At

NSa

b

a

ARR1AA

KRNPFH

1

itHarmcoEoadnfoa

s

0d

Journal of Pharmaceutical and Biomedical Analysis 62 (2012) 96– 104

Contents lists available at SciVerse ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

j ourna l ho me pa ge: www.elsev ier .com/ locate / jpba

nthraquinone derivatives based natural dye from Rheum emodi as a probe forhermal stability of proteins: Spectroscopic and chromatographic studies�

andini Sharmaa, Rajesh Kumara, Arun K. Sinhaa,∗, Peram B. Reddyb,hahid M. Nayeemb, Shashank Deepb,∗∗

Natural Plant Products Division, CSIR – Institute of Himalayan Bioresource Technology, Post Box No. 6, Palampur 176061, HP, IndiaDepartment of Chemistry, Indian Institute of Technology, New Delhi 110016, India

r t i c l e i n f o

rticle history:eceived 13 September 2011eceived in revised form4 December 2011ccepted 14 December 2011vailable online 23 December 2011

a b s t r a c t

Rheum emodi is a storehouse of a large number of anthraquinone derivatives which are known for alarge number of biological activities of significant potency. In this work, a study on the interactionsbetween anthraquinone derivatives based natural dye isolated from R. emodi and different proteins hasbeen reported for the first time, revealing the use of dye as an extrinsic probe to determine the stabilityof these proteins alone and in the presence of additives. The stability parameters have been evaluatedas a change in the fluorescence intensity of the dye as a function of temperature due to the differential

eywords:heum emodiatural dyerotein stabilityluorescence spectroscopy

interaction of the dye with various conformations of a protein. Also, the effect of the change in polarityof the solvent on the fluorescence emission spectra of dye was studied where high quantum yield wasobserved in alcohol as compared to water. The RP-HPLC characterization of the dye revealed the presenceof anthraquinones glycosides as main compounds in it. Thus, natural dyes may be used as biosensors tofollow the conformational changes in proteins.

igh performance liquid chromatography

. Introduction

Natural dyes have been an integral part of human life since timemmemorial. These dyes have been used extensively throughouthe world for coloring food, textiles, in printing, painting etc. [1–3].owever, with the influx of synthetic dyes, natural dyes suffered

setback. During the last few decades, questions have been raisedegarding the use of synthetic dyes due to the increased environ-ental awareness and serious health hazards like allergenicity and

arcinogenicity associated with their use and disposal [4]. More-ver, a recent ban imposed all over the world including Europeanconomic Community (EEC), Germany, USA and India on the usef some synthetic dyes (e.g. azo dyes) triggered an active researchnd development initiative to revive age old art and traditional wis-om of employing safer natural dyes [4,5]. The advantages of usingatural colorants are manifold as these dyes are ecofriendly, safe

or body contact, unsophisticated, harmonized with nature [6], andbtained from renewable sources. Also, the preparation involves

minimum possibility of chemical exposure. However, the use

� IHBT communication no. 2114.∗ Corresponding author. Tel.: +91 1894 230426; fax: +91 1894 230433.

∗∗ Corresponding author. Tel.: +91 11 26596596; fax: +91 11 26581102.E-mail addresses: aksinha08@rediffmail.com (A.K. Sinha),

deep@chemistry.iitd.ac.in (S. Deep).

731-7085/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.jpba.2011.12.017

© 2011 Elsevier B.V. All rights reserved.

of natural dyes for protein folding/unfolding study has not beenexplored in detail so far.

Recombinant proteins and antibodies are necessary for thetreatment and prevention of numerous diseases but are oftenunstable for therapeutic or pharmaceutical application. A sta-ble, correctly folded protein is an absolute requirement for asuccessful biotherapeutic and the stability of a desired conforma-tion is achieved through suitable formulation [7,8]. In order toassure protein integrity during bioprocessing, formulation, stor-age and handling, the use of analytical techniques to check thestability and quality of protein is inevitable. A high throughputscreening method, based on the thermal stability of protein, canbe used to identify optimum formulation conditions for crystal-lization and purification of several proteins with respect to pH,buffer, excipients and ligands. Increased stability is indicated byan increase in the protein’s denaturation temperature (Tm) whichis obtained by numerous methods, including differential scanningcalorimetry and optical methods (circular dichroism, fluorescenceor absorbance spectroscopy).

In fluorescence spectroscopy, extrinsic fluorescent dyes haveemerged as highly sensitive and versatile tools for the charac-terization of structural and physical properties of proteins [9].

Extrinsic dyes are independent of the presence and position ofaromatic amino acids within the protein and the possibility tomeasure fluorescence parameters for small concentration of pro-tein samples makes these dyes highly suitable for high throughput

cal an

siah

bb(tAaHposfirfeebe

omfioadwe

2

2

SecoAssacodfuRa

2

Sprofipp4w

N. Sharma et al. / Journal of Pharmaceuti

creening studies. Furthermore, these dyes had been used to mon-tor the aspects which are not necessarily probed by tryptophannd tyrosine intrinsic fluorescence such as changes in surfaceydrophobicity.

