Study on the interaction between amphiphilic drug and bovine serumalbumin: A thermodynamic and spectroscopic description

Malik Abdul Rub a,n, Javed Masood Khan b, Abdullah M. Asiri a,c,Rizwan Hasan Khan b, Kabir -ud-Din d

a Chemistry Department, Faculty of Science, King Abdulaziz University, Jeddah-21589, Saudi Arabiab Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, Indiac Center of Excellence for Advanced Materials Research, King Abdulaziz University, Jeddah-21589, Saudi Arabiad Department of Applied Chemistry, Aligarh Muslim University, Aligarh-202002, India

a r t i c l e i n f o

Article history:Received 6 March 2014Received in revised form28 May 2014Accepted 7 June 2014Available online 14 June 2014

Keywords:Bovine serum albuminAmphiphilic drugStatic quenchingThermodynamic parametersCircular dichroism

a b s t r a c t

Herein we report the interaction of amphiphilic drug clomipramine hydrochloride (CLP—a tricyclicantidepressant) with bovine serum albumin (BSA) studied by fluorescence, UV–vis, and circulardichroism (CD) spectroscopic techniques. Clomipramine hydrochloride is used to treat a variety ofmental health problems. The quenching rate constant (kq) values, calculated according to thefluorescence data, decrease with increase in temperature indicating the static quenching procedurefor the CLP–BSA interaction. The association binding constants (KA), evaluated at different conditions,and the thermodynamic parameters (free energy, enthalpy and entropy changes) indicate that thehydrophobic forces play a major role in the binding interaction of drug. The interaction of BSA with CLPwas further confirmed by UV absorption spectra. Blue shift of position was detected due to the complexformation between the BSA–CLP. The molecular distance, r0, between donor (BSA) and acceptor (CLP)was estimated by fluorescence resonance energy transfer (FRET) whose value (4.47 nm) suggests highprobability of static quenching interaction. The CD results prove the conformational changes in the BSAon binding with the drug. Thus, the results supply qualitative and quantitative understanding of thebinding of BSA to CLP, which is important in understanding their effect as therapeutic agents.

& 2014 Elsevier B.V. All rights reserved.

1. Introduction

The interaction between proteins and various kinds of pharma-ceuticals is imperative for wide range of pharmacological, biolo-gical, and clinical applications. Understanding the mechanism andrelated parameters of this kind of interaction, such as number andlocation of binding sites and binding constant(s), is essential forgaining insight regarding the pharmacodynamics and pharmaco-kinetics of a drug [1–3]. This includes providing informationconcerning the influence of binding to proteins on the absorption,elimination, distribution, and metabolic pathway of a drug. It isnoteworthy mentioning here that this influence appears chieflyduring transporting the drug in blood plasma to the targetedtissues via carrier proteins, such as serum albumins [4,5], whosestructures are vulnerable to pH, temperature, ionic strength, etc.

Many drugs, particularly those with local anesthetic, tranquil-lizer, antidepressant, and antibiotic, exercise their action by inter-actions with biological membranes. These compounds must becarried to their site of action and, usually, this function is achievedby globular protein serum albumins (blood carrier proteins) atwhich they bind/attach with different affinities. As such, strongbinding vis-à-vis weak binding plays a crucial role: the formercan lessen the concentration of free drug in plasma, while thelatter can lead to a low circulation time or poor distribution.In addition, binding processes modify the fragile equilibrium ofthe marginal stability of the native protein conformation [6],which is a delicate balance of various interactions such as vander Waals, electrostatic, hydrogen bonds, hydrophobic, and dis-ulfide bridges. There is, therefore, noteworthy need to furtherresearch and have a full knowledge of the extent and intensity ofthe interactions between plasma proteins and amphiphilic drugsin order to resolve the optimal dose of administration of thesecompounds and also to avoid irreversible structural changes inprotein molecules, which can lead to a deprivation of theirbiological activity and to side effects as well [7–9].

Contents lists available at ScienceDirect

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

Journal of Luminescence

http://dx.doi.org/10.1016/j.jlumin.2014.06.0090022-2313/& 2014 Elsevier B.V. All rights reserved.

n Corresponding author. Tel.: þ966 563671946.E-mail addresses: malikrub@gmail.com (M.A. Rub),

rizwanhkhan1@gmail.com (R.H. Khan).

