplementary Fig. S9).

Accessible surface areas (ASA) have been calculated for BSA–carmoisine (Supplementary Table T2) system with the help of low-est energy docked conformations. A considerable loss of accessiblesurface area (ASA) of Trp 213 and Trp 134 for BSA upon docking ofcarmoisine indicates binding near the respective tryptophan resi-dues. Similar change in the accessible surface area of Trp 214 ofHSA has also been found. Also the lowest energy pose indicatesthat there is a significant positive charge density around the bind-ing site in both the proteins. Importantly, in the docked pose we donot find any His residue in close proximity of the dye(s) in both theserum albumins (Fig. 15a and b and Supplementary Fig.S9) andhence histidine side chain should not be important in the electro-static interaction of dye and protein, which we have alreadypointed out earlier. Therefore, the presence of amino acids residueslike Arg and Lys with positively charged side chains near the bind-ing site may also clarify the pH insensitivity of binding as theseside chains have pKa > 10, so within the range of studied pH theircharged state remains unaltered.

4. Conclusions

In this paper the binding between food dye carmoisine andserum proteins (HSA and BSA) has been explored by absorption,fluorescence spectroscopy, SFS, time resolved study, circulardichroism spectroscopy, and molecular docking simulations. Thebinding of carmoisine with HSA and BSA leads to quenching offluorescence intensity of HSA and BSA. The interaction is specific

to ligand binding site in both the cases and it shows saturation asa function of ligand concentration. In case of HSA the value ofbinding constant is comparatively lower than with BSA. In bothcases the binding is found to be insensitive to pH of the medium.This is possibly because the dye binds in a site having side chainpositive charge density arising out of Lys and Arg residues. Thehigher binding site number (1.2–1.3) with BSA indicates involve-ment of an additional binding site. Site markers experiment andethanol variation study also indicates that binding of carmoisineis closer to Trp 214 of HSA which is also the binding site for war-farin, while for BSA the dye molecules bind closer to both thetryptophan residues.

The decrease of binding of carmoisine with ionic strength canbe well exploited in purifying a similar charged dye bound to pro-tein by mere leaching with saline solutions. This can have greatpharmaceutical significance as to purification of proteins. Similarstudies are underway to arrive at a generalized model of bindingof charged azo dyes with serum proteins.

Also the present study is significant with respect to the toxico-logical profile of similar azo dyes. It should provide significant in-sight on the interaction of protein with model azo dyes. Thetoxicity of pollutants such as dyes, introduced into the blood ofstream in the process of environmental exposure with industrialpollutants, is a real concern now-a-days. Thus investigation ofinteraction of plasma proteins with model dyes, which are of par-ticular interest in therapeutic/toxicological investigations, is in-deed relevant.

Carmoisine possesses a high binding ability (Kb � 106) with ser-um proteins which are although reduced to �105 with increasingionic strength of the medium but still is significantly high in thephysiological ionic strength. Since binding of carmoisine with ser-um protein is insensitive to pH, in the two extremes of pH the dyeshould remain strongly bound to the carrier protein. So it is likelythat with change of pH from its physiological to a significantly lowvalue, e.g., inside stomach, carmoisine is likely to be retained in thefood stuff and gets absorbed. High binding carmoisine can replaceanticoagulant drugs like warfarin from its binding site in serumprotein (see competitive binding section). Therefore it is possiblethat contamination of foodstuff with carmoisine is likely to affectthe absorption of such drugs in blood plasma and may resultimpairment of their activity. Thus while selecting such food color-ing agents one must take into consideration of the above men-tioned issues on possible health hazard ground.

S. Datta et al. / Journal of Photochemistry and Photobiology B: Biology 124 (2013) 50–62 61

5. Abbreviations

HSA

human serum albumin BSA bovine serum albumin CD circular dichroism

Acknowledgments

We thank DST-India (Fund No. SR/FTP/CS-97/2006), CSIR-India(Fund No. 01/(2177)/07 EMR-II, dated 24/10/2007) and IIT-Kharag-pur (ISIRD-EEM grant) for financial support. S.D. thanks IIT Kharag-pur for fellowship. N.M. thanks CSIR-India for individualfellowship. We thank Prof. S. Basak of SINP, Kolkata for single pho-ton counting measurements and Prof. S. Nanda of IIT Kharagpur forhelping us with purification of some chemicals. S.D. also thanks P.Bolel for help in various instances during experiments.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jphotobiol.2013.04.004.

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