There are two important considerations for an extrinsic dye foreing used as a probe for thermal denaturation study (a) it shoulde highly sensitive to the different conformations of proteins andb) the binding with protein should be weak so that denaturationemperature may not get affected in the presence of the dye. 8-nilino-1-naphthalene sulfonic acid (ANS) is quite often used asn extrinsic dye to probe the thermal denaturation of proteins.owever, ANS is known to affect the denaturation temperature ofroteins such as bovine serum albumin (BSA) significantly. Becausef this limitation and some other drawbacks with available extrin-ic dyes as mentioned by Hawe et al. [9], efforts are being put up tond a dye which can, at least, complement the existing dyes, if noteplace them, for characterizing the stability of protein in differentormulations. In this context, a colored fraction isolated from Rheummodi has been studied for its use in characterization of proteins. R.modi, an important medicinal plant, is a storehouse of a large num-er of anthraquinone derivatives such as physcion, chrysophanol,modin, aloe emodin, rhein, and their glycosides [10–12].

During the past years, several studies have been conductedn the binding of anthraquinone derivatives with serum albu-ins [13–15]. However, to the best of our knowledge, this is the

rst spectroscopic study on the interaction between a natural col-red fraction (rich in anthraquinone derivatives) from R. emodind different proteins. Further, utilization of the dye as a probe toetermine the stability of proteins using fluorescence spectroscopyas explored. These data can be used for calculation of enthalpy,

ntropy and free energy of folding.

. Experimental

.1. Chemicals and reagents

ANS and BSA (min 96% electrophoresis) were purchased fromigma Aldrich (Banglore, India), whereas lysozyme (extracted fromgg white), chymotrypsin, trypsin (extracted from bovine pan-reas) and pepsin (extracted from porcine stomach mucosa) werebtained from Sisco Research Laboratories Pvt. Ltd. (Mumbai, India)cetic acid, acetonitrile, hexane, chloroform, ethyl acetate, ethanol,odium sulfate and methanol were purchased from Merck (Darm-tadt, Germany). Silica gel (60-120) for column chromatographynd XAD-7 resin (product of M/s. Dow Chemicals, USA) were pur-hased from SD fine-chem limited (Mumbai, India). Componentsf buffer such as sodium hydrogen phosphate (Na2HPO4), sodiumihydrogen phosphate (NaH2PO4) and Tris–HCl were purchasedrom Sisco Research Laboratories Pvt. Ltd. (India). HCl and NaOHsed to maintain pH were also of analytical reagent grade from Siscoesearch Laboratories (India). Milli-Q grade water was produced by

Millipore system (Bedford, MA, USA).

.2. Isolation of colored fraction from R. emodi

Powder of R. emodi roots was purchased from Alps Industriesahibabad, India (sold commercially under the name desert). Theowder (10 g) was dissolved in water at 70 ◦C (powder to wateratio of 1:20), cooled and filtered. The filtrate was passed slowlyver 100 × g of pre-activated XAD-7 resin column. The column wasrst washed with water to remove the highly water soluble com-

onents and finally eluted with 500 ml of ethanol [16]. The ethanolart was dried over sodium sulfate and concentrated in vacuo at5 ◦C to obtain 3 g of yellowish-brown solid fraction (yield = 30%,/w), which was coded as IHBT-dye 4. A dye was also prepared from

d Biomedical Analysis 62 (2012) 96– 104 97

the dried and powdered roots of R. emodi collected from WesternHimalayan region (India) by above described process and comparedwith the former using HPLC and UV spectra (see Supplementary Fig.S1, S2).

2.3. Isolation of marker compounds from R. emodi

The dried and powdered rhizomes of R. emodi (500 g) wereextracted successively with hexane and chloroform and concen-trated extracts were subjected to column chromatography oversilica gel [17,18]. Column purification of hexane extract withethyl acetate–hexane (5:95, v/v) yielded physcion (1) and chryso-phanol (2) whereas column purification of chloroform extract withethyl acetate–hexane (30:70, v/v) yielded emodin (3). Similarly,chrysophanol glycoside and emodin glycoside were isolated as perprevious reports [17]. Structure of all the compounds was con-firmed on the basis of their NMR and comparison with the reportedNMR data of these compounds [19]. Isolated compounds were usedas standards in HPLC analysis.

