list of publications -...

Publications and Conferences

204

List of Publications

1. Study of the interaction between fluoxetine hydrochloride and bovine

serum albumin in the imitated physiological conditions by

multi-spectroscopic methods.

K. Umesha, J. Seetharamappa, S. K. Shankara

Journal of Luminescence 130 (2010) 211-216.

2. Interaction of Bioactive Coomassie Brilliant Blue G with Protein: Insights

from Spectroscopic Methods.

K. Umesha, S. K. Shankara, J. Seetharamappa

Scientia Pharmaceutica 78 (2010) 869-880.

3. Probing the binding of fluoxetine hydrochloride to human serum albumin

by multispectroscopic techniques.

K. Umesha, J. Seetharamappa, S. K. Shankara

Spectrochimica Acta Part A 75 (2010) 314-319.

4. The effect of anti-tubercular drug, ethionamide on the secondary structure

of serum albumins: A biophysical study.

K. Umesha, A. K. Veerendra Kumar, J. Seetharamappa

Journal of Pharmaceutical and Biomedical Analysis 59 (2012) 102-108.

Publications and Conferences

205

Conferences/Seminars/Workshops Participated

1. Presented a paper entitled “Mechanistic and conformational studies of the

interaction of nortriptyline hydrochloride with protein” at the 45th

Annual

Convention of Chemists 2008 and International Conference on “Recent

Advances in Chemistry, held at the Department of Studies in Chemistry,

Karnatak University, Dharwad, during 23rd

- 27th

November 2008.

2. Presented a paper entitled “Interaction studies of fluoxetine with BSA; A

spectroscopic approach” at the 27th

Annual Conference of Indian Council

of Chemists, held at Gurukul Kangri Vishwavidyalaya, Haridwar, during

26th

- 28th

December 2008.

3. Presented a paper entitled “Probing the binding of coomassie brilliant blue

G to protein by optical spectroscopy under imitated physiological

conditions” at the International Conference on Current Trends in

Chemistry and Biochemistry (ICCTCB-2009), held at Bangalore

University, Bangalore, during 18th

- 19th

December 2009.

4. Presented a paper entitled “Synthesis, Characterization and Biological

activities of coumarin moieties containing fused triazoles and tetrazoles” at

the 29th

Annual Conference of Indian Council of Chemists, held at

Punjab University, Chandigarh, during 19th

- 21st December 2010.

5. Attended the UGC Sponsored National Seminar on “Recent Trends in

Chemistry” at the Department of Studies in Chemistry, Karnatak

University, Dharwad, on 18th

February 2008.

6. Attended the UGC Sponsored National Seminar on “Recent Advances

in Chemistry - 2010” at the Department of Studies in Chemistry, Karnatak

University, Dharwad, on 18th

March 2010.

7. Attended the “Winter School in Crystallography” at the Central

University of Hyderabad, Hyderabad, during 22nd

November - 4th

December

2010.

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Spectrochimica Acta Part A 75 (2010) 314–319

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

journa l homepage: www.e lsev ier .com/ locate /saa

Probing the binding of fluoxetine hydrochloride to human serum albumin bymultispectroscopic techniques

Umesha Katrahalli, Seetharamappa Jaldappagari ∗, Shankara S. KalanurDepartment of Chemistry, Karnatak University, Pavate Nagar, Dharwad 580 003, Karnataka, India

a r t i c l e i n f o

Article history:Received 7 June 2009Received in revised form 6 October 2009Accepted 21 October 2009

Keywords:Human serum albuminFluoxetine hydrochlorideQuenching mechanismBinding constantThermodynamic parameters

a b s t r a c t

The interaction between human serum albumin (HSA) and fluoxetine hydrochloride (FLX) have been stud-ied by using different spectroscopic techniques viz., fluorescence, UV–vis absorption, circular dichroismand FTIR under simulated physiological conditions. Fluorescence results revealed the presence of statictype of quenching mechanism in the binding of FLX to HSA. The values of binding constant, K of FLX-HSAwere evaluated at 289, 300 and 310 K and were found to be 1.90 × 103, 1.68 × 103 and 1.45 × 103 M−1,respectively. The number of binding sites, n was noticed to be almost equal to unity thereby indicatingthe presence of a single class of binding site for FLX on HSA. Based on the thermodynamic parameters,�H0 and �S0 nature of binding forces operating between HSA and FLX were proposed. Spectral resultsrevealed the conformational changes in protein upon interaction. Displacement studies indicated the siteI as the main binding site for FLX on HSA. The effect of common ions on the binding of FLX to HSA wasalso investigated.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Fluoxetine (N-methyl-c-[4-(trifluoromethyl)phenoxy]benzen-epropanamine) (FLX) (Fig. 1) is a derivative of phenoxyphenylpropylamines [1]. It is a psychotropic drug and is employed for thetreatment of premenstrual dysporic disorder. It affects chemicals inthe brain that may become unbalanced and cause depression, panic,anxiety, or obsessive–compulsive symptoms. It is used to treatmajor depressive disorder, bulimia nervosa (an eating disorder)obsessive–compulsive disorder, panic disorder, and premenstrualdysphoric disorder.

Human serum albumin (HSA) is the most abundant protein inblood plasma, accounting for about 60% of the total protein corre-sponding to a concentration of 42 g/l and providing about 80% ofthe osmotic pressure of blood [2]. It consists of 585 amino acidswith 17 tyrosyl residues and one tryptophan residue located inposition 214 (Trp-214) [3,4]. The crystal structure analyses haverevealed that the drug binding sites are located in sub-domains IIAand IIIA [5]. HSA binds to a variety of hydrophobic ligands due toits well-established secondary structure, stability, water solubility,and versatile binding capacity [6–9]. Many drugs and other bioac-tive small molecules bind reversibly to HSA that then function ascarriers [2–10].

Protein binding, playing a potential role in distribution, excre-tion and therapeutic effectiveness, has long been considered one of

∗ Corresponding author. Tel.: +91 836 2215286/29; fax: +91 836 2747884.E-mail address: [email protected] (S. Jaldappagari).

the most important physicochemical characteristics of drugs [11].The investigation of small molecules with respect to albumin bind-ing is of imperative and fundamental importance and has been aninteresting research field in life sciences, chemistry and clinicalmedicine [1–12].

The literature survey revealed that the attempts have not beenmade to investigate the interaction of FLX with HSA. Hence, we havecarried out the detailed binding studies of FLX with HSA by differ-ent spectroscopic techniques viz., fluorescence, circular dichroism,UV–vis absorption and FTIR.

2. Experimental

2.1. Materials

Essentially fatty acid free human serum albumin was obtainedfrom Sigma Chemical Company (St. Louis, USA). Fluoxetinehydrochloride was obtained as gift sample from Micro Labs Limited,India. The solutions of FLX and HSA were prepared in 0.1 M phos-phate buffer of pH 7.4 containing 0.15 M NaCl. HSA solution wasprepared based on its molecular weight of 66,000. All other mate-rials were of analytical reagent grade. Millipore water was usedthroughout the study.

2.2. Instrumental

Fluorescence measurements were performed on a spectroflu-orimeter Model F-7000 (Hitachi, Japan) equipped with a 150 WXenon lamp and a slit width of 5/5 nm. A 1.00 cm quartz cell was

1386-1425/$ – see front matter © 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.saa.2009.10.031


U. Katrahalli et al. / Spectrochimica Acta Part A 75 (2010) 314–319 315

Fig. 1. Structure of fluoxetine hydrochloride.

used for measurements. The CD measurements were made on aJASCO-715 spectropolarimeter (Tokyo, Japan) using a 0.1 cm cell at0.2 nm intervals, with 3 scans averaged for each CD spectrum inthe range of 200–250 nm. The absorption spectra were recordedon a double beam CARY 50-BIO UV–vis spectrophotometer (Var-ian, Australia) equipped with a 150 W Xenon lamp and a slit widthof 5 nm. A quartz cell of 1.00 cm was used for measurements. FTIRspectra were acquired on a Thermo Nicolet-5700 FTIR spectrometer(Waltham, MA, USA).

2.3. Procedures

2.3.1. Fluorescence and UV–vis absorption spectral studiesBased on preliminary investigations, the concentration of HSA

was kept constant at 2.5 �M and that of the drug was varied from10 to 100 �M. The fluorescence intensity was recorded at 340 nmupon excitation at 280 nm. The investigations were carried out atdifferent temperatures (289, 300 and 310 K).

The UV–vis spectra were obtained by scanning the solution onthe spectrophotometer with the wavelength range of 250–300 nm.The operations were carried out at room temperature.

2.3.2. Circular dichroism (CD) studiesThe CD measurements of HSA in the presence and absence of FLX

were made in the range of 200–250 nm. A stock solution of 250 �MHSA was prepared in 0.1 M phosphate buffer of pH 7.4 containing0.15 M NaCl. The HSA to drug concentration was varied (1:2, 1:5,1:8 and 1:10) and the CD spectrum was recorded.

2.3.3. FTIR measurementsThe FTIR spectra of HSA in the presence and absence of

drug, FLX at room temperature were recorded in the range of1500–1700 cm−1. A stock solution of 250 �M of HSA and 1000 �Mof drug was prepared in 0.1 M phosphate buffer of pH 7.4 containing0.15 M NaCl.

2.3.4. Displacement studiesThe displacement experiments were performed using different

site probes viz., warfarin, ibuprofen and digitoxin for sites I, II andIII, respectively [13] by keeping the concentration of HSA and theprobe constant (2.5 �M each). The fluorescence quenching titrationwas used as before to determine the binding constant of FLX–HSAsystem in the presence of these site probes.

2.3.5. Effect of common ionsThe effect of some common ions was carried out using K+, Cu2+,

Zn2+, Ni2+, Co2+ and Ca2+ on FLX–HSA interactions. For this, the fluo-rescence spectra of FLX–HSA system were recorded in the presenceof the above ions at 340 nm upon excitation at 280 nm. The overallconcentration of HSA and that of the common ions were fixed at2.5 �M.

Fig. 2. Fluorescence spectra of HSA in the presence of FLX. HSA concentration wasmaintained at 2.5 �M and FLX concentration was at 0 (a), 10 (b), 20 (c), 30 (d), 40(e), 50 (f), 60 (g) and 70 �M (h).

3. Results and discussion

3.1. Fluorescence quenching spectra

The fluorescence quenching spectra of HSA with varying con-centrations of FLX at 27 ◦C are shown in Fig. 2. FLX was observed toquench the fluorescence of HSA and noticeable change in �max oftryptophan fluorescence in HSA occurred. This indicated that theFLX interacted with HSA. Further, the maximum emission wave-length of HSA was shifted from 331 to 328 nm indicating that theprotein was placed in a more hydrophobic environment after theaddition of FLX [14]. Thus, the microenvironment of tryptophanresidue was changed after the addition of FLX. It is known that thebinding of small molecules to HSA could induce the conformationalchanges in HSA, because the intramolecular forces involved tomaintain the secondary structure could be altered [15]. For macro-molecules, the fluorescence measurements can give informationof the binding of small molecules to protein, such as the bind-ing mechanism, binding mode, binding constants, binding sites,intermolecular distances. HSA has three intrinsic fluorophores viz.,tryptophan, tyrosine and phenylalanine. In fact, the intrinsic fluo-rescence of HSA is almost contributed by tryptophan alone, becausephenylalanine has a very low quantum yield and the fluorescence oftyrosine is almost totally quenched if it is ionized, or near an aminogroup, a carboxyl group, or a tryptophan. This viewpoint was wellsupported by the experimental observations made by Sulkowska[16]. That is, the change in intrinsic fluorescence intensity of HSAis that of fluorescence intensity of tryptophan residue when smallmolecules are bound to HSA. The binding of FLX may change thefluorescence characteristics of protein.

The fluorescence quenching of protein by the drug is usuallyanalyzed by the Stern–Volmer equation [17]:

F0

F= 1 + KSV[Q ] = 1 + Kq�0[Q ] (1)

where F0 and F are respectively, the fluorescence intensity ofprotein in the absence and presence of the quencher, KSV is theStern–Volmer quenching constant, Kq is the quenching rate con-stant of biomolecule and �0 is the average life-time of biomoleculewithout the quencher. Obviously,

KSV = Kq�0 (2)

The slope of linear regressions of Stern–Volmer plot (Fig. 3)yielded the values of KSV (Table 1). Using the reported value of �0for a biopolymer of 10−8 s [18], the values of Kq were calculated andare listed in Table 1.


