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

Journal of Luminescence 155 (2014) 39–46

Contents lists available at ScienceDirect

Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin

Study on the interaction between amphiphilic drug and bovine serum albumin: 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 Arabia Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India c Center of Excellence for Advanced Materials Research, King Abdulaziz University, Jeddah-21589, Saudi Arabia d Department of Applied Chemistry, Aligarh Muslim University, Aligarh-202002, India b

art ic l e i nf o

a b s t r a c t

Article history: Received 6 March 2014 Received in revised form 28 May 2014 Accepted 7 June 2014 Available online 14 June 2014

Herein we report the interaction of amphiphilic drug clomipramine hydrochloride (CLP—a tricyclic antidepressant) with bovine serum albumin (BSA) studied by fluorescence, UV–vis, and circular dichroism (CD) spectroscopic techniques. Clomipramine hydrochloride is used to treat a variety of mental health problems. The quenching rate constant (kq) values, calculated according to the fluorescence data, decrease with increase in temperature indicating the static quenching procedure for 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 the hydrophobic forces play a major role in the binding interaction of drug. The interaction of BSA with CLP was further confirmed by UV absorption spectra. Blue shift of position was detected due to the complex formation 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 high probability of static quenching interaction. The CD results prove the conformational changes in the BSA on binding with the drug. Thus, the results supply qualitative and quantitative understanding of the binding of BSA to CLP, which is important in understanding their effect as therapeutic agents. & 2014 Elsevier B.V. All rights reserved.

Keywords: Bovine serum albumin Amphiphilic drug Static quenching Thermodynamic parameters Circular dichroism

1. Introduction The interaction between proteins and various kinds of pharmaceuticals is imperative for wide range of pharmacological, biological, and clinical applications. Understanding the mechanism and related parameters of this kind of interaction, such as number and location of binding sites and binding constant(s), is essential for gaining insight regarding the pharmacodynamics and pharmacokinetics of a drug [1–3]. This includes providing information concerning the influence of binding to proteins on the absorption, elimination, distribution, and metabolic pathway of a drug. It is noteworthy mentioning here that this influence appears chiefly during transporting the drug in blood plasma to the targeted tissues via carrier proteins, such as serum albumins [4,5], whose structures are vulnerable to pH, temperature, ionic strength, etc.

n

Corresponding author. Tel.: þ 966 563671946. E-mail addresses: [email protected] (M.A. Rub), [email protected] (R.H. Khan). http://dx.doi.org/10.1016/j.jlumin.2014.06.009 0022-2313/& 2014 Elsevier B.V. All rights reserved.

Many drugs, particularly those with local anesthetic, tranquillizer, antidepressant, and antibiotic, exercise their action by interactions with biological membranes. These compounds must be carried to their site of action and, usually, this function is achieved by globular protein serum albumins (blood carrier proteins) at which they bind/attach with different affinities. As such, strong binding vis-à-vis weak binding plays a crucial role: the former can lessen the concentration of free drug in plasma, while the latter can lead to a low circulation time or poor distribution. In addition, binding processes modify the fragile equilibrium of the marginal stability of the native protein conformation [6], which is a delicate balance of various interactions such as van der Waals, electrostatic, hydrogen bonds, hydrophobic, and disulfide bridges. There is, therefore, noteworthy need to further research and have a full knowledge of the extent and intensity of the interactions between plasma proteins and amphiphilic drugs in order to resolve the optimal dose of administration of these compounds and also to avoid irreversible structural changes in protein molecules, which can lead to a deprivation of their biological activity and to side effects as well [7–9].

40

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

Various studies on serum albumins involving binding of small molecules, in particular fatty acids and amphiphiles/drugs, based on different techniques (fluorescence spectroscopy, UV–vis absorption spectroscopy, FTIR, Raman spectroscopy, CD spectroscopy, electrochemistry, NMR, etc.) have been described [10–13]; when these molecules bind to a serum albumin, the intramolecular forces creditworthy for sustaining the secondary structure can be altered, developing conformational changes in the protein [14]. Herein, we focus on studying the biophysical interactions of 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]azepine monohydrochloride) (Scheme 1) is an antiobsessional drug that belongs to the class dibenzazepine of pharmacologic agents known as tricyclic antidepressants. The bovine serum albumin (BSA) presents 76% sequence resemblance with the human serum albumin (HSA) [15,16]. One of the main differences within the two proteins is that BSA has two tryptophan residues (Trp-134 and Trp-212) whereas HSA has only one (Trp-212). The additional tryptophan residue in BSA is buried in a hydrophobic sack, which lies near the surface of the albumin molecule in the second α-helix of the first domain [15]. Like others, BSA possesses a wide range of physiological functions associated with the binding, transport and distribution of biologically active compounds. Drug interactions at protein binding level notably affect important factors such as drug availability, drug efficacy, drug transport, elimination rate, etc. Hence, the studies on this aspect can furnish information of the structural features that influence the therapeutic effectiveness of drug, and have been an interesting research field in life sciences, chemistry and clinical medicine [17]. In particular, our investigation is focused on quenching of BSA by CLP, determination of the binding constant and number of binding sites of the CLP–BSA system, energy transfer and binding distance between BSA and CLP, thermodynamic analysis and conformation changes of BSA upon binding to CLP.