2.4. Preparation of stock solution

Stock solutions of IHBT-dye 4 were prepared by dissolving1 mg of the dye in 1 ml of water and removing any insolublecontents by filtration. Stock solution of ANS (8.6 mM) was madein ethanol. Solutions of BSA (84 �M), chymotrypsin (40 �M), andtrypsin (86 �M) were prepared in phosphate buffer (25 mM, pH7.0), whereas solutions of lysozyme (68 �M) and pepsin (87 �M)were made using Tris–HCl buffer (50 mM, pH 7.4). Phosphate buffersolution (25 mM) at pH 7.0 was prepared by dissolving appropri-ate amount of sodium hydrogen phosphate (Na2HPO4) and sodiumdihydrogen phosphate (NaH2PO4) in milli-Q water.

2.5. Preparation of working solutions

The sample solutions of protein + dye in buffer were prepared byadding 10–100 �l of protein stock solution in a mixture of the 1.5 mlof dye stock solution and 1.5 ml of buffer. Sample chosen to obtainthe thermal denaturation profiles of protein was the one with max-imum emission intensity. The sample solutions of ANS + proteinwere obtained by adding varying amount of protein stock solutionto a mixture of 20 �l of stock ANS solution and 2980 �l of buffersolution such that ANS:protein ratio was 50:1 in the solution.

2.6. Estimation of concentration of protein and dye

IHBT-dye 4 has A364nm = 3.0 for the concentration of 1 mg/ml.The concentration of proteins and ANS solutions was calculatedby using reported molar absorption coefficients (ε). The ANS con-centration was determined using a ε375 value of 8000 M−1cm−1.The concentration of the protein solutions was estimated spec-trophotometrically by using the ε280 value of 43,824 M−1 cm−1

for BSA, 37,000 M−1 cm−1 for lysozyme, 52,000 M−1 cm−1 for chy-motrypsin, 37,650 M−1 cm−1 for trypsin and 38,600 M−1 cm−1 forpepsin. All the absorbance measurements were performed on Var-ian Cary 100 spectrophotometer (Varian Inc., the Netherland).

2.7. Fluorescence measurements

The interaction of the IHBT-dye 4 with proteins was observedwith a Cary Eclipse fluorescence spectrophotometer (Varian Inc.,

the Netherland). The fluorescence measurements were carried outin optically transparent quartz cells of 10 mm path length andsample temperature was maintained using peltier based thermalcontroller (Varian Inc., the Netherland). Fluorescence spectra were

9 cal an

or

oowtTwTplmimwwer

2

t

y

�

wo�eytaeu

2

oiP(

TF

(

8 N. Sharma et al. / Journal of Pharmaceuti

btained with excitation and emission slits set to 5 nm and 10 nm,espectively.

Emission spectra of IHBT-dye 4 in aqueous solution werebtained at different excitation wavelengths. The maximum flu-rescence intensity was observed for the IHBT-dye 4 at excitationavelength of 380 nm and thus 380 nm was chosen as the exci-

ation wavelength for the dye solution (Supplementary Fig. S3).he protein–dye mixtures were also excited at different excitationavelengths and emission spectra of the mixtures were obtained.

his yields the information about fluorescence intensity of dye inresence of protein as a function of excitation and emission wave-

engths. The excitation, at which the fluorescence intensity wasaximum, was chosen as the excitation maxima. Thermal exper-

ments were performed with excitation wavelength at excitationaxima of the dye–protein mixture. The dye–protein solutionsere equilibrated at 20 ◦C for 3–5 min, and fluorescence intensityas measured as a function of temperature. Temperature scan for

ach experiment was carried out between 20 ◦C and 99 ◦C at a scanate of 1 ◦C/min.

.8. Analysis of thermal denaturation data

The thermodynamic parameter (Tm) was extracted from thehermal profile by a non-linear least square analysis using Eq. (A.1).

(T) = yN + yDe−�G

RT

1 + e−�G

RT

(A.1)

G = �Hm

(1 − T

Tm

)− �CP

((Tm − T) + T ln

(T

Tm

))(A.2)

here y(T) is the experimentally observed spectroscopic propertyf protein at temperature T, �G is the free energy of denaturation,CP is the specific heat capacity of denaturation and �Hm is the

nthalpy change at Tm, the mid-point of the thermal denaturation.N and yD are the spectroscopic properties of the native and dena-ured state. For non-linear fitting of these profiles using Eq. (A.1),

good estimate of initial value of various thermodynamic param-ters is needed. Initial values of other parameters were obtainedsing method reported by Saini et al. [20].

.9. HPLC measurements

HPLC analysis of the crude powder/extract and isolated col-

red fraction (IHBT-dye 4) was performed on Shimadzu LC-20nstrument (Shimadzu Corporation, Kyoto, Japan) equipped withDA detector (CBM 20A) using Purospher®-Star RP-18e column4.6 mm × 250 mm, 5 �m, Merck, Darmstadt, Germany) and Class

able 1luorescence spectral properties of IHBT-dye 4 and ANS with different proteins.