316 U. Katrahalli et al. / Spectrochimica Acta Part A 75 (2010) 314–319

Fig. 3. The Stern–Volmer plot for the quenching of HSA by FLX at 289 K (�), 300 K(�) and 310 K (�).

Static quenching and dynamic quenching are the two quench-ing mechanisms which are operating in the interaction process.These are differentiated according to the variation of the quenchingconstant with varying temperatures. The quenching rate constantsdecrease with increasing temperature for static quenching, butthe reverse effect is observed for the case of dynamic quenching[19]. In the present study, the Kq values decreased with increasein temperature. Hence, the quenching mechanism was proposedto be static. The maximum scatter collision quenching constant,Kq of various quenchers with the biopolymer was reported to be1 × 1010 L mol−1 s−1 [17]. In the present work, the order of magni-tude of the quenching constant was noticed to be 1011. With this,we proposed that the quenching was not initiated by dynamic col-lision, but originated from the formation of a complex, FLX–HSA.The static quenching equation can be expressed as shown below[20]:

log(F0 − F)F

= log K + n log[Q ] (3)

where K is the binding constant and n is the number of binding sites.From the plot of log [(F0 − F)/F] versus log [Q], the values of K and nwere obtained from the intercept and slope, respectively. The val-ues of K were found to be 1.90 × 103, 1.68 × 103 and 1.45 × 103 M−1

and those of n were noticed to be 0.99, 1.00 and 1.01 respectively,at 289, 300 and 310 K. The values of binding sites close to unityindicated that there was only one independent class of binding siteon HSA for FLX. The decreased binding constant with increase intemperature resulted possibly due to the reduction of the stabilityof FLX–HSA complex.

3.2. Determination of the acting forces

The acting forces between a drug and a biomolecule may includehydrogen bond, van der Waals forces, electrostatic forces andhydrophobic interaction forces [20,21]. Because the temperatureeffect is very small, the enthalpy change for the interaction canbe regarded as a constant provided the temperature range is toowide. The thermodynamic parameters, enthalpy change (�H0) andentropy change (�S0) of a reaction are important for confirming

binding mode. For this purpose, the temperature dependence of thebinding constant was studied. HSA did not undergo any structuraldegradation at the selected temperatures (289, 300 and 310 K).

Ross and Subramanian [22] have characterized the nature ofbinding forces that are operative in drug–protein interactions basedon the signs and magnitudes of above thermodynamic parame-ters. These thermodynamic parameters were calculated using thefollowing equations:

log K = −�H0

2.303RT+ �S0

2.303R(4)

and

�G0 = −2.303RT log K (5)

From the slopes and intercepts of the plot of log K versus 1/T, thevalues of �H0 and �S0 were calculated and are shown in Table 1.For typical hydrophobic interactions, both �H0 and �S0 are posi-tive, while these are negative for van der Waals forces and hydrogenbond formation in low dielectric media [22]. Further, the specificelectrostatic interaction between ionic species in an aqueous solu-tion is characterized by positive �S0 value and negative �H0 value(small). In the present study, the main source of �G0 value wasderived from a large contribution of �S0 term with a little contri-bution from �H0 factor. So, the main interaction between FLX andHSA was believed to be hydrophobic. However, the electrostaticinteraction could not be ruled out. Negative �G0 values revealedthe spontaneity of the interaction process.

3.3. The distance between the FLX and HSA

The fluorescence studies indicated the formation of FLX–HSAcomplex. The fluorescence of HSA mainly comes from tryptophan(Trp-214). So, the distance between Trp-214 and bound FLX couldbe estimated based on the Förster’s non-radiative energy trans-fer theory (FRET) [23]. The non-radiative energy transfer takesplace from the donor (protein) to acceptor (drug) if the emissionspectrum of donor overlaps with the absorption spectrum of theacceptor. According to FRET, the energy transfer efficiency, E isrelated not only to the distance (r0) between acceptor and donor,but also to the critical energy transfer distance (R0), that is

E = 1 − F

F0= R6

0

R60 + r6

0

(6)

where R0 is a characteristic distance, called the Förster distance orcritical distance, at which the efficiency of the transfer is 50%. Thevalue of R0 is calculated using the following equation:

R60 = 8.8 × 10−25 K2N−4˚J (7)

where K2 is the spacial orientation factor describing the relativeorientation in the space of the transition dipole of the donor andacceptor, N is the refractive index for the medium, Ф is the fluores-cence quantum yield of the donor in the absence of the acceptor andJ is the overlap integral between the emission spectrum of donorand the absorbance spectrum of acceptor. J can be given by

J = ˙F(�)ε(�)�4��

˙F(�)��(8)

Table 1KSV, Kq and thermodynamic parameters of FLX–HSA system.

Temperature (K) KSV (M−1) Kq (L mol−1 s−1) �G0 (kJ mol−1) �H0 (kJ mol−1) �S0 (J mol−1 K−1)

289 1.87 × 103 1.87 × 1011 −18.2300 1.64 × 103 1.64 × 1011 −18.5 −9.53 29.85310 1.36 × 103 1.36 × 1011 −18.8



Fig. 4. The overlap of the fluorescence spectrum of HSA (a) with the absorptionspectrum of FLX (b).

where F(�) is the fluorescence intensity of the fluorescence donorat wavelength �, ε(�) is the molar absorption coefficient of theacceptor at wavelength, � and its unit is cm−1 mol−1 L.

The overlap of the absorption spectrum of FLX with the emissionspectrum of HSA is shown in Fig. 4. The value of J could be calculatedby integrating the spectrum shown in Fig. 4 and was observed tobe 5.16 × 10−16 cm3 L mol−1. For ligand–HSA interaction, K2 = 2/3,N = 1.36 and Ф = 0.118 [24]. The values of R0, r0 and E were calcu-lated to be 2.23, 2.73 and 0.23 nm, respectively. Larger HSA–FLXdistance, r0 compared to that of R0 observed in the present studyalso revealed the presence of static quenching mechanism betweendrug and protein [25,26]. The value of r0 < 8 nm [27] also indicatedthat the energy transfer has occurred from HSA to FLX with highpossibility.

3.4. Analysis of HSA conformation after FLX binding

In order to examine the conformation of HSA upon binding withFLX, we have employed three spectroscopic methods viz., UV–visabsorption, FTIR and circular dichroism. UV–vis absorption study isa simple method and applicable to explore the structural changes[20] and to know the complex formation [28]. UV–vis absorp-tion spectra of FLX, HSA and FLX–HSA are shown in Fig. 5. It wasobserved that the absorption of HSA increased regularly with theincreasing amounts of drug and the maximum peak position ofFLX–HSA complex was noticed to be shifted slightly towards thehigher wavelength region. This indicated the change in polarityaround the tryptophan residue and the change in peptide strandof HSA molecule and hence changes in hydrophobicity [29,30]. So,the binding of FLX to HSA molecule might lead to changes in confor-mation of protein [31]. This also indicated that the peptide strands

Fig. 5. UV absorption spectra of HSA–FLX system: HSA was maintained at 2.5 �M(a); in HSA–FLX, the concentrations of FLX were maintained at 10, 20, 30, 40, 50, 60,70, 80, 90 and 100 �M (b–k); FLX only (10 �M) (x).

Fig. 6. CD spectra of HSA–FLX system. HSA concentration was fixed at 2.5 �M (a);in HSA–FLX system, the FLX concentration was at 5 (b), 12.5 (c), 20 (d) and 25 �M(e).

of protein molecule extended further more upon the addition ofFLX to HSA [29].

CD study was employed to monitor the conformational changesin the protein. The CD spectra of HSA in the presence and absenceof FLX are shown in Fig. 6. The CD spectrum of HSA exhibited twonegative bands in the UV region at 208 and 222 nm, which arecharacteristic of an �-helical structure of protein [32]. By using thefollowing equation, the CD results were expressed in terms of meanresidue ellipticity (MRE) in deg cm2 dmol−1.

MRE = observed CD (m deg)CPnl × 10

(9)

where CP is the molar concentration of the protein, n is the numberof amino acid residues and l is the path length. The �-helical con-tents of free and combined HSA were calculated from MRE valuesat 208 nm using the following equation [32]:

˛-Helix (%) = −MRE208 − 400033, 000 − 4000

× 100 (10)

where MRE208 is the observed MRE value at 208 nm, 4000 is theMRE of the �-form and random coil conformation cross at 208 nmand 33,000 is the MRE value of a pure �-form at 208 nm. Using theabove equation, the �-helicity in secondary structure of HSA wascalculated and was found to be decreased from 59.63% in free HSAto 43.86% in FLX–HSA complex. This was indicative of the loss of�-helical content upon interaction. The CD spectra of HSA in thepresence and absence of FLX were observed to be similar in shape,indicating thereby that the structure of HSA was predominantly�-helical even after binding [33].

Further evidence regarding the interaction of FLX with HSA wasobtained from FTIR results. IR spectrum of the protein exhibits anumber of amide bands, which represent different vibrations ofthe peptide moiety. Of all the amide bands of the peptide group,the single most widely used one in studies of protein secondarystructures is the amide I. The amide I and II peaks occur in theregion of 1600–1700 and 1500–1600 cm−1, respectively. Amide Iband is more sensitive to the changes in protein secondary struc-ture compared to amide II and hence, it is more useful [34–37].In the amide I region, the observed stretching frequency of C Ohydrogen bonded to NH moieties is dependent upon the secondarystructure adopted by the peptide chain. Changes in the structure ofthe protein are reflected by changes in the component band posi-tions of the amide I region [38]. The FTIR spectra of free HSA (Fig. 7a)and its complex with FLX (Fig. 7b) in phosphate buffer solution wererecorded at 299 K. The peak positions of amides I and II were foundto be shifted upon the addition of FLX to HSA. The amide I bandwas shifted from 1670.1 to 1643.1 cm−1, while the amide II peak


318 U. Katrahalli et al. / Spectrochimica Acta Part A 75 (2010) 314–319

Fig. 7. FTIR spectra and difference spectra of HSA: (a) the FTIR spectra of free HSA(subtracting the absorption of the buffer solution from the spectrum of the proteinsolution) and (b) the FTIR difference spectra of HSA (subtracting the absorption of theFLX-free form from that of FLX–HSA bound form); CHSA = 2.5 �M and CFLX = 10 �M.

was shifted from 1554.4 to 1567.8 cm−1. These results indicatedthat the FLX interacted with the C O and CN groups in the proteinpolypeptides. This interaction resulted in the rearrangement of thepolypeptide carbonyl hydrogen-bonding network and finally thereduction of �-helicity in the protein.

3.5. Location of binding site

HSA has a limited number of binding sites for endogenous andexogenous ligands that are typically bound reversibly. The prin-cipal regions of ligand binding sites of albumin are located inhydrophobic cavities in sub-domains IIA and IIIA, which exhibitsimilar chemistry. Sudlow et al. [39] have suggested two maindistinct binding sites (sites I and II) in HSA. Site I has affinity for war-farin, phenylbutazone, etc. while site II shows affinity for ibuprofen,flufenamic acid, etc. It is reported that digitoxin binding is inde-pendent of sites I and II [40] and binds to site III. In order to knowthe exact binding site for FLX on HSA, the displacement studieswere carried out using different site probes, viz., warfarin, ibupro-fen and digitoxin. For this, emission spectra of ternary mixtures ofHSA, FLX and site probes were recorded separately and the bindingconstant values were calculated. The value K for FLX–HSA withoutthe site probe was observed to be 1.68 × 103 M−1. However, the Kvalues of FLX–HSA in the presence of warfarin, ibuprofen and dig-itoxin were found to be 2.03 × 102, 1.71 × 103 and 1.67 × 103 M−1,respectively. It is evident that the drug competes with warfarin forsite I in the protein. This resulted in decreased binding constant.However, the binding constant values remained almost same inthe presence of ibuprofen and digitoxin. Hence, FLX is most likelybound to the hydrophobic pocket located in sub-domain IIA; thatis to say, Trp-214 is near or within the binding site.