2. Experimental 2.1. Materials All starting materials were of analytical grade and double distilled water was used throughout. Clomipramine hydrochloride (CLP) with purity Z98% and bovine serum albumin (BSA, fatty acid free) were purchased from Sigma and used without further purification. A 20 mM Tris-hydrochloride buffer (pH ¼7.40) was prepared by dissolving the required amount of Tris in water and adjusting pH with hydrochloric acid.

2.2. Apparatus Fluorescence measurements were carried out with ShimadzuRFPC5301 spectrofluorimeter equipped with a computer. The fluorescence spectra were deliberated using a 1 cm path length cell and a thermostatically controlled cell holder attached to Neslab’s RTE—110 water bath with an accuracy of70.1 K. The UV absorption measurements were carried out with a double-beam Perkin Elmer Lambda 25 spectrophotometer using a cuvette of 1 cm path length. A Jasco spectropolarimeter, model J-720, equipped with a microcomputer was used for recording the circular dichroism (CD) spectra in the Far UV-region.

.HCl

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

2.3. Procedures 2.3.1. CLP–BSA interactions The steady-state fluorescence experiments were performed at a constant concentration of BSA (2 mM) wherein the CLP concentration was varied from 0 to 18 mM. The fluorescence spectra were recorded at three temperatures (298, 310 and 318 K) in the range of 300–400 nm upon excitations at 280 and 295 nm.

2.3.2. UV measurements The UV spectroscopic measurements of BSA in the absence and presence of CLP were made in the range of 245–310 nm. BSA concentration was fixed at 10 mM while the drug concentration was varied from 20 to 135 mM.

2.3.3. Energy transfer between CLP and BSA The absorption spectrum of CLP was recorded in the range of 300–450 nm. The emission spectrum of BSA was also recorded in the range of 300–450 nm. Then, the overlap of the UV absorption spectrum of CLP with the fluorescence emission spectrum of BSA was used to calculate the energy transfer.

2.3.4. Circular dichroism (CD) measurements The CD measurements of BSA in presence and absence of CLP were made in the range of 200–250 nm. Using a stock solution of 5 mM BSA, solutions of molar ratios (BSA:CLP) 1:5, 1:10, 1:15 and 1:20 were prepared for recording the CD spectra. The instrument was calibrated with d-10-camphorsulfonic acid. All the CD measurements were carried out at 303 K with a thermostatically controlled cell holder attached to Neslab’s RTE-110 water bath. The spectra were collected with scan speed of 20 nm/min and response time of 1 s. All the experiments were performed in Tris-hydrochloride buffer of pH 7.4 (20 mM). The concentration of protein was determined spectrophotometrically using Є11%cm of 6.5/M/cm at 280 nm.

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

3. Results and discussion

equation [22].

3.1. Measurement of fluorescence spectra

F0 ¼ 1 þ K SV ½Q  ¼ 1 þ kq τ0 ½Q  F

Because of its outstanding sensitivity, selectivity, reproducibility, easy implication and vast theoretical foundation, fluorescence spectroscopy is an appropriate method to investigate interactions between drugs and proteins [18], which can reveal the accessibility of drugs to fluorophore group of the protein and supply an understanding of binding mechanism to drugs, and yield clues to the chemistry of the binding phenomena. Alterations of protein molecule can be followed via intrinsic (by emission of tryptophan residue) or extrinsic (fluorescent probe) emitters bound to the protein. The fluorescence quenching has been widely studied both as a fundamental phenomenon and also for studying structural dynamics of biochemical problems.

3.2. Quenching mechanism of BSA fluorescence by CLP An useful feature of the intrinsic fluorescence of proteins is the high sensitivity of tryptophan and its local environment. Changes in the emission spectra of tryptophan are common in response to protein conformational transitions, subunit associations, substrate binding, or denaturation [19]. Therefore, the intrinsic fluorescence of proteins can provide considerable information on their structure and dynamics and is often utilized in the study of protein folding and association reactions. The effect of CLP on fluorescence intensity of BSA is shown in Fig. 1, which indicates the fluorescence emission of BSA with different amounts of CLP following the excitations at 280 and 295 nm. BSA shows strong fluorescence emission at 338 or 339 nm. On titration of the serum albumin with the drug solution (Fig. 1), the fluorescence intensity of BSA decreases regularly with the increasing concentration of CLP due to a variety of molecular interactions, viz., excited-state reactions, molecular rearrangements, energy transfer, ground state complex formation and collisional quenching; such decrease in fluorescence intensity is known as quenching which indicates that there are interactions between CLP and BSA. On exciting the protein at 280 nm, light excites both tryptophan and tyrosine while 295 nm excites only tryptophan groups; because of this the quenching of the fluorescence intensity of tryptophan residues is higher than that of the tyrosine residues, proposing that tryptophan residues contribute significantly to the quenching of intrinsic serum albumin fluorescence. For further confirming the structural change of BSA by addition of CLP, we measured the UV absorbance spectra of BSA with various amounts of CLP having peak at 280 nm (Fig. 2). The absorption intensity of BSA was enhanced as CLP increased, and there was a distinct blue shift of the CLP–BSA spectrum. The evidence from UV spectra recommended that the interaction between CLP and BSA changed the microenvironment around BSA. The change in λmax points the change in polarity around the tryptophan residue and the change in peptide strand of serum albumin molecules and therefore the change in hydrophobicity [20]. These above observations mean that with the addition of CLP, the peptide strands of serum albumin molecules were extended more and the hydrophobicity was decreased. As is well known, dynamic quenching only affects the excited state of fluorophores but did not change the absorption spectrum. However, the formation of a nonfluorescence ground state of CLP induced a change in the absorption spectrum of fluorophores and, therefore, the possible quenching mechanism of BSA by CLP is a static quenching process [21]. For further elucidation of the quenching mechanism, fluorescence quenching data were analyzed using the Stern–Volmer