Proteins Emission maximaa (nm)IHBT-dye 4

1st 2nd

�em Intensity (a.u) �em Intensity (

Without protein 538e 102.9 760 34.9

BSA 531e 99.9 760 38.5

Lysozyme 529 136.4 761e 424.3

Trypsin 527 111.0 760e 326.0

Chymotrypsin 532e 134.5 760 43.5

Pepsinc 538e 104.7 760 21.1

a Emission wavelengths of maximum fluorescence intensity at excitation wave length o50 mM of phosphate buffer having pH 7.0, 50 mM of Tris–HCl buffer having pH 7.4) of pr

b Emission wavelength of maximum fluorescence intensity at excitation wavelength of

c A third emission maxima is also observed at 516 nm wavelength with fluorescence ind At �ex = 300.e Shows the wavelength maxima of the emission peak with greater fluorescence intens

d Biomedical Analysis 62 (2012) 96– 104

VP software. Acetonitrile–methanol (95:5, v/v) (solvent A) andwater–acetic acid (99.9:0.1, v/v, pH 3.5) (solvent B) were usedas mobile phase with a linear gradient elution profile as follows:0–15 min, 20% A; 15–25 min, 50% A; 25–30 min, 70% A; 30–40 min,100% A. The flow rate was kept at 0.8 ml/min [21]. The detectionwavelength was set at 290 nm. The column temperature was 30 ◦C,and the injection volume of samples was 20 �l.

2.10. Hydrolysis of IHBT-dye 4

Acidic hydrolysis of above IHBT-dye 4 was performed in wateras described below: 50 mg dye was added to 10 ml of 2 N HCl andthe solution was refluxed for 6 h. The contents were extracted withethyl acetate and organic layer was washed with water till neu-tral pH is obtained. The resulting ethyl acetate part was dried oversodium sulfate and concentrated in vacuo to obtain a solid productwhich was subjected to HPLC analysis as discussed above.

3. Results and discussion

Initially, crude extract of R. emodi (desert, powder form), chryso-phanol, emodin and the water soluble colored fraction preparedfrom R. emodi extract (coded as IHBT-dye 4) were subjected toexperiments with lysozyme. The results obtained for crude extract(probably has some constituents which can act as a quencher),chrysophanol and emodin indicated either complete absence or avery lesser interaction with proteins. However, the interaction ofproteins with IHBT-dye 4 resulted in enhancement of protein flu-orescence intensity. Thus, dye fraction (IHBT-dye 4) was subjectedfor detailed spectroscopic studies.

3.1. Fluorescence properties of the dyes in the free form and inpresence of protein

IHBT-dye 4 shows two emission maxima at 530 nm and 760 nm(second order scattering peak) in the absence of protein whenexcited at 380 nm (Table 1). In the presence of proteins, a small blueshift of 2–5 nm was observed for the emission maxima at 530 nm(except pepsin where red shift was noticed) with concomitantincrease in fluorescence intensity, whereas other emission maxima(760 nm) remained unchanged. ANS shows two emission maximaat 525 nm and 691 nm (second order scattering peak) in the absenceof protein when excited at 345 nm.

A few representative spectra of different dyes (IHBT-dye 4 andANS) alone and in the presence of proteins are shown in Fig. 1Aand B. It is evident from Fig. 1A and B that both the intensity andposition of the fluorescence emission spectra of ANS and dye are

Emission maximab (nm)ANS

1st 2nd

a.u) �em Intensity (a.u) �em Intensity (a.u)

525e 140.9 691 36.2474e 177.2 691 7.6523 109.7 691 175.3e

529e 120.4 693 55.5525e 119.4 693 43.59354d,e 227.2 601 150.1

f 380 nm for IHBT-dye 4 in water (two maxima was observed). IHBT-dye 4 in bufferotein have almost same emission wave length values.345 nm for ANS, excitation and emission slit width for all experiments set to 10 nm.tensity of 98.6 a.u.

ity.

N. Sharma et al. / Journal of Pharmaceutical and Biomedical Analysis 62 (2012) 96– 104 99

Fig. 1. (A) Representative spectra of IHBT-dye 4 with trypsin, chymotrypsin and lysozyme; (B) representative spectra of ANS with trypsin, chymotrypsin and lysozyme.Excitation wavelengths are 380 of dye 4 and 345 for ANS.

Table 2Denaturation temperatures of proteins in presence of extrinsic dyes.

Denaturation temperature Tm (◦C)

Proteins pH IHBT-dye 4 ANS Reported value (◦C)

�ex �emm Tm (◦C) �ex �emm Tm (◦C)Scanned Scanned

BSA 7.0 380 760 49 345 474 71 52.5 [24]Lysozyme 7.4 380 761 79 345 691 55 77.0 [25]Trypsin 7.0 380 760 49 345 693 64 55.0 [26]Chymotrypsin 7.0 380 760 54 345 693 55 53.6 [27]Pepsin 7.4 380 760 47 300 354 65 52.0 [28]

Fig. 2. (a) Intrinsic tryptophan fluorescence intensity vs. temperature profile of BSA; (b) extrinsic ANS fluorescence intensity vs. temperature profile of ANS-BSA solution(50:1 molar ratio); (c) extrinsic fluorescence intensity vs. temperature profile of dye 4–BSA solution. Excitation and emission wavelength used for different fluorophores aregiven in Table 2.