3.6. The effect of common ions on the binding constant

In plasma, there are some metal ions, which can affect the reac-tions of the drugs and serum albumins. These trace metal ions,

Table 2Effect of common ions on binding constant of FLX–HSA.

System Association constant (M−1)

HSA + FLX 1.68 × 103

HSA + FLX + K+ 9.90 × 103

HSA + FLX + Co2+ 9.12 × 103

HSA + FLX + Cu2+ 3.53 × 103

HSA + FLX + Ni2+ 1.48 × 102

HSA + FLX + Ca2+ 1.15 × 102

HSA + FLX + Zn2+ 1.13 × 102

especially the bivalent are essential in the human body and exhibitstructural and functional roles in many biomolecules. It is reported[41] that K+, Cu2+, Zn2+, Ni2+, Co2+ and Ca2+ and other metal ionscan form complexes with serum albumins. Hence, it was felt thatresearch on this aspect was necessary. Under the experimentalconditions, none of the cations gave the precipitate in phosphatebuffer.

The binding constants of FLX–HSA were determined in the pres-ence of various metal ions at 300 K and the corresponding resultsare shown in Table 2. The concentrations of HSA and various metalions were kept constant (2.5 �M), while that of the drug was var-ied from 10 to 100 �M. The binding constant of FLX–HSA systemdecreased slightly in the presence of Ni2+, Ca2+ and Zn2+. This waslikely to be caused by a conformational change in the vicinity ofthe binding site. The decrease in the binding constant value wouldshorten the storage time of the compound in the blood plasma [26].In such cases, there was a need for more doses of drug to achieve thedesired therapeutic effect in the presence of above ions. The bind-ing constant increased slightly in the presence of K+, Co2+ and Cu2+,thereby indicating the strong binding between the drug and HSAand availability of more drug for action. This indicated the require-ment of less dose of drug for desired therapeutic effect. In this way,the trace metal ions influence the binding of drug to protein and inturn the dose limits.

4. Conclusions

The proposed spectroscopic methods are highly sensitive andconvenient to investigate the interactions of drug with proteincompared to classical techniques (gel filtration, dialysis, ultra-centrifugation, etc.) which are laborious, time consuming and attimes the results are not reproducible. The interaction between FLXand HSA was established by fluorescence spectroscopy combinedwith UV–vis, CD and FTIR spectroscopic techniques under simula-tive physiological conditions for the first time. The study showedthat FLX bound to protein most likely at the hydrophobic pocketlocated in sub-domain IIA of site I. The spectral results also revealedthat the binding of FLX to HSA induced a conformational changein HSA, which was further proved by the quantitative analysis ofCD spectral data. The biological significance of this work is evidentsince albumin serves as a carrier molecule for multiple drugs.

Acknowledgements

We are grateful to the Council of Scientific and Indus-trial Research, New Delhi, for financial assistance (No.01(2279)/08/EMR-II). Umesha K. thank the University GrantsCommission, New Delhi, for awarding the meritorious Fellowship.Thanks are also due to the authorities of the Karnatak University,Dharwad, for providing the necessary facilities.

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Journal of Pharmaceutical and Biomedical Analysis 59 (2012) 102– 108

Contents lists available at SciVerse ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

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

The effect of anti-tubercular drug, ethionamide on the secondary structure ofserum albumins: A biophysical study�

Umesha Katrahalli, Veerendra Kumar A. Kalalbandi, Seetharamappa Jaldappagari ∗

Department of Chemistry, Karnatak University, Dharwad 580 003, India


Article history:Received 15 July 2011Received in revised form 22 August 2011Accepted 18 September 2011Available online 22 September 2011

Keywords:Serum albuminEthionamideBinding mechanismEnergy transferSite probesSecondary structure

a b s t r a c t

Serum albumin (SA) is the principal extra cellular protein with higher concentration in the blood plasmaand acts as a carrier for many drugs to different molecular targets. The present work is designed to inves-tigate the mechanism of interaction between the protein and an anti-tubercular drug, ethionamide (ETH)at the physiological pH by different molecular spectroscopic techniques viz., fluorescence, UV absorp-tion, CD and FTIR. The interaction of SA with ETH was studied by following the quenching of intrinsicfluorescence of protein by ETH at different temperatures. The Stern–Volmer quenching constant, bindingconstant and the binding site numbers were calculated from fluorescence results. The results indicatedthe presence of static quenching mechanism in both HSA–ETH and BSA–ETH systems. The distances ofseparation between the acceptor and donor were calculated based on the theory of fluorescence reso-nance energy transfer and were found to be 2.35 nm and 2.18 nm for HSA–ETH and BSA–ETH systems,respectively. The conformational changes in protein were confirmed from UV absorption, CD and FTIRspectral data. Displacement experiments with different site probes revealed that the site I was the mainbinding site for ETH in protein. Effect of some metal ions was also investigated.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Spectroscopic techniques such as fluorescence, UV absorption,FTIR and circular dichroism are useful in the study of interactions ofsmall molecules with protein [1]. Binding of a drug with the albu-min affects its pharmacological effect since only the free fraction ofdrug exhibits therapeutic activity. In the body of an animal, serumalbumin is the main transporting protein which has the ability tobind and transport the various exogenous and endogenous ligandsby forming complexes. A great deal of attention has been paid toinvestigate the interaction of SA with a number of active natural orsynthetic ligands because it has its own importance in clinical andpharmaceutical fields [2]. Among serum albumins, bovine serumalbumin (BSA) and human serum albumin (HSA) are involved intransportation of small ligands including drugs. The structure ofBSA has 76% similarity with that of HSA [3]. HSA has only one tryp-tophan (Trp-214) which is located in sub domain IIA, where as BSAhas two tryptophans moieties (Trp-135 and Trp-214), located insub-domain IA and IIA, respectively [4].

� Part of this work was presented at the XXI Annual Conference of Indian Council ofChemists held at the Punjab University, Chandigarh, India, during 19–21 December2010.

∗ Corresponding author. Tel.: +91 836 2215286/27; fax: +91 836 2747884.E-mail address: [email protected] (S. Jaldappagari).

Tuberculosis continues to be a major worldwide epidemicdisease and drugs such as isoniazid (INH) and rifampicin havehistorically been successful in the treatment of this disease [5].Ethionamide (ETH, Fig. 1), 2-ethylpyridine-4-carbothioamide, acommonly used antibiotic, is used in the treatment of tuberculosis.This drug presents strong bacteriostatic properties against somemycobacteria and is rather more active against isoniazid-resistantmutants [6]. Like INH, this drug is thought to be a prodrug, whichmust be converted to its active form by the bacterial cell. In thepresent study, we have investigated the mechanism of interactionof ETH with protein (HSA and BSA) employing different spectro-scopic methods.

2. Materials and methods

2.1. Materials

HSA (fatty acid free), BSA (Fraction V, approximately 99%; pro-tease free and essentially �-globulin free), warfarin, ibuprofen anddigitoxin were purchased from Sigma–Aldrich Chemical Co., St.Louis, USA. Pure sample of ethionamide was gifted by Medopharm,Bengalooru, India. All the investigations were carried out in 0.1 Mphosphate buffer solution of pH 7.4. All the chemicals used were ofanalytical reagent grade and Millipore water was used throughoutthe experiment.

0731-7085/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.jpba.2011.09.013


U. Katrahalli et al. / Journal of Pharmaceutical and Biomedical Analysis 59 (2012) 102– 108 103

N

NH2S

Fig. 1. Structure of ethionamide (ETH).

2.2. Methods

2.2.1. Fluorescence studiesThe fluorescent measurements were carried out on a Hitachi

Instrument F-7000 spectrofluorometer provided with a 1 cm quartzcell equipped with a 150 W Xenon lamp at 301 K. The fluorescenceintensity of both HSA and BSA at increasing molar ratios of ETH toSA was recorded upon excitation at 280 nm for HSA and at 296 nmfor BSA keeping 5 nm as excitation and emission slit widths. Forthis, the concentration of protein was fixed at 2.5 �M while that ofthe drug was varied from 5 to 50 �M. Fluorescence measurementswere carried out at 294 K, 301 K and 310 K. For this, the instrumentwas thermostatically controlled by a Cyberlab CB2000 circulatingwater bath.

2.2.2. Energy transfer between ETH and proteinThe emission spectrum of protein (each of 2.5 �M) upon exci-

tation at 280 nm for HSA and at 296 nm for BSA was recorded. Theabsorption spectrum of 2.5 �M ETH was also recorded in the wave-length range similar to that of emission spectrum. The overlap ofthese two spectra was used to calculate the energy transfer basedon the Förster’s theory.

2.2.3. Absorption studiesThe absorption measurements of protein were recorded on a

CARY BIO-50 double beam spectrophotometer with a 1 cm quartzcell equipped with a 150 W Xenon lamp at 301 K. Absorbance valuesof protein in the absence and presence of ETH were recorded in therange of 250–350 nm. The concentration of HSA/BSA was fixed at2.5 �M while that of the drug was varied from 5 to 50 �M.

2.2.4. Circular dichroism studiesCircular dichroism measurements were made on a JASCO-810

spectropolarimeter (Tokyo, Japan) using a quartz cell of 0.1 cmwith three scans averaged for each CD spectrum in the range of200–250 nm. The molar ratios of protein to drug concentrationswere maintained at 1:2, 1:4 and 1:6 and CD spectra were recorded.

2.2.5. FTIR spectroscopic measurementsThe infrared spectra of protein solutions were obtained on a

Thermo Nicolet-5700 FTIR spectrophotometer via the attenuatedtotal reflection (ATR) method with resolution of 4 cm−1 and 60scans.

Spectra processing procedure: IR spectra of protein and buffersolutions were recorded separately under similar conditions. Theabsorbance of buffer solution was then subtracted from that ofprotein solution (to get the FTIR spectrum of the protein alone).Later, the spectrum of drug, and protein–drug system (along withbuffer solution) was noted down under similar conditions. Fur-ther, the absorbance of drug solution was subtracted from thatof protein–drug solution to get the FTIR spectrum of protein–ETHcomplex. The subtraction criterion was that the original spectrumof protein solution between 2200 and 1800 cm−1 was not depictedany significant signal in this region [7].

Fig. 2. Fluorescence spectra of HSA (a) and BSA (b) (2.5 �M) in presence of ETH: (a)0, (b) 5, (c) 10, (d) 15, (e) 20, (f) 25, (g) 30, (h) 35, (i) 40, (j) 45 and (k) 50 �M.

2.2.6. Competitive binding studiesThe competitive binding studies were performed using different

competitors, warfarin for site I, ibuprofen for site II and digitoxinfor site III by keeping the concentration of protein and the competi-tor, constant (each of 2.5 �M). The fluorescence quenching titrationwas performed as before to determine the binding constant ofETH–protein in the presence of the above said site probes.


3.1. Fluorescence quenching studies

In order to understand the quenching mechanism operatingbetween ETH and protein, the fluorescence quenching experimentswere performed. The fluorescence spectra of HSA (Fig. 2a) and BSA(Fig. 2b) were recorded in the presence of increasing amounts ofETH, separately. In these spectra, we noticed that the fluorescenceintensity of protein decreased regularly with increasing concen-trations of ETH indicating the interaction between the drug andprotein.

The fluorescence results were preliminarily analyzed using theStern–Volmer equation [8] shown below:(1) F0

F = 1 + KSV[Q] = 1 +kq�0[Q]where F and F0 are the fluorescence intensities with andwithout the quencher, respectively, kq is the bimolecular quenchingrate constant, KSV is the Stern–Volmer quenching constant, �0 is


104 U. Katrahalli et al. / Journal of Pharmaceutical and Biomedical Analysis 59 (2012) 102– 108

Table 1Stern–Volmer quenching constants and modified Stern–Volmer association constants for HSA–ETH and BSA–ETH systems at different temperatures.