Journal of Luminescence 155 (2014) 39–46

Various studies on serum albumins involving binding of smallmolecules, in particular fatty acids and amphiphiles/drugs, based ondifferent techniques (fluorescence spectroscopy, UV–vis absorptionspectroscopy, FTIR, Raman spectroscopy, CD spectroscopy, electro-chemistry, NMR, etc.) have been described [10–13]; when thesemolecules bind to a serum albumin, the intramolecular forcescreditworthy for sustaining the secondary structure can be altered,developing conformational changes in the protein [14].

Herein, we focus on studying the biophysical interactionsof clomipramine hydrochloride (CLP) with bovine serum albumin(BSA) using the fluorescence, UV absorption and circular dichroism(CD) techniques. Clomipramine hydrochloride (3-chloro-5-[3-(dimethylamino)propyl]-10,11-dihydro-5H-dibenz[b,f]azepinemonohydrochloride) (Scheme 1) is an antiobsessional drug thatbelongs to the class dibenzazepine of pharmacologic agentsknown as tricyclic antidepressants. The bovine serum albumin(BSA) presents 76% sequence resemblance with the human serumalbumin (HSA) [15,16]. One of the main differences within the twoproteins is that BSA has two tryptophan residues (Trp-134 andTrp-212) whereas HSA has only one (Trp-212). The additionaltryptophan residue in BSA is buried in a hydrophobic sack, whichlies near the surface of the albumin molecule in the second α-helixof the first domain [15]. Like others, BSA possesses a wide range ofphysiological functions associated with the binding, transport anddistribution of biologically active compounds.

Drug interactions at protein binding level notably affect impor-tant factors such as drug availability, drug efficacy, drug transport,elimination rate, etc. Hence, the studies on this aspect can furnishinformation of the structural features that influence the therapeu-tic effectiveness of drug, and have been an interesting researchfield in life sciences, chemistry and clinical medicine [17].In particular, our investigation is focused on quenching of BSAby CLP, determination of the binding constant and number ofbinding sites of the CLP–BSA system, energy transfer and bindingdistance between BSA and CLP, thermodynamic analysis andconformation changes of BSA upon binding to CLP.

2. Experimental

2.1. Materials

All starting materials were of analytical grade and doubledistilled water was used throughout. Clomipramine hydrochloride(CLP) with purity Z98% and bovine serum albumin (BSA, fattyacid free) were purchased from Sigma and used without furtherpurification. A 20 mM Tris-hydrochloride buffer (pH¼7.40) wasprepared by dissolving the required amount of Tris in water andadjusting pH with hydrochloric acid.

2.2. Apparatus

Fluorescence measurements were carried out with Shimadzu-RFPC5301 spectrofluorimeter equipped with a computer. Thefluorescence spectra were deliberated using a 1 cm path lengthcell and a thermostatically controlled cell holder attached toNeslab’s RTE—110 water bath with an accuracy of70.1 K.

The UV absorption measurements were carried out with adouble-beam Perkin Elmer Lambda 25 spectrophotometer using acuvette of 1 cm path length.

A Jasco spectropolarimeter, model J-720, equipped with amicrocomputer was used for recording the circular dichroism (CD)spectra in the Far UV-region.

2.3. Procedures

2.3.1. CLP–BSA interactionsThe steady-state fluorescence experiments were performed at a

constant concentration of BSA (2 mM) wherein the CLP concentra-tion was varied from 0 to 18 mM. The fluorescence spectra wererecorded at three temperatures (298, 310 and 318 K) in the rangeof 300–400 nm upon excitations at 280 and 295 nm.

2.3.2. UV measurementsThe UV spectroscopic measurements of BSA in the absence

and presence of CLP were made in the range of 245–310 nm. BSAconcentration was fixed at 10 mM while the drug concentrationwas varied from 20 to 135 mM.

2.3.3. Energy transfer between CLP and BSAThe absorption spectrum of CLP was recorded in the range of

300–450 nm. The emission spectrum of BSA was also recorded inthe range of 300–450 nm. Then, the overlap of the UV absorptionspectrum of CLP with the fluorescence emission spectrum of BSAwas used to calculate the energy transfer.