100 N. Sharma et al. / Journal of Pharmaceutical and Biomedical Analysis 62 (2012) 96– 104

F nsity (p tempf

ss

tetsaec(boac

Fsg

ig. 3. (a) Lysozyme fluorescence emission spectra at excitation of �ex 295 nm, Interofile of IHBT-dye 4–lysozyme solution; (c) extrinsic ANS fluorescence intensity vs.or different fluorophores are given in Table 2.

ensitive to changes in the dye environment and, to the proteintructure.

The emission maxima of ANS do not change in presence of pro-eins except in presence of BSA and pepsin (Table 1). For BSA,mission maxima at 525 nm shifts to 474 nm. The emission spec-ra of pepsin–ANS complex at excitation wavelength of 300 nmhow an extra band at 354 nm of high fluorescence intensitypart from 525 and 691 nm bands. (Supplementary Fig. S4). Inter-stingly, IHBT-dye 4 seems to show poor to no response withhymotrypsin, pepsin and BSA while it shows a good responseenhancement in fluorescence signal of the longer wavelength

and) with lysozyme and trypsin (Table 1). The different responsef dye to different proteins is probably related to the solventccessible surface area of the proteins. NACCESS was used to cal-ulate the solvent accessible surface area (SASA) of these five

ig. 4. (a) Intrinsic tryptophan fluorescence intensity vs. temperature profile of trypsin;olution; (c) extrinsic fluorescence ANS intensity vs. temperature profile of ANS–trypsiniven in Table 2.

a.u.) vs. wavelength (nm); (b) extrinsic dye 4 fluorecsnce intensity vs. temperatureerature profile of ANS-lysozyme solution. Excitation and emission wavelength used

proteins. The sum of SASA of all non-polar residues varies asBSA > pepsin > chymotrypsin > trypsin > lysozyme. The fact that thedye-4 shows fluorescence enhancement only with proteins of lownon-polar SASA points towards the role of exposed non-polar sur-face patches in the quenching of the fluorescence.

3.2. Thermal denaturation experiments

All the thermal denaturation experiments were monitored byobserving the change in fluorescence intensity as a function oftemperature. Temperature was varied between 20 ◦C and 99 ◦C.

Concentrations of dye used were similar as mentioned above.The protein concentration was 10 �M in 3 ml of respective buffer.Intrinsic tryptophan fluorescence intensity of proteins decreasedwith increase in the temperature. Recorded emission maxima and

(b) extrinsic dye 4 fluorescence intensity vs. temperature profile of dye 4–trypsin solution. Excitation and emission wavelength used for different fluorophores are

N. Sharma et al. / Journal of Pharmaceutical and Biomedical Analysis 62 (2012) 96– 104 101

F hymo4 e prod

Tropeottoa

Fsg

ig. 5. (a) Intrinsic tryptophan fluorescence intensity vs. temperature profile of c–chymotrypsin solution; (c) extrinsic ANS fluorescence intensity vs. temperaturifferent fluorophores are given in Table 2.

m (denaturation temperature) are mentioned in Table 2. The fluo-escence intensity of ANS and dye in presence of proteins was alsobtained as a function of temperature. Thermal experiments wereerformed with excitation wavelength at maximum fluorescencemission of the dye, and emission wavelength at emission maximaf both bands of dye–protein mixture. However, Fig. 2 shows onlyhe change in emission maxima of the band which is most sensitive

o the changes in environment of proteins. For example, in the casef BSA denaturation, the thermal denaturation profile was shownt the emission wavelength of 760 nm rather at 531 nm since the

ig. 6. (a) Intrinsic tryptophan fluorescence intensity vs. temperature profile of pepsin;olution; (c) extrinsic ANS fluorescence intensity vs. temperature profile of ANS-pepsiniven in Table 2.

trypsin; (b) extrinsic dye 4 fluorescence intensity vs. temperature profile of dyefile of ANS–chymotrypsin solution. Excitation and emission wavelength used for

760 nm band is more sensitive to the change in the environment(Supplementary Fig. S5). Tm thus obtained is also given in Table 2.

In the absence of any extrinsic fluorophore, BSA and chy-motrypsin show a typical two-state thermally induced unfoldingwith a mid-point of denaturation (Tm) at 55 ◦C (pH 7.0) and 52 ◦C,respectively, whereas lysozyme, trypsin and pepsin do not show atypical sigmoidal thermally induced unfolding, making it impossi-

ble to get the mid point transition temperature.