System T (K) KSV × 10−4 (L mol−1) Kq × 10−12 (L mol−1 s−1) R2 Ka × 10−4 (L mol−1) R2

HSA294 2.13 ± 0.041 4.26 ± 0.041 0.997 1.01 ± 0.031 0.999301 1.90 ± 0.024 3.80 ± 0.024 0.999 1.33 ± 0.026 0.999310 1.49 ± 0.034 2.98 ± 0.034 0.999 1.54 ± 0.028 0.991

BSA294 2.72 ± 0.011 5.44 ± 0.011 0.999 1.03 ± 0.022 0.999301 2.31 ± 0.036 4.62 ± 0.036 0.999 2.01 ± 0.041 0.999310 2.14 ± 0.023 4.28 ± 0.023 0.999 3.15 ± 0.027 0.999

the average life time of biomolecule without the quencher and itsvalue was reported to be 5 ns [9] and [Q] is the concentration ofthe quencher. The quenching rate constant, kq could be calculatedusing the equation shown below:

KSV = kq�0 (2)

The Stern–Volmer plots (Fig. 3a and b) revealed the presenceof static quenching as evident from decreased KSV values (Table 1)with increase in temperature [10,11]. Further, the higher valuesof kq compared to that reported for the maximum quenching rateconstant of bimolecular diffusion collision (2.0 × 1010 L mol−1 s−1)supported the static quenching mechanism between the drug andprotein [12–14]. Therefore, the quenching data were further ana-lyzed using the modified Stern–Volmer equation shown below[8,15]:

F0

F0 − F= 1

faKa[Q]+ 1

fa(3)

where F and F0 are the fluorescence intensities of protein with andwithout ETH, respectively; Ka is the Stern–Volmer quenching con-stant of the accessible fraction, [Q] is the concentration of quencherand fa is the fraction of the initial fluorescence of protein that isaccessible to the quencher. The plot of F0/(F0 − F) versus 1/[Q] (fig-ures not shown) yielded f −1

a as the intercept on y axis and (faKa)−1

as the slope. The values of fa for ETH–HSA and ETH–BSA were foundto be 1.25 and 1.27, respectively, indicating that only 80.31% and

Fig. 3. The Stern–Volmer curves for quenching of ETH with HSA (a) and BSA (b) atdifferent temperatures.

78.88% of the initial fluorescence of HSA and BSA was accessible forquenching. The results are listed in Table 1.

The fluorescence data was also used to evaluate the binding con-stant, K and the number of binding sites, n for drug–protein complexusing the equation shown below [16]:

log(F0 − F)F

= log K + n log[Q] (4)

From the intercept and slope of the graph of log[(F0 − F)/F]versus log[Q] (figures not shown), the values of K werefound to be (3.95 ± 0.052) × 104, (2.72 ± 0.035) × 104 and(2.42 ± 0.032) × 104 M−1 for HSA–ETH and (1.30 ± 0.024) × 105,(6.43 ± 0.054) × 104 and (5.06 ± 0.046) × 104 M−1 for BSA–ETH at294, 301 and 310 K, respectively. The values of n (1.04–1.16) werefound to be close to unity indicating the presence of single classof binding site for ETH in both HSA and BSA. The decreased Kvalues with increase in temperature suggested the lesser stabilityof ETH–protein complex at higher temperature.

3.2. Binding mode

Thermodynamic parameters, �H0 and �S0 were evaluatedusing the van’t Hoff’s equation shown below:

log K = −�H0

2.303RT+ �S0

2.303R(5)

where K is the binding constant and R is the gas constant. Thevalues of �H0 and �S0 were calculated from the slope and inter-cept, respectively, of the plot of log K versus 1/T (figures notshown). The value of free energy change was evaluated using theGibbs–Helmholtz equation given below [17]:

�G0 = �H0 − T�S0 (6)

The corresponding values for ETH–HSA and ETH–BSA are summa-rized in Table 2. Both processes were found to be spontaneous asevident from negative �G0 values. The negative �H0 and positive�S0 values indicated that both hydrogen bonding and hydrophobicforces played a major role in the formation of ETH–HSA complex.Where as the negative values of both �H0 and �S0 for ETH–BSArevealed that the van der Waals forces and hydrogen bondingplayed a significant role in the formation of ETH–BSA complex [18].

Table 2The relative thermodynamic parameters of HSA–ETH and BSA–ETH systems at dif-ferent temperatures.

System T (K) �G0 (kJ mol−1) �H0 (kJ mol−1) �S0 (J mol−1 K−1)

HSA294 −25.88 ± 0.008

−23.11 ± 0.012 9.27 ± 0.016301 −25.85 ± 0.014310 −26.02 ± 0.011

BSA294 −28.80 ± 0.022

−44.80 ± 0.022 −54.5 ± 0.014301 −28.33 ± 0.008310 −27.92 ± 0.016



0

50

100

150

200

250

460410360310Wavelength, nm

0

0.02

0.04

0.06

0.08b

1

2

0

50

100

150

200

250

300

500450400350300

Wavelength, nm

Inte

nsit

y

0

0.02

0.04

0.06

0.08

Abs

orba

nce

Inte

nsit

y

Abs

orba

nce

a

1

2

Fig. 4. The overlap of fluorescence spectrum of (a) HSA/BSA (1) with the absorptionspectrum of ETH (2); {[HSA/BSA]:[ETH] = 1:1}.

3.3. Fluorescence resonance energy transfer

The spectral studies revealed that both HSA and BSA formedcomplexes with ETH. The distance r between the protein residueand acceptor was calculated based on fluorescence resonanceenergy transfer [19]. This distance of separation and the extent ofspectral overlap determine the extent of energy transfer. The over-lap of fluorescence emission spectrum of protein and the absorptionspectrum of ETH is shown in Fig. 4a and b. The efficiency of energytransfer, E is related to r as shown below:

E = 1 − F

F0= R6

0

R60 + r6

(7)

where R0 is the critical distance when the efficiency of energy trans-fer is 50%, which is calculated using the equation given below:

R60 = 8.8 × 10−25k2�−4˚J (8)

where k2 is the spatial orientation factor of the dipole, � is therefractive index of the medium, Ф is the fluorescence quantumyield of the donor and J is the overlap integral of the fluorescenceemission spectrum of the donor and the absorption spectrum of theacceptor. The value of J was evaluated using the equation shownbelow:

J =∑

F(�)ε(�)�4��∑F(�)��

(9)

where F(�) is the fluorescence intensity of the fluorescent donorof wavelength, � and ε(�) is the molar absorption co-efficient ofthe acceptor at wavelength, �. In the present case, K2 = 2/3, � = 1.36and Ф = 0.118 for HSA–ETH system [20] and K2 = 2/3, � = 1.336 andФ = 0.15 for BSA–ETH system [10]. From Eqs. (7) to (9), we were

0

0.1

0.2

0.3

0.4

0.5

250 27 0 29 0 31 0 33 0 35 0Wav elength, nm

Abs

orba

nce

a

Con centratio n of E TH

X

0

0.1

0.2

0.3

0.4

0.5

250 270 290 310 330 350Wavelength, nm

Abs

orba

nce

b

Concentratio n of E TH

X

Fig. 5. Absorption spectra of HSA (a), BSA (b), ETH and HSA/BSA–ETH system. Proteinconcentration was maintained at 2.5 �M and that of ETH at 5 �M. (x) refers to ETHonly.

able to calculate that J = 2.33 × 10−15 and 2.14 × 10−15 cm3 L mol−1,R0 = 2.86 and 3.11 nm, E = 0.23 and 0.28 and r = 3.50 and 3.40 nm forHSA–ETH and BSA–ETH systems, respectively. The value of r < 8 nm[21,22] observed in the present study revealed the presence of staticquenching mechanism [20] and the transfer of energy between theprotein and drug.

3.4. UV absorption studies

UV absorption spectra of protein in the presence of increasingamounts of ETH are shown in Fig. 5a and b. It could be noticed fromthe figure that the intensity of absorption of HSA/BSA increasedwith successive addition of ETH. Further, a red shift in the maxi-mum peak of protein was observed probably due to the formationof complex between ETH and HSA/BSA [10,23].

3.5. Conformational changes in protein upon interactionwith ETH

3.5.1. Circular dichroism (CD) analysisCircular dichroism, a sensitive technique to monitor the confor-

mational changes in the protein [24], was employed in the presentstudy. For this, the CD spectra of HSA and BSA in the absence andpresence of different concentrations of ETH were recorded (Fig. 6aand b, respectively). The CD spectra of protein (HSA/BSA) showedtwo minima at 208 nm and 222 nm, characteristics of an �-helicalstructure of the protein. The negative peaks at 208–209 nm and222–223 nm were attributed n → �* transition for peptide bond ofthe �-helix [25]. The helical content of free and bound protein was



-250 00

-200 00

-150 00

-100 00

-50 00

0

200 210 220 230 240 250

λ, nm

MRE

a

1

4

-2500 0

-2000 0

-1500 0

-1000 0

-500 0

0

200 21 0 220 230 240 250

λ, nm

MRE

1

4

b

Fig. 6. CD spectra of HSA–ETH (a) and BSA–ETH (b) systems. HSA/BSA concentrationwas fixed at 2.5 �M (1). In HSA/BSA–ETH system, the ETH concentration was keptat 5 (2), 10 (3), and 15 �M (4).

calculated from the mean residue elipticity (MRE) values at 208 nmusing the equation shown below [26]:

MRE = �obs

Cpnl × 10(in units of degcm2 dmol−1) (10)

�obs is the observed CD (in milli degrees), Cp is the concentrationof the protein, n is the number of amino acid residues and l is thepath length of the cell (in cm).

˛-Helix (%) = −MRE208 − 400033, 000 − 4000

× 100 (11)

where MRE208 is the observed MRE value at 208 nm, 4000 is theMRE of �-form and random coil conformation cross at 208 nm and33,000 is the MRE value of a pure �-helix at 208 nm.

The �-helicity decreased gradually from (58.9 ± 0.9)% in freeHSA to (42.7 ± 0.5)% in bound HSA and from (62.6 ± 0.8)% in freeBSA to (47.1 ± 0.4)% in bound BSA thereby indicating that the ETHhas altered the hydrogen bonding networks of protein. Further, italso revealed the changes in secondary structure of the respectiveprotein. The shapes of CD spectra of HSA and BSA in the absenceand presence of ETH were observed to be similar indicating thatthe structure of protein was predominantly �-helical even afterbinding with ETH [27].

3.5.2. FTIR analysisIn order to get more information on binding mechanism and

changes in conformation of the protein upon interaction withETH, FTIR spectra of protein alone and protein–drug systems wererecorded. Amide I band is generally more sensitive compared toamide II band for the changes in secondary structure of the protein[28]. The amide I band occurs in the region of 1600–1700 cm−1

(mainly C O stretching vibrations of amide groups) while amideII occurs around 1500–1600 cm−1 (mainly C–N stretch couplewith N–H bending frequency). Fig. 7a and b shows that the peakposition of amide I and amide II bands are shifted from 1648.5to 1644.1 cm−1 and from 1561.3 to 1552.8 cm−1 in HSA–ETHsystem, and from 1651.2 to 1647.4 cm−1 and from 1558.5 to1551.7 cm−1 in BSA–ETH system, respectively. This indicated that

Fig. 7. FTIR spectra and difference spectra of HSA/BSA; the FTIR spectra of free HSA (a1)/BSA (b1) (subtracting the absorption of buffer solution from the absorption of proteinsolution) and the FTIR difference spectra of HSA (a2)/BSA (b2) (subtracting the absorption of ETH-free form from that of ETH–HSA/BSA bound form) in phosphate buffer;CHSA/BSA = 5 �M and CETH = 5 �M.



Table 3Binding constant values of HSA–ETH and BSA–ETH systems in presence of site probes at 301 K.

System K (M−1) without site probe K (M−1) with warfarin K (M−1) with ibuprofen K (M−1) with digitoxin

HSA + ETH 2.72 × 104 ± 0.022 1.67 × 103 ± 0.028 2.70 × 104 ± 0.034 2.72 × 104 ± 0.012BSA + ETH 6.43 × 104 ± 0.012 5.16 × 103 ± 0.023 6.40 × 104 ± 0.014 6.42 × 104 ± 0.018

the secondary structure of protein was changed upon interactionwith ETH. The ETH has interacted with C O and C–N groups inprotein polypeptides and resulted in rearrangement of polypeptidecarbonyl hydrogen bonding network [29].