2.3.4. Circular dichroism (CD) measurementsThe CD measurements of BSA in presence and absence of CLP

were made in the range of 200–250 nm. Using a stock solution of5 mM BSA, solutions of molar ratios (BSA:CLP) 1:5, 1:10, 1:15 and1:20 were prepared for recording the CD spectra. The instrumentwas calibrated with d-10-camphorsulfonic acid. All the CD mea-surements were carried out at 303 K with a thermostaticallycontrolled cell holder attached to Neslab’s RTE-110 water bath.The spectra were collected with scan speed of 20 nm/min andresponse time of 1 s.

All the experiments were performed in Tris-hydrochloride bufferof pH 7.4 (20 mM). The concentration of protein was determinedspectrophotometrically using Є1%

1 cm of 6.5/M/cm at 280 nm.

.HCl

Scheme 1. Molecular model of clomipramine hydrochloride (CLP).

M.A. Rub et al. / Journal of Luminescence 155 (2014) 39–4640

3. Results and discussion

3.1. Measurement of fluorescence spectra

Because of its outstanding sensitivity, selectivity, reproducibil-ity, easy implication and vast theoretical foundation, fluorescencespectroscopy is an appropriate method to investigate interactionsbetween drugs and proteins [18], which can reveal the accessi-bility of drugs to fluorophore group of the protein and supply anunderstanding of binding mechanism to drugs, and yield clues tothe chemistry of the binding phenomena. Alterations of proteinmolecule can be followed via intrinsic (by emission of tryptophanresidue) or extrinsic (fluorescent probe) emitters bound to theprotein. The fluorescence quenching has been widely studied bothas a fundamental phenomenon and also for studying structuraldynamics of biochemical problems.

3.2. Quenching mechanism of BSA fluorescence by CLP

An useful feature of the intrinsic fluorescence of proteins is thehigh sensitivity of tryptophan and its local environment. Changesin the emission spectra of tryptophan are common in response toprotein conformational transitions, subunit associations, substratebinding, or denaturation [19]. Therefore, the intrinsic fluorescenceof proteins can provide considerable information on their struc-ture and dynamics and is often utilized in the study of proteinfolding and association reactions. The effect of CLP on fluorescenceintensity of BSA is shown in Fig. 1, which indicates the fluores-cence emission of BSA with different amounts of CLP following theexcitations at 280 and 295 nm. BSA shows strong fluorescenceemission at 338 or 339 nm. On titration of the serum albumin withthe drug solution (Fig. 1), the fluorescence intensity of BSAdecreases regularly with the increasing concentration of CLP dueto a variety of molecular interactions, viz., excited-state reactions,molecular rearrangements, energy transfer, ground state complexformation and collisional quenching; such decrease in fluores-cence intensity is known as quenching which indicates that thereare interactions between CLP and BSA. On exciting the protein at280 nm, light excites both tryptophan and tyrosine while 295 nmexcites only tryptophan groups; because of this the quenching ofthe fluorescence intensity of tryptophan residues is higher thanthat of the tyrosine residues, proposing that tryptophan residuescontribute significantly to the quenching of intrinsic serum albu-min fluorescence.

For further confirming the structural change of BSA by addition ofCLP, we measured the UV absorbance spectra of BSA with variousamounts of CLP having peak at 280 nm (Fig. 2). The absorptionintensity of BSA was enhanced as CLP increased, and there was adistinct blue shift of the CLP–BSA spectrum. The evidence from UVspectra recommended that the interaction between CLP and BSAchanged the microenvironment around BSA. The change in λmax

points the change in polarity around the tryptophan residue and thechange in peptide strand of serum albumin molecules and thereforethe change in hydrophobicity [20]. These above observations meanthat with the addition of CLP, the peptide strands of serum albuminmolecules were extended more and the hydrophobicity wasdecreased. As is well known, dynamic quenching only affects theexcited state of fluorophores but did not change the absorptionspectrum. However, the formation of a nonfluorescence ground stateof CLP induced a change in the absorption spectrum of fluorophoresand, therefore, the possible quenching mechanism of BSA by CLP is astatic quenching process [21].

For further elucidation of the quenching mechanism, fluores-cence quenching data were analyzed using the Stern–Volmer

equation [22].