As seen from Table 2, Tm obtained for proteins with IHBT-dye4 is similar to the one obtained by other methods which is not

(b) extrinsic dye 4 fluorescence intensity vs. temperature profile of dye 4–pepsin solution. Excitation and emission wavelength used for different fluorophores are

102 N. Sharma et al. / Journal of Pharmaceutical an

tbrI

in temperature when they were in solution with BSA, pepsin

Fig. 7. Fluorescence spectra of IHBT-dye 4 in ethanol.

he case with ANS. The binding of ANS increases the thermal sta-ility of BSA. At saturation condition (50:1 of ANS: protein molaratio), Tm increases by 16 ◦C as compared with free protein (Fig. 2).n case of IHBT-dye 4, dye–BSA complex showed a mid-point

Mi

(a)

(b)

(c)

0 5 10 15 20

mAU

0

500

1000

1500

2000

2500

3000

Emodin

Chry

M

0 5 10 15 20

mAU

0

500

1000

1500

2000

Chrys

Emod

M

0 5 10 15 20

mAU

0

1000

2000

3000

4000

Fig. 8. (a) HPLC chromatogram of IHBT-dye 4; (b) HPLC chromatogram of crud

d Biomedical Analysis 62 (2012) 96– 104

denaturation (Tm) at 49 ◦C which is close to the denaturation tem-perature of BSA obtained by DSC. Tm of lysozyme was 78 ◦C in thepresence of IHBT-dye 4, similar to that of free lysozyme. However, adecrease in Tm was observed in the presence of ANS. Protein denat-uration temperature of trypsin was extracted to be 49 ◦C in thepresence of IHBT-dye 4, where as it was 64 ◦C in the presence ofANS (also see Supplementary Fig. S6, S7). In the presence of IHBT-dye 4, denaturation temperature of chymotrypsin was recordedto be 54 ◦C, whereas it was 55 ◦C in the presence of ANS (similarto the denaturation temperature obtained by other methods). Inthe presence of IHBT-dye 4, denaturation temperature obtained forpepsin was 47 ◦C, and with ANS mid- point denaturation centeredat 65 ◦C.

Fluorescence intensity of both dyes decreased with an increase

and chymotrypsin. On the other hand, the fluorescence inten-sity of IHBT-dye 4 decreased with an increase in the temperaturewhile that of ANS increased with an increase in the temperature

nutes

25 30 35 40 45

glycosi de

sophanol glycoside

inutes

25 30 35 40 45

ophanol glycosi de

Emodin

Chrysophano l

Physcion

in glycosi de

inutes

25 30 35 40 45

Emodin

Chrysophano l

Physcion

e extract; (c) HPLC chromatogram of IHBT-dye-4 after acidic hydrolysis.

cal an

(Tdi

mtiecdtlii

3

ecesfliitabimpiieecc

dtmoctpa[grcweoda(dtmccss

[

[

[

N. Sharma et al. / Journal of Pharmaceuti

Figs. 3–6) when the dyes are in solution with trypsin and lysozyme.hus, the fluorescence intensity of IHBT-dye 4 always decreased onenaturation whereas the increase and decrease in the fluorescence

ntensity of the ANS was dependent on the protein.ANS binds strongly to the native state of BSA, pepsin and chy-

otrypsin since large hydrophobic surface patches are available inhe native state as indicated in the higher SASA of non-polar residuen these proteins. Binding of ANS to hydrophobic patch leads tonhancement of the fluorescence intensity. Any conformationalhange in these proteins leads to loss of binding with concomitantecrease in fluorescence intensity. In case of lysozyme and trypsin,he ANS does not bind to the native state of proteins because ofack of hydrophobic patches. Instead, it is denatured state whichnteracts preferentially to the ANS. Thus, the fluorescence intensityncreases on the denaturation of proteins.

.3. Characterization of IHBT-dye 4 by RP-HPLC

Based on thermal denaturation experiments of proteins in pres-nce of dyes, IHBT-dye 4 seems to be a good candidate for theharacterization of protein denaturation temperature. For this, theffect of change in polarity of the solvent on the fluorescence emis-ion spectra of IHBT-dye 4 was studied. It was observed that theuorescence intensity of IHBT-dye-4 decreased with the increase

n the polarity of solvent i.e. low quantum yield of dye was recordedn water in comparison to organic solvent (Fig. 7). Interestingly,here is an inverse correlation between the intensities of 534 nmnd 760 nm peaks with changing solvent polarity. Two bands cane correlated to two populations of dye. Decrease in one will result

n increase of the other. The higher wavelength band at 760 nmay be attributed to second order scattering of dye–ethanol com-

lex due to the polar interaction between dye and ethanol. Increasen the ethanol content decreases this interaction due to the increasen hydrophobic character of solvent resulting into a decrease in themission at higher wavelength with concomitant increase in themission at lower wavelength since it leads to an increase in theoncentration of uncomplexed dye. This data of dye in ethanol isonsistent with the data of protein with the dye.