3.6. Site specific probe

In order to locate the binding site of ETH in protein, the com-petitive binding experiments were performed using different sitecompetitors viz. warfarin, ibuprofen and digitoxin for sites I, II andIII respectively as per Sudlow et al. [30] and Sjoholm et al. [31] clas-sification of binding sites. For this, emission spectra of ETH–proteinand ternary mixtures of protein–ETH-site probe were recorded,separately. The corresponding binding constant values were eval-uated and are shown in Table 3. In both cases (ETH–HSA andETH–BSA systems), the binding constant values decreased remark-ably in the presence of warfarin while this value remained almostsame in the presence of ibuprofen and digitoxin. These resultsrevealed that the warfarin displaced ETH from the binding sitewhile ibuprofen and digitoxin had a little effect on the binding ofETH to HSA/BSA. Hence, we concluded that the ETH bound to site I ofHSA/BSA, which is located in the hydrophobic pocket of subdomainIIA.

3.7. Effect of metal ions

In plasma, there are some of the metal ions which can affect thebinding of drugs to serum albumins. Hence, the effects of some ofcommon metal ions viz., K+, Mn2+, Ni2+, Co2+, Zn2+, and Cu2+ onthe binding of ETH to protein was investigated at 301 K by fluo-rescence technique. For this, the concentration of both protein andmetal ion was kept constant (2.5 �M) while that of the drug solu-tion was varied (5–50 �M). The corresponding results are given inTable 4. The decreased binding constant of ETH–HSA in the presenceof Cu2+, Co2+, Ni2+ and K+ might cause the drug to be quickly clearedfrom the blood [32]. This might lead to the need of higher doses ofETH to achieve the desired therapeutic effect. However, the bindingconstant increased in the presence of Zn2+ and Mn2+ thereby mak-ing the ETH to be retained for longer time in the blood [33]. Thiswould lead to lesser doses of drug to achieve the intended thera-peutic effect. Further, it was noticed that the binding constant ofETH–BSA decreased in the presence of all of the above ions therebycausing ETH to be quickly cleared from the blood. This might neces-sitate for higher doses of ETH in the presence of above metalions.

Table 4The effect of metal ions on the binding of ETH to protein at 301 K.

Metal ions Binding constant (M−1)

HSA + ETH BSA + ETH

Without metal ion 2.72 × 104 ± 0.022 6.43 × 104 ± 0.022With Cu2+ 2.22 × 104 ± 0.012 1.93 × 104 ± 0.026With Co2+ 2.14 × 104 ± 0.018 4.59 × 104 ± 0.014With Zn2+ 3.85 × 104 ± 0.027 3.48 × 104 ± 0.031With Ni2+ 2.52 × 104 ± 0.011 2.44 × 104 ± 0.024With Mn2+ 3.73 × 104 ± 0.017 2.13 × 104 ± 0.015With K+ 1.51 × 104 ± 0.032 5.29 × 104 ± 0.033

4. Conclusions

In this paper, the interaction between ethionamide and serumalbumin was investigated employing different spectroscopic tech-niques. The results revealed that the secondary structure of proteinwas affected upon interaction with the drug. Fluorescence resultsindicated the presence of static quenching mechanism in the bind-ing of ETH to protein. Based on spectral data we have concludedthat the ETH bound to site I of protein, which is located in thehydrophobic pocket of subdomain IIA.

Acknowledgements

We gratefully acknowledge financial support of the Coun-cil of Scientific and Industrial Research, New Delhi (Grant No.01(2279)/08/EMR-II, dated 20–11–2008). One of the authors (Ume-sha K.) thanks the University Grants Commission, New Delhi, forawarding the Research Fellowship in Science for meritorious Stu-dents. Thanks are also due to the authorities of the KarnatakUniversity, Dharwad, for providing the necessary facilities.

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Sci Pharm www.scipharm.at

Research article Open Access Interaction of Bioactive Coomassie

Brilliant Blue G with Protein: Insights from Spectroscopic Methods

Umesha KATRAHALLI, Shankara Sharanappa KALANUR, Jaladappagari SEETHARAMAPPA *

Department of Chemistry, Karnatak University, Dharwad 580 003, India.

* Corresponding author. E-mail: [email protected] (J. Seetharamappa)

Sci Pharm. 2010; 78: 869–880 doi:10.3797/scipharm.1008-15

Published: November 6th 2010 Received: August 26th 2010 Accepted: November 6th 2010

This article is available from: http://dx.doi.org/10.3797/scipharm.1008-15

© Katrahalli et al.; licensee Österreichische Apotheker-Verlagsgesellschaft m. b. H., Vienna, Austria.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract The binding of coomassie brilliant blue G (CBB) to bovine serum albumin (BSA) was investigated under simulative physiological conditions employing different optical spectroscopic techniques viz., fluorescence emission, UV–visible absorption and FTIR. Fluorescence quenching data obtained at different temperatures suggested the presence of dynamic type of quenching mechanism. The binding constant of CBB-BSA and the number of binding sites (n) for CBB in BSA were calculated and found to be 4.20 x 104 M−1 and 0.96 respectively, at 302 K. The value of n close to unity indicated that the protein has a single class of binding sites for CBB. The thermodynamic parameters revealed that the hydrophobic forces played a major role in the interaction of CBB to BSA. The distance between the CBB and protein was calculated using the theory of Föster’s Resonance Energy Transfer (FRET). The conformational change in the secondary structure of BSA upon interaction with dye was investigated by synchronous fluorescence and FTIR techniques. Competitive binding studies were also carried out to know the location of binding of CBB on BSA.

Keywords Coomassie brilliant blue G • Physiological condition • Optical spectroscopy • Binding parameter • Thermodynamic parameter

http://creativecommons.org/licenses/by/3.0/

870 U. Katrahalli, S. S. Kalanur and J. Seetharamappa:

Sci Pharm. 2010; 78: 869–880.

Introduction Coomassie brilliant blue G (CBB) dye (Fig. 1), [also known as coomassie dye] is employed to stain proteins in sodium dodecyl sulfate and blue native polyacrylamide gel in electrophorosis gels. It is used in the quantification of electrophoretically separated protein [1]. The CBB has also been popularly used in biochemical and clinical laboratories for the purification and quantification of proteins [2]. Recently, CBB was used in the treatment of spinal injuries in rats [3]. It was also used as a stain to assist surgeons in retinal surgery [4].

Serum albumin (SA), often simply referred as albumin, is the most abundant plasma protein in humans as well as in other mammals which plays a major role in the transport and deposition of many drugs in the blood. This albumin is essential for the maintenance of osmotic pressure which is needed for proper distribution of body fluids between intravascular components and body tissues. SA consists of three homologous domains (I–III), each of them is composed of two subdomains (A and B) [5, 6]. In the IIA and IIIA pocket, the majority of small ligands like drugs are bound and carried to the target site [7–10]. Therefore, it is important to study the interaction of the drug with the serum albumin, which plays an important role in pharmacology, pharmacokinetics and pharmaco-dynamics. In this regard, bovine serum albumin (BSA) has been studied extensively, partly because of its structural homology with human serum albumin (HSA) [11].

NHO3S

N+ SO3

-

NH

O

Fig. 1. Structure of Coomassie brilliant blue G.

In the present report, we have focused on the interaction of CBB with bovine serum albumin and obtained the information with regard to binding constant, number of binding sites, thermodynamic parameters, and conformational changes in the protein employing spectroscopic techniques. Among these, fluorescence technique is a well known practical method for studying protein interactions with various ligands [12–14] as it yields a vast amount of information on binding characteristics and the microenvironment surrounding the protein residues.

Materials and Methods Materials Bovine serum albumin (fatty acid free, fraction V) and Brilliant blue G–250 were purchased from Sigma Chemicals Co. Millipore water was used throughout the experiment. All other chemicals used in the present study are of analytical reagent grade.

Interaction of Bioactive Coomassie Brilliant Blue G with Protein: Insights from Spectroscopic … 871

Sci Pharm. 2010; 78: 869–880.

Equipment and spectral measurements The fluorescence spectra were recorded on a spectrofluorimeter model F–7000 (Hitachi, Japan) equipped with a 1.0 cm quartz cell and a thermostat bath. Inner filter effect was not observed during fluorescence measurements. Necessary corrections have been made. The widths of both excitation and emission slit were set to 5 nm in the experiment. The UV–vis spectra were recorded on a double beam CARY 50–BIO UV–vis spectro-photometer (Varian, Australia) equipped with a 1.0 cm quartz cell and a slit width of 5 nm. FTIR spectra were acquired on a Thermo Nicolet–5700 FTIR spectrometer (Waltham, MA, USA).

Procedures The fluorimetric titrations were carried out and the fluorescence intensities of protein were recorded at around 340 nm upon excitation at 296 nm. Based on preliminary studies, the concentration of BSA was fixed at 2.5 μM while that of the dye was varied from 2.5 to 25 μM. The interaction studies were carried out at 293, 302 and 309 K.

The site probe studies were performed using different probes viz., warfarin, ibuprofen and digitoxin respectively for site I, II and III by keeping the concentration of both protein and the probe constant (each at 2.5 μM) and varying the concentration of the dye from 2.5 to 25 μM.

The absorption spectra were recorded by scanning the binary mixture of dye and the protein in the wavelength range of 250 to 300 nm.

The FTIR spectrum of BSA in the absence of CBB was obtained by subtracting the IR spectrum of buffer. The FTIR spectrum of BSA in presence of CBB was obtained by subtracting the IR spectrum of free CBB in buffer from that of the bound CBB to protein in the range of 1500 to 1700 cm−1.

Fig. 2. Fluorescence spectra of BSA. Concentration of BSA was fixed at 2.5 μM (1)

and that of CBB was varied in the range of 2.5 to 25 μM (2–11).

1

11


Sci Pharm. 2010; 78: 869–880.

Results and discussion CBB–induced quenching studies of bovine serum albumin The fluorescence of SA mainly resides in the emission from the tryptophan, Trp (~ 340 nm) and tyrosine, Tyr (~ 315 nm) residues. Fluorescence spectra of BSA were recorded in the presence and absence of CBB upon excitation at 296 nm. The fluorescence emission of protein was observed to be quenched (around 340 nm) in a concentration dependent manner by CBB (Fig. 2). Quenching of the intrinsic fluorescence of protein can be used to retrieve information on ligand–protein binding. The fluorescence quenching data were analyzed using the Stern–Volmer equation shown below [15]:

Eq. 1. ][10 QKFF

SV+=

where F0 and F are the steady–state fluorescence intensities in the absence and presence of the quencher (CBB), respectively; Q is the quencher concentration and KSV is the Stern–Volmer quenching constant.

The plot of the fluorescence intensity ratio of BSA in the absence and presence of quencher (CBB) as a function of the quencher concentration showed a linear dependence (Fig. 3). The values of KSV for BSA–CBB system were calculated from the slope of the Stern–Volmer plot and the corresponding values are given in Table 1.

Fig. 3. The Stern–Volmer plot for quenching of BSA.

Tab. 1. KSV, Kq, binding and thermodynamic parameters of CBB–BSA. Temp. (K)

KSV (mol−1)

K (mol−1)

n ΔG0 (kJ mol−1)

ΔH0

(kJ mol−1) ΔS0 (J mol−1K−1)

293 5.29 x 104 2.81 x 104 0.94 −24.96 302 6.52 x 104 4.20 x 104 0.96 −26.73 309 8.74 x 104 7.95 x 104 0.99 −28.99

48.0 2.49 x 102

♦ 293 K ▲ 302 K ■ 309 K


Sci Pharm. 2010; 78: 869–880.

The quenching mechanisms are usually classified as either dynamic quenching or static quenching. Dynamic and static quenching can be differentiated by their differing dependence on temperature [15]. From the results of Stern–Volmer plots, we noticed that the KSV values increased with rise in temperature indicating the presence of dynamic quenching mechanism in the interaction of the protein with CBB. Further, it was also noticed that the fluorescence intensity of BSA was decreased in the presence of CBB with a slight blue shift of the maximum emission wavelength indicating that the chromophores of the protein were placed in a more hydrophobic environment after the addition of CBB. The blue shift could also be explained by a preferential quenching of the Trp residues that would leave only the Tyr residues to contribute to protein fluorescence. Moreover, we observed a concomitant increase in the fluorescence intensity at 401 nm, which is the characteristic wavelength of the bound BSA. This phenomenon might be the result of the radiationless energy transfer between CBB and BSA. Further, the existence of an isoactinic point at 375 nm indicated the presence of bound and unbound CBB at equilibrium.