F0F¼ 1þKSV ½Q � ¼ 1þkqτ0½Q � ð1Þ

kq ¼ KSV

τ0ð2Þ

where F0 and F are the steady-state fluorescence intensities in theabsence and presence of quencher, respectively, KSV is the Stern–Volmer quenching constant, kq stands for bimolecular quenchingconstant, τ0 for the life time of flurophore in the absence ofquencher and [Q] the concentration of quencher (i.e., drug).The graphs plotted as per the Stern–Volmer equation are shownin Fig. 3. The dynamic Stern–Volmer quenching constants (Ksv)were achieved by the slope of regression curves in the linearrange, and quenching rate constants kq were calculated based onthe fluorescence lifetime of biopolymers (about 10�8 s [23]). Theresults are recorded in Table 1.

There are two potential reasons for the small molecule quench-ing the serum albumin fluorescence—the static quenching and thedynamic quenching. Dynamic and static quenching are the differentmechanisms of quenching and they can be distinguished by theirdiffering dependence on temperature [22]. For the static quench-ing, quenching rate constants decrease with increasing tempera-ture, but the contrary effect is seen in the case of dynamicquenching [24].

It can be seen from Table 1 that the kq of BSA decreases with arise in temperature, thereby suggesting the presence of staticquenching [25]. In general, the maximum collision quenchingconstant (kq) of various kinds of biomolecules is 2.0�1010 L/mol/s.Moreover, the values of kq (which are in the order of 1012 forCLP–BSA) are far larger than 2.0�1010 L/mol/s, the maximumvalue reported for diffusion quenching rate constant of variousquenchers with the biopolymers [26]. So we propose the presenceof the occurrence of a static quenching interaction between BSAand CLP. This is an indication that quenching occurs via theformation of complex(es).

For BSA–CLP complexes, KSV values were slightly more at280 nm than at 295 nm. These results suggest that for BSA–CLPcomplexes the quenching of fluorescence intensity by tyrosineresidues is more effective in comparison to tryptophan residues.The noticed blue shift at maximum emission wavelength of theserum albumin is likely because of the loss of compact structure ofhydrophobic sub-domains where tryptophan was identified [19].Nevertheless, the micro-environment about the tyrosine residuesdid not undergo evident changes during the binding process.

3.3. Association binding constants and number of binding sites

On the basis of the above conclusion, it is postulated that thefluorescence quenching of BSA is a static quenching process.The static quenching can then be mathematically expressed byLineweaver–Burk formula [27]:

1F0�F

¼ 1F0

þ KD

F0½Q � ð3Þ

where KD is dissociation binding constant. The Lineweaver–Burkdouble-reciprocal plots were constructed based on the relation-ship (3) (see Fig. 4). From the regression equation of curves,association constants (KA¼1/KD) between CLP and BSA wereobtained (see Table 1). It is evident that the association constantvalues are high, indicating high affinity of CLP to BSA.

The Scatchard equation can be used to estimate the number ofthe binding sites between organic micromolecule and biologicalmacromolecule based on the above conclusion that the fluores-cence quenching is caused by the static quenching of compound

M.A. Rub et al. / Journal of Luminescence 155 (2014) 39–46 41

formation. It is described as follows [28]:

logF0�FF

¼ log KAþn log ½Q � ð4Þ

where KA represents the static association binding constant andn the number of binding sites, respectively. From a plot oflog F0�F=F versus log [Q], the KA of CLP with BSA and the bindingsites n can be obtained from the intercept and the slope (Table 1).The value of KA is significant to understand the distribution of thedrug in plasma since a weak binding can lead to a short lifetime or

poor distribution, as strong binding can lessen the concentrationof free drug in plasma. Larger values of KA observed in the presentstudy propose the presence of strong binding between CLP andBSA. Further, the association binding constant value increases withthe increase in temperature indicating the increased instabilityof the CLP–BSA complex with temperature. Also, the bindingcapability is more at 295 nm with respect to 280 nm. From thevalue of n, it was found that there is one independent class ofbinding sites on BSA for CLP, which indicates that a CLP molecule isbound to one BSA molecule.

Fig. 1. Fluorescence emission spectra of native BSA and BSA–CLP complexes, excited at 280 and 295 nm at different temperatures (298.15 K (A), 311.15 K (B), 318.15 K (C)).