Both the Rheum extract and the dye prepared from it (IHBT-ye 4) were subjected to HPLC analysis for an insight into theype of compounds responsible for protein interaction. The chro-

atogram obtained for the dye showed only few peaks in the regionf 12–25 min (Fig. 8a) in contrast to the chromatogram obtained forrude extract (Fig. 8b); showing a large number of peaks spread overhe entire polar to non-polar region. The UV maxima values for theeaks in the region of 12–25 min were recorded at 225, 257, 280nd 420 nm which is characteristic of anthraquinone derivatives17]. Many reports have indicated Rheum to possess anthraquinonelycosides, especially of emodin and chrysophanol [17,18] in thisegion. Thus, the identity of these peaks could be assigned to aboveompounds, which was confirmed by comparing some of the peaksith available standard compounds i.e. chrysophanol glycoside and

modin glycoside (Fig. 8a). Further confirmation of the presencef glycoside group was done by disappearance of the above peaksuring acid hydrolysis of the dye (Fig. 8c). Among all the peaksppearing in non-polar region, three were identified as emodin1), chrysophanol (2) and physcion (3) when compared with stan-ard marker compounds. In the light of above, it is presumed thathe anthraquinone glycosides present in the IHBT-dye-4 might be

ainly responsible for protein interactions; thus bringing about a

hange in their conformation (measured as change in the fluores-ence intensity of the dye). These findings corroborated well withome earlier studies on protein binding of anthraquinone glyco-ides [22,23].

[

[

d Biomedical Analysis 62 (2012) 96– 104 103

4. Conclusions

The research work described herein indicates a promising appli-cation of natural dye isolated from R. emodi for thermal shift assay;by exploiting the fluorescence properties of anthraquinone deriva-tives for their interactions with relevant target proteins. The dyehas several characteristics that are particularly useful for the studyof proteins such as enhanced solubility in water and good sensi-tivity to conformational changes of protein. The dye binds weaklyto proteins and thus does not affect the overall stability of pro-teins. These findings would open a new arena in the applicabilityof natural dyes towards replacement of carcinogenic syntheticdyes.

Contribution to the field

This study is the first report where natural fractions/dyes(anthraquinone derivatives) have been explored in the area of pro-tein binding, revealing the use of dye as an extrinsic probe todetermine the stability of proteins. We are of the view that ourfindings would open a new arena in the applicability of naturaldyes towards replacement of some carcinogenic synthetic dyes.

Acknowledgements

N. Sharma and R. Kumar are indebted to CSIR New Delhi forthe award of research fellowships. The authors are grateful to CSIRDelhi for financial support (IHBT Nos. MLP0008 and COR003), andthe Director, CSIR-IHBT, Palampur, for his kind cooperation andencouragement. S. Deep is grateful to CSIR for the financial sup-port and Department of Science and Technology (DST), Delhi forfunding of Cary Eclipse Fluorimeter.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jpba.2011.12.017.

References

[1] A. Downham, P. Collins, Colouring our foods in the last and next millennium,Int. J. Food Sci. Technol. 35 (2000) 5–22.

[2] T.S. Nayar, S. Binu, P. Pushpagandhan, Uses of plants and products in traditionalIndian mural paintings, Econ. Bot. 53 (1999) 41–50.

[3] L.G. Angelini, A. Bertoli, S. Rolandelli, L. Pistelli, Agronomic potential of Resedaluteola L. as new crop for natural dyes in textiles production, Ind. Crops Prod.17 (2003) 199–207.

[4] J.K. Kumar, A.K. Sinha, Resurgence of natural colourants: a holistic view, Nat.Prod. Res. 18 (2004) 59–84.

[5] R. Bhuyan, C.N. Saikia, Isolation of colour components from native dye-bearingplants in northeastern India, Bioresour. Technol. 96 (2005) 363–372.

[6] G. Brian, Doing what comes naturally in the dye house, J. Soc. Dyers Color. 114(1998) 4–7.

[7] E.Y. Chi, S. Krishnan, T.W. Randolph, J.F. Carpenter, Physical stability of pro-teins in aqueous solution: mechanism and driving forces in nonnative proteinaggregation, Pharm. Res. 20 (2003) 1325–1336.

[8] M.C. Manning, K. Patel, R.T. Borchardt, Stability of protein pharmaceuticals,Pharm. Res. 6 (1989) 903–918.

[9] A. Hawe, M. Sutter, W. Jiskoot, Extrinsic fluorescent dyes as tools for proteincharacterization, Pharmaceut. Res. 25 (2008) 1487–1499.

10] L.D. Kapoor, Handbook of Ayurvedic Medicinal Plants, CRC Press, Boca Raton,FL and London, 1990, p. 487.