Determination of binding constant and binding capacity CBB induced fluorescence quenching data of BSA was analyzed to obtain the binding parameters like binding constant (K) and the number of binding sites (n) from the equation shown below [16, 17]:

Eq. 2. ][logloglog 0 QnKF

FF+=⎟

⎠⎞

⎜⎝⎛ −

Fig. 4 depicts the linear plot of log [(F0−F)/F] versus log [Q] and the corresponding results of K and n are given in Table 1. The slope registered in this plot was noticed to be close to unity revealing that one ligand molecule bound to a molecule of protein. This means, BSA has a single class of binding site for CBB. Hence, we propose that CBB most likely binds to the hydrophobic pocket located in subdomain IIA, that is to say, Trp–214 is near or within the binding site.

Fig. 4. The plot of log[(F0−F)/F] versus log[Q] for quenching of BSA by CBB at different

temperatures.

-1.2

-0.8

-0.4

0

0.4

-5.7 -5.4 -5.1 -4.8 -4.5

log [Q]

log

[(Fo-

F)/F

]

♦ 293 K ▲ 302 K ■ 309 K


Sci Pharm. 2010; 78: 869–880.

The interacting force between CBB and protein There are essentially four different types of interacting forces, viz., hydrogen bonds, van der Waals forces, electrostatic forces and hydrophobic interactions, which could play a major role in ligand binding to serum albumins. The signs and magnitudes of thermo-dynamic parameters determine the nature of interacting forces, which are taking part in drug–protein interactions [18]. The ΔH0 and ΔS0 were calculated using the van’t Hoff’s equation shown below [19]:

Eq. 3. R

SRT

HK303.2Δ

303.2Δlog

00

+−

=

The values of ΔG0 for the binding process at different temperatures were calculated using the equation shown below and the corresponding results are given in Table 1.

Eq. 4. KRTG log303.2Δ 0 −=

The negative values of ΔG0 indicated spontaneity of the binding process. The positive values of both ΔH0 and ΔS0 observed in CBB–BSA system showed revealed that the hydrophobic forces played a major role in the binding process between CBB and BSA [18].

Exchange of energy between protein and dye The fluorescence quenching of BSA by CBB revealed the occurrence of energy transfer between the protein and dye. According to the Förster’s non–radiative energy transfer theory [15, 20], the energy transfer between protein and dye depends on (a) the overlap of the fluorescence emission spectrum of the donor with UV–vis absorption spectrum of the acceptor and (b) the distance of approach between the donor and acceptor. The efficiency, E of a FRET process depends on the inverse sixth–distance between donor and acceptor (r) as well as on the critical energy transfer distance or Förster radius (R0) under the condition of 1:1 situation of donor to acceptor concentrations and can be expressed by the following equation:

Eq. 5. 66

0

60

0

1rR

RFFE

+=−=

where E is the efficiency of energy transfer; F and F0 are the fluorescence intensities of the donor in the presence and absence of the acceptor, respectively. The critical distance, R0 when the transfer efficiency is 50% can be calculated using the equation:

Eq. 6. JNkR 422560 Φ108.8 −−×=

where k2 is the spatial orientation factor of the dipole, Ф is the fluorescence quantum yield of the donor, N is the refractive index of the medium and J is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor.

The term J is expressed as;


Sci Pharm. 2010; 78: 869–880.

Eq. 7. ( ) ( )

( ) ( )λελFλλλελFJ

∑∑

=Δ4

where F(λ) is the fluorescence intensity of the donor at wavelength λ and ε(λ) is the molar absorptivity of the acceptor at wavelength λ.

Fig. 5 depicts the overlap between the fluorescence emission spectrum of free protein and the absorption spectrum of dye. The efficiency of energy transfer and overlapping integration values were obtained from equations 5 and 7, respectively. In order to evaluate the Förster’s critical distance using equation 6, we have used k2 = 2/3; Ф = 0.15; N =1.36 for BSA [21, 22]. By using the equations 5–7, we have calculated the values of E, r, R0 and J and these values were found to be 0.28 nm, 1.9 nm, 1.63 nm and 7.15 x 10−16 cm3 m−1 for BSA-CBB system. In the present study, r is considered as the average value between the bound ligand and two tryptophan residues (Trp–135 and Trp–214) [23]. The value of r less than 7 nm indicated the non–radiative energy transfer between BSA and CBB [15].

Fig. 5. The overlap of BSA fluorescence spectrum (1) and CBB absorption spectrum

(2). [BSA] : [CBB] = 1 : 1.

Effect of CBB on the Conformation of protein Synchronous fluorescence measurements Synchronous fluorescence measurements were carried out in order to get the information on the molecular environment in the vicinity of the fluorophores (Tyr and Trp) of protein. Synchronous fluorescence spectra of BSA (Fig. 6) were obtained by simultaneously scanning the excitation and emission monochromater maintaing Δλ = 15 nm (Tyr excitation) and Δλ = 60 nm (Trp excitation) between them. Fig. 6 shows the effect of CBB on the synchronous spectrum of protein when Δλ = 15 nm (Fig. 6a) or Δλ = 60 nm (Fig. 6b). As it is evident, the intensity of the tryptophan and tyrosine decreased in the presence of CBB; but no significant shift was noticed in the signals. This supported the preferential quenching of Trp residues over Tyr residues. This indicated that the binding between CBB and the protein did not lead to a change in the polarity of the microenvironment of the tryptophan and tyrosine residues, however, the internal packing of the protein changed.

0

50

100

150

200

250

300 350 400 450 500Wavelength, nm

Inte

nsity

0

0.005

0.01

0.015

0.02

Abs

orba

nce

2

1


Sci Pharm. 2010; 78: 869–880.

Fig. 6. Synchronous fluorescence spectra of BSA: (a) Δλ = 15 nm; (b) Δλ = 60 nm.

CBSA = 2.5 μM (1), CCBB = 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 20.0 and 22.5 μM (2–10).

UV–visible absorption measurements We have recorded the absorption spectra of BSA in the absence and presence of CBB (Figure not shown). The absorption peaks observed around 280 nm were noticed to be shifted to the lower wavelength with increase in the concentration of CBB indicating the extension of the peptide strands of protein molecules [24, 25].

FTIR measurements FTIR spectroscopy is a well defined tool for the determination of protein’s secondary structure [26, 27]. The conformational sensitivity of amide bands is governed by the two most important factors viz., hydrogen bonding and the coupling between the transition dipoles. Both the amide I (noticed around 1653 cm−1 due to C=O stretching) and amide II bands (observed around 1548 cm−1 due to C–N stretch coupled with N–H bending mode) of the protein have the relationship with secondary structure of protein [28]. It is evident from Fig. 8a and Fig. 8b that the secondary structure of BSA was changed. The amide I band was noticed to be shifted from 1650.8 cm−1 (in free BSA) to 1646.9 cm−1 (in CBB–BSA) while amide II band was shifted from 1548.1 cm−1 (in free BSA) to 1554.6 cm−1(in CBB–BSA complex).

Location of binding site The principal regions of the ligand binding sites of albumin are located in the hydrophobic cavities of subdomain IIA and IIIA. Sudlow et al [29, 30] have suggested two distinct binding sites (site I and site II) in SAs. Warfarin, phenyl butazone etc., have high affinity towards site I while ibuprofen, flufenamic acid etc., exhibit affinity towards site II. Zhang et al [31] have suggested another site (site III) that has the affinity for digitoxin. In order to locate the binding site in BSA, we have recorded the fluorescence intensity of CBB–BSA in the presence and absence of site probe and in turn calculated the values of binding constant. The corresponding values are given in Table 2. It is clear from Table 2 that there is a significant decrease in the binding constant of CBB–BSA in presence of warfarin. However, the binding constant values remained almost same in presence of ibuprofen and digitoxin. These results revealed that the site I was the main binding site for CBB on

0

20

40

60

80

100

120

140

260 285 310 335Wavelength, nm

Inte

nsity

0

20

40

60

80

100

120

140

300 325 350 375Wavelength, nm

Inte

nsity

a b1

10

1

10


Sci Pharm. 2010; 78: 869–880.

protein. Therefore, the site I located in the hydrophobic pocket of subdomain IIA was proposed to be the binding site for CBB in protein.

Tab. 2. The comparison of binding constants of CBB-BSA before and after the addition of site probes.

K without the site probe (mol−1)

K with warfarin (mol−1)

K with ibuprofen (mol−1)

K with digitoxin(mol−1)

BSA 4.20 x 104 7.73 x 103 4.22 x 104 4.19 x 104 Fig. 8. FTIR spectra and difference spectra of BSA: (a) free BSA (subtracting the

absorption of the buffer solution from the spectrum of the protein solution) and (b) the difference spectra of BSA (subtracting the absorption of the CBB–free form from that of CBB–BSA bound form); CBSA = CCBB = 2.5 μM.

Conclusions In this paper, the interaction between coomassie brilliant blue G and bovine serum albumin was investigated for the first time by fluorescence spectroscopy, UV–visible absorption and FTIR spectroscopy under simulative physiological conditions. The fluorescence data indicated the strong interaction as characterized by binding constant value of 4.20 x 104 M−1 at 302 K. Further, the fluorescence quenching mechanism was observed to be dynamic process. Hydrophobic force played the major force in the interaction process.

Acknowledgements This work is supported by Council of Scientific and Industrial Research, New Delhi, India (No. 01(2279)/08/EMR–II, dated – 20/11/2008). One of the authors (Umesha K.) is also grateful to the University Grant Commission, New Delhi, India for awarding RFSMS. Thanks are also due to the authorities of the Karnatak University, Dharwad, for providing the necessary facilities.

Authors' Statement The authors declare no conflict of interest.

1554.6

1646.9

ba

1650.8

1548.1


Sci Pharm. 2010; 78: 869–880.

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[16] Zhou N, Liang YZ, Wang P. 18β–Glycyrrhetinic acid interaction with bovine serum albumin. J Photochem Photobiol A Chem. 2007; 185: 271–276. doi:10.1016/j.jphotochem.2006.06.019

[17] Chakraborty B, Basu S. Interaction of BSA with proflavin: A spectroscopic approach. J Lumin. 2009; 129: 34–39. doi:10.1016/j.jlumin.2008.07.012

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[19] Zhang YZ, Xiang X, Dai PMJ, Zhang LL, Liu Y. Spectroscopic studies on the interaction of Congo Red with bovine serum albumin. Spectrochim Acta A Mol Biomol Spect. 2009; 72: 907–914. doi:10.1016/j.saa.2008.12.007

[20] Sklar LA, Hudson BS, Simoni RD. Conjugated polyene fatty acids as fluorescent probes: binding to bovine serum albumin. Biochemistry. 1977; 16: 5100–5108. doi:10.1021/bi00642a024

[21] Epps DE, Raub TJ, Caiolfa V, Chiari A, Zamai M. Determination of the affinity of drugs toward serum albumin by measurement of the quenching of the intrinsic tryptophan fluorescence of the protein. J Pharm Pharmacol. 1999; 51: 41–48. doi:10.1211/0022357991772079

[22] Valeur B, Brochon JC. New trends in fluorescence spectroscopy. 6th edn. Springer, Berlin, 1999, p 25.

[23] Liu J, Tian JN, Zhang J, Hu Z, Chen X. Interaction of magnolol with bovine serum albumin: a fluorescence–quenching study. Anal Bioanal Chem. 2003; 376: 864–867. doi:10.1007/s00216-003-2017-8

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ARTICLE IN PRESS

Journal of Luminescence 130 (2010) 211–216

Contents lists available at ScienceDirect

Journal of Luminescence

0022-23

doi:10.1

� Corr

E-m

journal homepage: www.elsevier.com/locate/jlumin

Study of the interaction between fluoxetine hydrochloride andbovine serum albumin in the imitated physiological conditionsby multi-spectroscopic methods

Umesha Katrahalli, Seetharamappa Jaldappagari �, Shankara S. Kalanur

Department of Chemistry, Karnatak University, Dharwad 580 003, India


Article history:

Received 21 April 2009

Received in revised form

20 July 2009

Accepted 31 July 2009Available online 7 August 2009

Keywords:

Spectroscopy

Bovine serum albumin

Fluoxetine hydrochloride

Quenching mechanism

Thermodynamic parameters

Displacement studies

13/$ - see front matter & 2009 Published by

016/j.jlumin.2009.07.033

esponding author. Tel.: +91836 2215286; fax

ail address: [email protected] (S. Ja

a b s t r a c t

The mechanism of interaction of an antidepressant, fluoxetine hydrochloride (FLX) with bovine serum

albumin (BSA) has been studied by different spectroscopic techniques under physiological conditions.