M.A. Rub et al. / Journal of Luminescence 155 (2014) 39–4642

3.4. Determination of the binding force

Considering the dependence of binding constant on tempera-ture, a thermodynamic process was believed to be liable for theformation of a complex. Hence, the thermodynamic parametersdependent on temperature were studied in order to further

characterize the acting forces between CLP and BSA. As statedearlier, the working forces between a small molecule and macro-molecule mainly let in hydrogen bonds, van der Waals forces,electrostatic forces and hydrophobic interaction forces. The ther-modynamic parameters, free energy change (ΔG), enthalpy change(ΔH), and entropy change (ΔS), are the main evidences todetermine the binding mode which were evaluated using thefollowing equations:

ln KA ¼ �ΔHRT

þΔSR

ð5Þ

and

ΔG¼ΔH�TΔS¼ �2:303RT log KA ð6Þ

where R and T are the gas constant and temperature in Kelvinscale, respectively. The results obtained are shown in Table 1. Thenegative values of free energy (ΔG) show that the interactionprocess is spontaneous. Free energy (ΔG) is more at 295 nm withrespect to 280 nm (Table 1). It means that BSA–CLP binding ismore spontaneous at 295 nm as compared to 280 nm. Ross andSubramanian [29] have quantified the sign and magnitude of thethermodynamic parameters related with various individual kindsof interactions that may take place in the protein associationprocess, which can be easily resolved as: (i) ΔH40 and ΔS40,hydrophobic force; (ii) ΔHo0 and ΔSo0, van der Waals’ forceand hydrogen bonding; (iii) ΔHo0 and ΔS40, electrostatic inter-actions. Therefore, from the thermodynamic characteristics sum-marized above, the positive ΔH and ΔS values suggest thathydrophobic force plays the major role in the BSA–CLP bindinginteraction. The positive values of enthalpy (ΔH) and entropy (ΔS)

250 260 270 280 290 300 3100.0

0.1

0.2

0.3

0.4

0.5

Abs

orba

nce

Native BSA /10 μM 20 M CLP 45 70 90 119 135

λ /nm

μμM CLPμM CLPμM CLP

μM CLPμM CLP

Fig. 2. Ultraviolet absorbance spectra of native BSA and BSA–CLP complexes.

0.0 0.5 1.0 1.5 2.01.0

1.1

1.2

1.3

1.4

1.5

280 nm

F 0/F

F 0/F

[CLP] x 10-5 mol/L

Temperature /K 298.15 310.15 318.15

0.0 0.5 1.0 1.5 2.0

1.0

1.1

1.2

1.3

1.4

1.5

295 nm

[CLP] x 10-5 mol/L

Temperature /K 298.15 310.15 318.15

Fig. 3. The Stern–Volmer plots for BSA–CLP system at different temperatures as in case of Fig. 1.

Table 1Binding and thermodynamic parameters of BSA–CLP system at different temperatures.

T (K) KSV � 10�4 (L/mol) kq �10�12 (L/mol/s) R2 N KD �105 (mol/L) KA � 10�5 (L/mol) �ΔG (kJ/mol) ΔH (kJ/mol) ΔS (J/mol/K)

280 nm298.15 2.75 2.75 0.9965 0.89 17.97 0.05 8.77311.15 1.58 1.58 0.9945 0.96 12.55 0.08 11.44 57.52 222.35318.15 1.27 1.27 0.9993 1.21 0.35 2.83 13.22

295 nm298.15 2.33 2.33 0.9963 1.01 1.08 0.93 12.26311.15 1.26 1.26 0.9921 1.10 0.36 2.75 14.15 34.67 157.46318.15 0.90 0.90 0.9911 1.24 0.13 7.32 15.41

M.A. Rub et al. / Journal of Luminescence 155 (2014) 39–46 43

indicate that the binding of CLP and BSA is mainly entropy-driven,and the enthalpy is unfavorable for it. Thus, it can be concludedthat the hydrophobic force enacted a major role in the interaction,but it did not mean that the electrostatic interaction was omitted.

3.5. Energy transfer distance between CLP and BSA

According to Förster’s non-radiation energy transfer theory[30], the rate of energy transfer depends on: (i) the relativeorientation of the donor and acceptor dipoles, (ii) the extent ofoverlap of the emission spectrum of the donor with the absorptionspectrum of the acceptor, and (iii) the distance between the donorand the acceptor.

Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction in which excitation energy is transferrednonradiatively from the donor molecule to the acceptor. Usually,FRET occurs whenever the emission spectrum of a fluorophore(donor) overlaps with the absorption spectrum of another mole-cule (acceptor) and the distance between the donor and theacceptor is no longer than 7 nm [31,32]. The distance betweenthe donor and acceptor and extent of spectral overlap determinethe extent of energy transfer. Fig. 5 shows this overlap for theinvestigated donor–acceptor pair. The molecular distance r0 wasthen calculated for the BSA–CLP complex which is actually theaverage value between the bound CLP and the two tryptophanresidues [17].

The energy transfer effect is related not only to the distancebetween the acceptor and the donor, but also on the critical energytransfer distance R0, i.e.,

E¼ R60

R60þr60

ð7Þ

where E is the energy transfer efficiency between the donor andthe acceptor, r0 is the distance between the acceptor and the donorand the R0 is the critical distance when the transfer efficiency is50%, which can be calculated by

R60 ¼ 8:79x10�25K2n�4ΦJ ð8Þ

In Eq. (8), K2 is the space factor of orientation, n is the refractiveindex of medium, Φ is the fluorescence quantum yield of thedonor, and J is the overlap integral between the donor fluores-cence emission spectrum and the acceptor absorption spectrum.

Its value can be estimated by the following equation:

J ¼∑FðλÞεðλÞλ4Δλ∑FðλÞΔλ ð9Þ

where F(λ) is the fluorescence intensity of the fluorescent donor inwavelength λ and is dimensionless, and ε(λ) is the molar extinctioncoefficient of the acceptor in wavelength λ. The energy transferefficiency is calculated from the relative fluorescence yield in thepresence (F) and absence of acceptor (F0):

E¼ 1� FF0

ð10Þ

In the present case, K2¼2/3 [33], n¼1.336, and Φ¼0.15 [34].According to Eqs. (7)–(10), the overlap integral J, R0, E, and r0 canbe evaluated. The value of Jwas found to be 2.87�10�14 cm3/L/moland R0 to be 2.52 nm. The efficiency of energy transfer E was0.02129. The actual distance r0 was 4.47 nm. It can be seen that thedistance r0 is less than 7 nm, which indicates that energy transferfrom BSA to CLP occurs with high probability. At the same time, itis proved again that the fluorescence quenching of BSA is caused

0 2 4 6 8 10 12 14 160

20

40

60

80

100

120

140

160

280 nmF 0

/(F0-F

)

1/[CLP] x 105 L/mol

Temperature /K 298.15 310.15 318.15

0 2 4 6 8 10 12 14 160

100

200

300

400

500295 nm

F 0/(F 0-F

)

1/[CLP] x 105 L/mol

Temperature /K 298.15 310.15 318.15

Fig. 4. The Lineweaver–Burk plots for BSA–CLP system at different temperatures as in case of Fig. 1.

300 320 340 360 380 400 420 4400

100

200

300

400

500

600

700

800

900

1000

(b)

(a)

λ /nm

Inte

nsity

(a)

0.00

0.01

0.02

0.03

0.04

0.05

Abs

orba

nce

(b)

Fig. 5. Spectral overlap of BSA fluorescence (a) with CLP absorption (b) (CBSA/CCLP¼1:1at pH¼7.40).

M.A. Rub et al. / Journal of Luminescence 155 (2014) 39–4644

by the static quenching of compound formation because the valueof r0 obtained in present study is less than R0 [35,36]. BSA has twotryptophan residues: trp-212 is located in a hydrophobic fold andthe additional tryptophan (trp-134) is located on the surface of themolecule. In this study, CLP was probably bound to the trp-212residue mainly through the hydrophobic interaction according tothe thermodynamic results. However, an interaction between trp-134 and CLP cannot be ruled out and, therefore, the distancecalculated here is actually the average distance between CLP andthe two tryptophan residues in BSA.

3.6. Circular dichroism (CD) studies

Circular dichroism (CD) spectroscopy is a well-establishedmethod in biological chemistry and structural biology, with aprogressively wide range of applications. It is a technique that playsan important role in the characterization of proteins, particularly forsecondary structure (e.g., α-helix, β-sheet) determination. Informa-tions related to the polypeptide backbone conformations of proteinsare provided by the spectra in the ultraviolet wavelength range(typically from 200 to 250 nm). The CD measurements for the drug-serum albumin complexes were made in the range of 200–250 nmusing a 0.1 cm path length cell. The molar ratio for all serumalbumin/drug complexes was in between 1:5 and 1:20.