11] L. Krenn, R. Pradhan, A. Presser, G. Reznicek, B. Kopp, Anthrone C-glucosidesfrom Rheum emodi, Chem. Pharm. Bull. 52 (2004) 391–393.

12] T.R. Kelly, N.S. Chandrakumar, N. Walters, J. Blancaflor, Practical procedurefor the isolation of emodin and chrysophanol, J. Org. Chem. 48 (1983)3573–3574.

13] Y. Li, X. Yao, J. Jin, X. Chen, Z. Hu, Interaction of rhein with human serum albu-min investigation by optical spectroscopic technique and modeling studies,Biochim. Biophys. Acta 1774 (2007) 51–58.

14] P. Sevilla, J.M. Rivas, F. García-Blanco, J.V. García-Ramos, S. Sánchez-Cortés,Identification of the antitumoral drug emodin binding sites in bovine serum

1 cal an

[

[

[

[

[

[

[

[

[

[

[

[

[aggregation of �-chymotrypsin: role of hydrophobic and electrostatic interac-

04 N. Sharma et al. / Journal of Pharmaceuti

albumin by spectroscopic methods, Biochim. Biophys. Acta 1774 (2007)1359–1369.

15] J.Q. Gao, Y.W. Guo, J. Wang, X.D. Jin, Z.Q. Wang, T.T. Fan, K. Li, Y.N. Xu, Spec-troscopic analysis of the interactions of anthraquinone derivatives (alizarin,alizarin-DA and alizarin-DA-Fe) with bovine serum albumin (BSA), J. SolutionChem. 40 (2011) 876–888.

16] A.K. Sinha, U.K. Sharma, A. Sharma, N. Sharma, A process for the prepara-tion of phenolic rich crystalline and non-hygroscopic natural colors from plantextracts, patent application WO/2010/109286.

17] S.C. Verma, N.P. Singh, A.K. Sinha, Determination and locational variations inthe quantity of hydroxyanthraquinones and their glycosides in rhizomes ofRheum emodi using high-performance liquid chromatography, J. Chromatogr.A 1097 (2005) 59–65.

18] N.P. Singh, A.P. Gupta, A.K. Sinha, P.S. Ahuja, High-performance thin layerchromatography method for quantitative determination of four majoranthraquinone derivatives in Rheum emodi, J. Chromatogr. A 1077 (2005)202–206.

19] I. Kubo, Y. Murai, I. Soediro, S. Soeetarno, S. Sastrodihardjo, Cytotoxicanthraquinones from Rheum pulmatum, Phytochemistry 31 (1992) 1063–1065.

20] K. Saini, U. Ahluwalia, S. Deep, Determination of heat capacity of unfolding formarginally stable proteins from a single temperature induced protein unfoldingprofile, Thermochim. Acta 506 (2010) 28–33.

21] S. Malik, N. Sharma, U.K. Sharma, N.P. Singh, S. Bhushan, M. Sharma, A.K. Sinha,P.S. Ahuja, Qualitative and quantitative analysis of anthraquinone derivatives

[

d Biomedical Analysis 62 (2012) 96– 104

in rhizomes of tissue culture-raised Rheum emodi Wall Plants, J. Plant Physiol.167 (2010) 749–756.

22] S. Eksborg, H. Ehrsson, B. Ekqvist, Protein binding of anthraquinone glycosides,with special reference to adriamycin, Cancer Chemother. Pharmacol. 10 (1982)7–10.

23] K. Toshima, Nonnatural glycosyl anthraquinones as DNA binding and photo-cleaving agents, Topics Curr. Chem. 282 (2008) 285–298.

24] C. Giancola, C. De Sena, D. Fessas, G. Graziano, G. Barone, DSC studies on bovineserum albumin denaturation effects of ionic strength and SDS concentration,Int. J. Biol. Macromol. 20 (1997) 193–204.

25] S. Singh, J. Singh, Effect of polyols on the conformational stability and bio-logical activity of a model protein lysozyme, AAPS PharmSciTech 4 (2003) 42(1–9).

26] A.M. Santos, M.A. Santana, F.T. Gomide, A.A. Miranda, J.S. Oliveira, F.A. Boas,A.B. Vasconcelos, M.P. Bemquerer, M.M. Santoro, Physical–chemical character-ization and stability study of alpha–trypsin at pH 3.0 by differential scanningcalorimetry, Int. J. Biol. Macromol. 42 (2008) 278–284.

27] N.R. Ghaleh, H. Ramshini, A.E. Habibi, A.A.M. Movahedi, M.N. Gorgani, Thermal

tions, Biophys. Chem. 132 (2008) 23–32.28] P.L. Privalov, P.L. Mateo, N.N. Khechinashvili, V.M. Stepanov, L.P. Revina, Com-

parative thermodynamic study of pepsinogen and pepsin structure, J. Mol. Biol.152 (1981) 445–464.