FLX was found to quench the intrinsic fluorescence of protein by static quenching mechanism. The

binding constant ‘K’ was found to be 7.06�103 M�1 at 296 K. The value of ‘n’ close to unity revealed that

the BSA has a single class of binding site for FLX. Based on thermodynamic parameters, hydrogen

bonding and van der Waals forces were proposed to operate between BSA and FLX. The change in

conformation of protein was noticed upon its interaction with the drug. From displacement studies it

was concluded that the FLX bound to protein at site I. The effects of various common metals ions on the

binding were also investigated.

& 2009 Published by Elsevier B.V.

1. Introduction

Serum albumin, the most abundant protein in the circulatorysystem, has been one of the most extensively studied of allproteins [1]. Serum albumins are prone to bind effectively manysmall organic molecules. Bovine serum albumin (BSA), a largeglobular protein (65,000 Daltons), consists of a single chain of 583amino acids residues [1]. It is known that the distribution, freeconcentration and metabolism of several drugs might be stronglyaffected by drug–protein interactions in the blood stream. Thistype of interaction can also influence the drug stability andtoxicity during the chemotherapeutic process. Therefore, thestudies on the binding of a drug with protein will facilitateinterpretation of the metabolism and transporting process ofdrug. In this regard, BSA has been studied extensively, partlybecause of its structural homology with human serum albumin(HSA) [2,3].

Fluoxetine hydrochloride (FLX) (Fig. 1), an antidepressant,belongs to a group of drugs called selective serotonin reuptakeinhibitors. It affects chemicals in the brain that may becomeunbalanced and cause depression, panic, anxiety, or obsessive–compulsive symptoms. It is used to treat major depressivedisorder, bulimia nervosa (an eating disorder) obsessive–

Elsevier B.V.

: +91836 2747884.

ldappagari).

compulsive disorder, panic disorder, and premenstrual dysphoricdisorder.

In view of the above, we planned to study the interaction ofFLX with BSA by using different spectroscopic techniques viz.,fluorescence, circular dichroism (CD), UV–vis absorption and FT-IR. The present study reveals the changes in the fluorescence andstructural properties of BSA upon binding with FLX, which havebeen utilized to characterize the interaction parameters.

2. Experimental

2.1. Reagents

Bovine serum albumin (BSA, Fraction V) was obtained fromSigma Chemical Company, St. Louis, USA. Fluoxetine hydrochlor-ide was obtained as gift sample from Micro Labs Ltd. The solutionsof FLX and BSA were prepared in 0.1 M phosphate buffer of pH 7.4containing 0.15 M NaCl. Millipore water was used throughout thestudy.

2.2. Apparatus

Fluorescence measurements were performed on a spectro-fluorimeter Model F-7000 (Hitachi, Japan) equipped with a 150 WXenon lamp and a slit width of 5 nm. The CD measurements were

www.elsevier.com/locate/jlumin

dx.doi.org/10.1016/j.jlumin.2009.07.033

mailto:[email protected]

ARTICLE IN PRESS

0

200

400

600

800

310Wavelength, nm

Inte

nsity

a

k

330 350 370 390 410

Fig. 2. Fluorescence spectra of BSA (5mM) in presence of FLX: (a) 0, (b) 5, (c) 10, (d)

15, (e) 20, (f) 25, (g) 30, (h) 35, (i) 40, (j) 45 and (k) 50mM.

O

F3C

NHCH3

. HCl

Fig. 1. The structure of fluoxetine hydrochloride.

U. Katrahalli et al. / Journal of Luminescence 130 (2010) 211–216212

made on a JASCO-810 spectropolarimeter (Tokyo, Japan) using a0.1 cm cell at 0.2 nm intervals, with three scans averaged for eachCD spectrum in the range of 200–250 nm. The absorption spectrawere recorded on a double beam CARY 50-BIO UV–vis spectro-photometer (Varian, Australia) equipped with a 150 W Xenonlamp and a slit width of 5 nm. FT-IR spectra were acquired on aThermo Nicolet-5700 FTIR spectrometer (Waltham, MA, USA).

2.3. Procedures

2.3.1. Fluorescence studies

Based on preliminary investigations, the concentration of BSAwas kept constant at 5mM while that of the drug was varied from5 to 50mM. The fluorescent intensity of BSA was recorded at340 nm upon excitation at 296 nm. The interactions were carriedout at three different temperatures (296, 303, and 308 K).

2.3.2. Ultraviolet absorption studies

The UV–vis spectra were obtained by scanning the solution onthe spectrophotometer in the wavelength region of 250–300 nm.

2.3.3. Circular dichroism (CD) measurements

The CD spectra of BSA in presence and absence of FLX wererecorded in the range of 200–250 nm. The BSA to drug concentra-tion was varied (1:1, 1:4, 1:7 and 1:9) and the CD spectra wererecorded. The content of a-helix was calculated using thefollowing equation [4]:

% a� helix ¼ fð�½y�208 � 4000Þ=ð33;000� 4000Þg � 100

2.3.4. FT-IR Measurements

The FT-IR spectra of BSA in presence and absence of FLX at296 K were recorded in the range of 1500–1700 cm�1.

2.3.5. Displacement studies

The displacement experiments were performed using differentsite probes viz., warfarin, ibuprofen and digitoxin for site I, II andIII, respectively [5] by keeping the concentration of BSA and theprobe constant (5mM each). The fluorescence quenching titrationwas used as before to determine the binding constant of FLX–BSAin presence of above site probes.

2.3.6. Effect of common ions

The effects of some common ions viz., K+, Co+2, Cu+2 Ni+2, Ca+2

and Zn+2 were investigated on FLX–BSA interactions. Thefluorescence spectra of FLX–BSA system were recorded inpresence of above ions at 340 nm upon excitation at 296 nm.The overall concentration of BSA and that of the common ions wasfixed at 5mM.


3.1. Fluorescence quenching of BSA by FLX

The fluorescence spectroscopy was used to determine thenature of interaction between FLX and BSA. In BSA, tryptophanand tyrosine residues contribute to fluorescence spectra. Thedecrease in fluorescence intensity of a compound by a variety ofmolecular reactions viz., energy transfer, ground state complexformation, excited state reactions, collisional quenching andmolecular rearrangements is called quenching. In order to knowthe binding of FLX to BSA, the fluorescence spectra were recordedupon excitation at 296 nm (Fig. 2).

The fluorescence intensity of BSA decreased regularly with asmall blue shift in presence of increasing concentrations of FLX.This suggested that a complex was possibly formed between FLXand BSA which was responsible for quenching of fluorescence ofBSA and the possible change in the microenvironment aroundBSA. All these results indicated that there were strong interactionsbetween FLX and BSA.

In order to predict the possible quenching mechanism, thefluorescence quenching data were subjected to Stern–Volmeranalysis using the equation [6]

F0

F¼ 1þ KSV½Q � ¼ 1þ Kqt0½Q � ð1Þ

where F and F0 are the fluorescence intensity with and withoutthe quencher, respectively, Kq the quenching rate constant of thebio-molecule, KSV the Stern–Volmer quenching constant, t0 theaverage life time of bio-molecule without the quencher and [Q]the concentration of the quencher. Obviously,

KSV ¼ Kqt0 ð2Þ

The Stern–Volmer plots for the results of interactions carriedout at different temperatures (296, 303 and 308 K) were observedto be linear with slopes (KSV values) decreasing with increasingtemperature (Fig. 3). This indicated the presence of staticquenching mechanism in the interaction between FLX and BSA.Further, the values of Kq were evaluated using the Eq. (2). Thevalues of KSV and Kq are given in Table 1. The maximum scattercollision-quenching constant of various kinds of quenchers to

ARTICLE IN PRESS

1

1.1

1.2

1.3

1.4

1.5

1.6

0

Fo/F

5 10 15 20 25 30Concentration, μM

Fig. 3. The Stern–Volmer curves for quenching of FLX with BSA at 296 K ( ), 303 K

( ) and 309 K ( ).

Table 1KSV, Kq and thermodynamic parameters of FLX–BSA.

Temp.

(K)

KSV�10�3

(L M�1)

Kq� 10�11

(M�1 s�1)

DG0

(kJ mol�1)

DH0

(kJ mol�1)

DS0

(J mol�1 K�1)

296 2.1070.012 2.1070.012 �21.8

�90.3 �2.30�102303 1.7470.017 1.7470.017 �21.2

308 1.4470.023 1.4470.023 �18.9

-2-1.8-1.6-1.4-1.2

-1-0.8-0.6-0.4-0.2

0

-5.7log [Q]

log

[(Fo-

F)/F

]

-5.2 -4.7

Fig. 4. The plot of log(F0�F)/F vs. log[Q] for quenching of BSA by FLX at 296 K ( ),

303 K ( ) and 308 K ( ) (lex ¼ 296 nm; lem ¼ 340 nm. c(FLX) ¼ 5�50mM;

c(BSA) ¼ 5mM.

3

3.2

3.4

3.6

3.8

4

0.00321/T

log

K

0.00325 0.0033 0.00335 0.0034

Fig. 5. van’t Hoff plot for the binding of FLX with BSA.

U. Katrahalli et al. / Journal of Luminescence 130 (2010) 211–216 213

biopolymer is reported to be 1.0�1010 L mol–1 s–1 [6]. The highervalues of Kq noticed in the present investigation revealed that thequenching was not initiated by dynamic collision but from theformation of a complex. This phenomenon might be the result ofthe radiation less energy transfer between FLX and BSA.

3.2. Binding constant and number of binding sites

Fluorescence intensity data can also be used to obtain thebinding constant, K and the number of binding sites, n. Whensmall molecules bind independently to a set of equivalent sites ona macromolecule, the equilibrium between free and boundmolecules is given by the equation [7]

logðF0 � FÞ

F¼ logK þ nlog½Q � ð3Þ

The values of K and n were obtained from the intercept andslope of the plot of log[(F0�F)/F] vs. log[Q] (Fig. 4). The values of K

were found to be (7.0570.024)�103, (4.5870.041)�103 and(1.6070.039)�103 M�1 and those of n were noticed to be0.9370.013, 0.9270.011 and 0.9070.008, respectively, at 296,303 and 308 K. The decreased binding constant with increase intemperature, resulted possibly due to the reduction of thestability of FLX–BSA complex. The values of binding sites closeto unity indicated that there was only one independent class ofbinding site on BSA for FLX.

3.3. Thermodynamic parameters and the nature of binding forces

Considering the dependence of binding constant on tempera-ture, a thermodynamic process was considered to be responsiblefor the formation of the complex. Therefore, the thermodynamicparameters (enthalpy change DH0, entropy change DS0 and free

energy change DG0) dependent on temperatures were analyzed inorder to further characterize the acting forces between FLX andBSA, as these are the main evidences to propose the binding mode.Primarily, four types of forces take part in drug–protein interac-tion, viz., electrostatic forces, hydrophobic forces, van der Waalsinteractions and hydrogen bonding [7]. Signs and magnitudes ofthermodynamic parameters determine the nature of forcesactually taking part in protein–drug interaction. The bindingstudies were carried out at three different temperatures viz., 296,303 and 308 K and thermodynamic parameters were evaluatedusing the following van’t Hoff equation and Gibbs–Helmholtzequation:

logK ¼ �DH0=2:303RTþ DS0=2:303R ð4Þ

and

DG0 ¼ DH0 � TDS0 ð5Þ

The values of DH0 and DS0 were obtained from the slope andintercept of the plot of logK vs. 1/T (Fig. 5). The values of DH0, DS0

and DG0 are shown in Table 1. Since, both DH0 and DS0 arenegative, we propose that the van der Waals interactions andhydrogen bonding [8] are operating between drug and protein.