The CD results are expressed in terms of mean residualellipticity (MRE) in deg/cm2/dmol according to the equation:

MRE¼ observed CD ðmdegÞCPnl� 10

ð11Þ

where Cp is the molar concentration of the protein, n the numberof amino acid residues and l is the path length of the cell.

The secondary structure was estimated from spectra recordedbetween 200 and 247 nm using K2d CD secondary structure server,which uses an unsupervised neutral network to predict secondarystructure [25].

In solution, far-UV CD spectrum of serum albumin is character-ized by the presence of two minima at 222 nm and at 208 nmassociated with the existence of predominant α-helical structure inthe protein molecule. The BSA concentration used in this studywas 5 mM. The acquired data indicate that the secondary structureof free BSA consists of �67% of α-helix, �8% of β-sheets, and�25% of random coils (RC) (Fig. 6). In all the BSA–CLP complexes at1:5, 1:10, 1:15 and 1:20 M ratios, α-helical structure is induced andβ-sheets reduced (Table 2). The values of the negative peaksslightly increase at lower concentration of drug (lower ratio),which denotes that the secondary structure is not greatly affected.At higher ratios (higher drug concentration), a more severeincrease of the ellipticity is noted which arises from a foldingprocess underwent by the protein in the presence of excess drugmolecules. This process involves an important gain of α-helixcontent in the secondary structure of the protein in contrast to alittle loss in β-sheet and an extensive ordered conformation for thedrug, as seen in Table 2 after deconvolution of CD spectra. Thiscorroborates that, as the concentration of drug increases, theprotein folding occurs, which can be facilitated by attractiveelectrostatic interactions between the drug molecule bound tothe exposed binding sites. Furthermore, the CD spectra of BSA inthe presence and absence of CLP molecules were observed to besimilar in shape indicating that the structure of bovine serumalbumin was also predominantly α-helical [37].

4. Conclusions

In present study the mechanism of CLP interacting with BSA wasinvestigated by various spectroscopic methods under physiological

conditions (i.e., pH 7.4). The knowledge of the binding character-istics of biomolecules and drugs plays an important role in under-standing the biological process. The biological significance of thiswork is evident since albumin serves as a carrier molecule formultiple drugs and the interaction of CLP with albumin is notcharacterized so far. Hence, this report has a great impact inpharmacology and clinical medicine as well as methodology.Experimental results indicated that CLP could bind with the serumalbumin and quench the fluorescence of serum albumin. Based onthe Stern–Volmer equation, the quenching rate constants wereevaluated and their values suggested that the fluorescence quench-ing was a static process. The negative value of ΔG implies thespontaneity of the complexation. In addition, the values of enthalpychange and the entropy change suggested that the hydrophobicforces played a major role in the interaction between CLP andBSA. Based on the theory of the FRET, the energy transfer efficiency(E) and the molecular distance (r0) between CLP and BSA werecalculated. The intensity of negative CD bands at 208 and 220 nmdiffered in the presence of drug due to changes in the chemicalenvironment of α-helices lying at the surface of the protein, whichsuggested change in secondary structure of the protein.

Acknowledgements

This work was funded by the Deanship of Scientific Research(DSR), King Abdulaziz University, Jeddah, under grant no. (130-059-D1434). The authors, therefore, acknowledge with thanks DSRtechnical and financial support.

205 210 215 220 225 230 235 240 245-35000

-30000

-25000

-20000

-15000

-10000

-5000

0

MR

E /d

eg c

m2 d

mol

-1

Native BSA 25 50 75 100

λ /nm

μM CLPμM CLPμM CLP

μM CLP

Fig. 6. CD spectra of native BSA and BSA–CLP complexes.

Table 2Secondary structural (i.e., α-helix, β-sheets, and random coil (RC)) analysis for thenative BSA and its complexes with CLP.

Molar ratio of drug: BSA Conc. of drug (mM) α (%) β (%) RC (%)

Native BSA (5 mM) 0 67 8 25BSA:CLP1:5 25 68 7 251:10 50 70 5 251:15 75 74 2 241:20 100 76 1 23

M.A. Rub et al. / Journal of Luminescence 155 (2014) 39–46 45

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