ARTICLE IN PRESS

0

100

200

300

400

500

600

310Wavelength, nm

Inte

nsity

0

0.01

0.02

0.03

0.04

0.05

0.06

Abs

oban

ce

a

b

360 410 460

Fig. 6. The overlap of fluorescence spectrum of BSA (a) and the absorbance

spectrum of FLX (b), {(BSA):(FLX) ¼ 1:1}.

0

0.04

0.08

0.12

0.16

250Wavelength, nm

Abs

orba

nce

a

d

x

260 270 280 290 300

Fig. 7. Absorption spectra of BSA, FLX and BSA–FLX system. BSA concentration was

at 5mM (a). FLX concentration for BSA–FLX system was at 5 (b), 10 (c) and 15mM

(d) A concentration of 5mM FLX (x) was used for FLX only.


Negative values of DG0 indicated the spontaneity of interaction atall the three temperatures.

3.4. Energy transfer between FLX and BSA

There is a considerable overlap between absorption spectrumof FLX (acceptor) and fluorescence spectrum of BSA (donor) whichforms the basis of fluorescence resonance energy transfer (FRET)(as shown in Fig. 6). Energy transfer phenomena have wideapplications in energy conversion process [9]. Photodynamicaction, which is often used in the treatment of cancer, is also aconsequence of energy transfer [10].

According to Forster’s non-radiative energy transfer theory[11], the energy transfer will happen under the followingconditions: (i) the donor can produce fluorescence light, (ii)fluorescence emission spectrum of the donor and UV absorptionspectrum of the acceptor have more overlap and (iii) the distancebetween the donor (BSA) and the acceptor (FLX) is lower than8 nm. The fluorescence quenching of BSA upon binding with FLXindicated the energy transfer between FLX and BSA. The efficiencyof energy transfer, E, was calculated using the equation

E ¼ 1�F

F0¼

R60

R60 þ r6

ð6Þ

where F and F0 are the fluorescence intensities of BSA in presenceand absence of FLX, r the distance between acceptor and donorand R0 the critical distance when the transfer efficiency is 50%.The value of R0 is calculated using the equation

R60 ¼ 8:8� 10�25k2N�4FJ ð7Þ

where k2 is the spatial orientation factor of the dipole, N therefractive index of the medium, F the fluorescence quantum yieldof the donor and J the overlap integral of the fluorescenceemission spectrum of the donor and the absorption spectrum ofthe acceptor. J is given by the equation

J ¼

PFðlÞeðlÞl4DlP

FðlÞDlð8Þ

where F(l) is the fluorescence intensity of the fluorescent donor ofwavelength, l, e(l) is the molar absorption coefficient of theacceptor at wavelength, l. For ligand–BSA interaction, K2

¼ 2/3,N ¼ 1.336 and F ¼ 0.15 [12]. The values of J, R0, E and r werecalculated to be 8.87�10�15 cm3 L mol�1, 1.70, 0.28 and 2 nm,respectively. Larger BSA–FLX distance, r compared to that of R0

observed in the present study also revealed the presence of staticquenching mechanism between drug and protein. [12,13]. Thevalue of ro8 nm [14] also indicated that the energy transfer hasoccurred from BSA to FLX with high possibility.

3.5. Absorption spectroscopic studies

UV–visible absorption spectroscopy is employed to explore thestructural change [7] and to know the formation of complexbetween the drug and protein [15]. The lmax of BSA observed ataround 280 nm was mainly due to the presence of tryptophan andtyrosine residues in BSA. It was evident from the spectrum of BSA(Fig. 7) that the absorption intensity of BSA increased regularlywith increasing concentration of FLX. Further, the blue shift inabsorption maximum indicated the change in polarity aroundtryptophan residue and changes in the peptide strand of BSAmolecule and hence the change in hydrophobicity [7,16].

3.6. Circular dichroism studies

Further evidence for conformational changes in BSA upon theaddition of FLX was obtained by circular dichroism spectroscopicstudies. CD spectra of BSA in presence and absence of variousamounts of FLX are shown in Fig. 8. CD spectrum of BSA exhibitedtwo bands at 208 and 222 nm, characteristic of a predominantlya-helical structure of BSA [17]. The addition of FLX, however, ledto a decrease in band intensity of CD spectra without anysignificant shift of the peaks, indicating thereby that theaddition of FLX changed the secondary structure of BSA.Moreover, the decrease in negative ellipticity meant that thepeptide strand extended even more, while the hydrophobicity wasdecreased.

The CD results were expressed in terms of mean residueelipticity (MRE) in deg cm2 dmol�1 according to the followingequation [18]:

MRE ¼observed CDðm degÞ

Cpnl� 10ð9Þ

where CP is the molar concentration of the protein, n the numberof amino acid residues and l the path length. The a-helicalcontents of free and bound BSA were calculated from MRE value at208 nm using the following equation [18]:

a-Helixð%Þ ¼�MRE208 � 4000

33;000� 4000� 100 ð10Þ

ARTICLE IN PRESS

-25000

-20000

-15000

-10000

-5000

0

5000

10000

200

Wavelength, nm

[θ] d

eg.c

m2 .

dmol

-1

a

e

210 220 230 240 250

Fig. 8. CD spectra of BSA–FLX system. BSA concentration was fixed at 5mM (a). In

BSA–FLX system, the FLX concentration was at 5 (b), 20 (c), 35 (d) and 45mM (e).

0.0015

0.0019

0.0023

0.0027

1500

Wavenumber, cm-1

Abs

orba

nce

1547

1653

0.0060

0.0068

0.0076

0.0084

1500Wavenumber, cm-1

Abs

orba

nce

1552

1664

1550 1600 1650 1700

1550 1600 1650 1700

Fig. 9. FT-IR spectra and difference spectra of BSA; (a) the FT-IR spectra of free BSA

(subtracting the absorption of the buffer solution from the spectrum of the protein

solution) and (b) the FT-IR difference spectra of BSA (subtracting the absorption of

the FLX-free form from that of FLX–BSA bound form) in phosphate buffer;

CBSA ¼ 5mM and CFLX ¼ 5mM.

Table 2The comparison of binding constants of FLX–BSA before and after the addition of

site probes.

K�10�3 without the

site probe (M�1)

K�10�2 with

warfarin (M�1)

K�10�3 with

ibuprofen (M�1)

K�10�3 with

digitoxin (M�1)

4.5870.041 2.7270.018 4.5070.024 4.1970.031

U. Katrahalli et al. / Journal of Luminescence 130 (2010) 211–216 215

where MRE208 is the observed MRE value at 208 nm, 4000 is theMRE of a-form and random coil conformation cross at 208 nm and33,000 is the MRE value of a pure a-helix at 208 nm. The a-helicityin the secondary structure of BSA was determined using Eq. (10).The a-helicity decreased from 60.87% in free BSA to 47.27% inBSA–FLX complex, which was indicative of the loss of a-helicityupon interaction. The decrease in a-helix structure indicated thatthe drug bound with the amino acid residues of the mainpolypeptide chain of protein and destroyed their hydrogenbonding networks [19]. The CD spectra of BSA in presence andabsence of FLX were observed to be similar in shape indicatingthat the structure of BSA was also predominantly a-helical [17]even after binding to FLX.

3.7. FT-IR spectroscopic studies

Additional evidence for FLX–BSA interaction was obtainedfrom FT-IR spectra. Infrared spectrum of protein exhibited anumber of amide bands due to different vibrations of the peptidemoiety. Of all the amide modes of the peptide group, the singlemost widely used one in studies of protein secondary structure isthe amide I. The amides I and II peaks occurred in the region of1600–1700 and 1500–1600 cm–1, respectively. Amide I band ismore sensitive to changes in protein secondary structurecompared to amide II. Hence, the amide I band is more usefulfor studies of secondary structure [20–23]. Hydrogen bonding andthe coupling between transition dipoles are amongst the mostimportant factors governing conformational sensitivity of amidebands. The protein amide I band appeared at 1653 cm�1 (mainlyC ¼ O stretch) and amide II band noticed at 1548 cm�1 (C–Nstretch coupled with N–H bending mode) have a relationship withthe secondary structure of the protein [24]. The FT-IR spectrum offree BSA in phosphate buffer solution and the difference spectraafter binding with FLX are given in Fig. 9. It was noticed that thepeak position of amide I was shifted from 1664 to 1653 cm�1,while that of amide II was shifted from 1552 to 1547 cm�1 in theIR spectrum of BSA upon interaction with FLX. This indicated thatthe FLX interacted with BSA and the secondary structure of BSAwas changed.

3.8. Site probe studies

Sudlow et al. [25] have suggested two main distinct bindingsites (sites I and II) in BSA. Site I of BSA has affinity for warfarin,

phenylbutazone, etc. and site II for ibuprofen, flufenamic acid, etc.It is reported that digitoxin binding is independent of sites I and II[26] and binds to site III. In order to establish the binding site inBSA for FLX, competitive binding studies were performed usingsite probes, warfarin, ibuprofen, and digitoxin. For this, emissionspectra of ternary mixtures of FLX, BSA and site probes wererecorded, separately. The corresponding binding constant valueswere evaluated and these are recorded in Table 2. The bindingconstant of FLX–BSA decreased remarkably in presence ofwarfarin while this value remained almost same in presence ofibuprofen and digitoxin. These results revealed that the warfarindisplaced FLX from the binding site while ibuprofen and digitoxinhad a little effect on the binding of FLX to BSA. Hence, we haveconcluded that the FLX was bound to site I of BSA.

3.9. Effect of metal ions on the interactions of FLX with BSA

In plasma, there are some metal ions, which can affect theinteractions of the drugs and serum albumins. Trace metal ions,especially the bivalent type are essential in the human body and

ARTICLE IN PRESS

Table 3Effect of common ions on binding constant of FLX–BSA.

System Association constant (M�1)

BSA+FLX 4.5870.011�103

BSA+FLX+K+ 2.6870.017�103

BSA+FLX+Co2+ 2.5170.025�104

BSA+FLX+Cu2+ 9.3670.022�103

BSA+FLX+Ni2+ 1.7470.012�103

BSA+FLX+Ca2+ 3.2470.016�103

BSA+FLX+Zn2+ 7.4170.004�103


play an important structural role in many proteins. It is reported[27] that K+, Cu2+, Zn2+, Ni2+, Co2+ and Ca2+ and other metal ionscan form complexes with serum albumins. Hence, the effects ofsome metal salt solutions viz., KCl, CuCl2, ZnCl2, NiCl2, CoCl2 andCaCl2 on the binding of FLX with BSA were investigated in thepresent study. Under the experimental conditions, none of thecation gave the precipitate in phosphate buffer. The bindingconstant of FLX–BSA in presence of above ions was evaluated andthe corresponding results are shown in Table 3. The bindingconstant of FLX–BSA system slightly decreased in presence of K+,Cu2+, Ni2+ and Ca2+. This was likely to be caused by aconformational change in the vicinity of the binding site. Thedecrease in the binding constant in presence of above metal ionswould shorten the storage time of the drug in blood plasma andhence more amount of free drug would be available in plasma[13]. This led to the need for more doses of drug to achieve thedesired therapeutic effect in presence of above ions. The bindingconstant increased slightly in presence of Co2+ and Zn2+, therebyindicating the strong binding between the drug and BSA andavailability of more drug for action. This led to the need for lessdose of drug for desired therapeutic effect.

4. Conclusions

In the present case, we have studied the interaction of FLX withBSA by employing various spectroscopic techniques. The resultsshowed that the BSA fluorescence was quenched by FLX throughthe static quenching mechanism. The thermodynamic parametersrevealed that the van der Waals forces and hydrogen bonding arethe main forces in the interaction process. The UV absorption, CDand FT-IR studies showed that there is a change in the secondarystructure of the protein after binding. The biological significanceof this work is evident since albumin serves as a carrier molecule

for multiple drugs and the interaction of FLX with albumin wasnot characterized earlier.

Acknowledgements

We are grateful to the Council of Scientific and IndustrialResearch, New Delhi, for financial assistance (No. 01(2279)/08/EMR-II). One of us (Umesha K) thanks the University GrantsCommission, New Delhi, for awarding the meritorious Fellowship.Thanks are also due to the authorities of the Karnatak University,Dharwad, for providing the necessary facilities.

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