kq ¼

K SV τ0

41

ð1Þ ð2Þ

where F0 and F are the steady-state fluorescence intensities in the absence and presence of quencher, respectively, KSV is the Stern– Volmer quenching constant, kq stands for bimolecular quenching constant, τ0 for the life time of flurophore in the absence of quencher and [Q] the concentration of quencher (i.e., drug). The graphs plotted as per the Stern–Volmer equation are shown in Fig. 3. The dynamic Stern–Volmer quenching constants (Ksv) were achieved by the slope of regression curves in the linear range, and quenching rate constants kq were calculated based on the fluorescence lifetime of biopolymers (about 10  8 s [23]). The results are recorded in Table 1. There are two potential reasons for the small molecule quenching the serum albumin fluorescence—the static quenching and the dynamic quenching. Dynamic and static quenching are the different mechanisms of quenching and they can be distinguished by their differing dependence on temperature [22]. For the static quenching, quenching rate constants decrease with increasing temperature, but the contrary effect is seen in the case of dynamic quenching [24]. It can be seen from Table 1 that the kq of BSA decreases with a rise in temperature, thereby suggesting the presence of static quenching [25]. In general, the maximum collision quenching constant (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 for CLP–BSA) are far larger than 2.0  1010 L/mol/s, the maximum value reported for diffusion quenching rate constant of various quenchers with the biopolymers [26]. So we propose the presence of the occurrence of a static quenching interaction between BSA and CLP. This is an indication that quenching occurs via the formation of complex(es). For BSA–CLP complexes, KSV values were slightly more at 280 nm than at 295 nm. These results suggest that for BSA–CLP complexes the quenching of fluorescence intensity by tyrosine residues is more effective in comparison to tryptophan residues. The noticed blue shift at maximum emission wavelength of the serum albumin is likely because of the loss of compact structure of hydrophobic sub-domains where tryptophan was identified [19]. Nevertheless, the micro-environment about the tyrosine residues did 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 the fluorescence quenching of BSA is a static quenching process. The static quenching can then be mathematically expressed by Lineweaver–Burk formula [27]: 1 1 KD ¼ þ F 0 F F 0 F 0 ½Q 

ð3Þ

where KD is dissociation binding constant. The Lineweaver–Burk double-reciprocal plots were constructed based on the relationship (3) (see Fig. 4). From the regression equation of curves, association constants (KA ¼1/KD) between CLP and BSA were obtained (see Table 1). It is evident that the association constant values are high, indicating high affinity of CLP to BSA. The Scatchard equation can be used to estimate the number of the binding sites between organic micromolecule and biological macromolecule based on the above conclusion that the fluorescence quenching is caused by the static quenching of compound

42

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

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)).

formation. It is described as follows [28]: log

F0  F ¼ log K A þn log ½Q  F

ð4Þ

where KA represents the static association binding constant and n the number of binding sites, respectively. From a plot of log F 0  F=F versus log [Q], the KA of CLP with BSA and the binding sites n can be obtained from the intercept and the slope (Table 1). The value of KA is significant to understand the distribution of the drug in plasma since a weak binding can lead to a short lifetime or

poor distribution, as strong binding can lessen the concentration of free drug in plasma. Larger values of KA observed in the present study propose the presence of strong binding between CLP and BSA. Further, the association binding constant value increases with the increase in temperature indicating the increased instability of the CLP–BSA complex with temperature. Also, the binding capability is more at 295 nm with respect to 280 nm. From the value of n, it was found that there is one independent class of binding sites on BSA for CLP, which indicates that a CLP molecule is bound to one BSA molecule.

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

3.4. Determination of the binding force Considering the dependence of binding constant on temperature, a thermodynamic process was believed to be liable for the formation of a complex. Hence, the thermodynamic parameters dependent on temperature were studied in order to further 0.5

43

characterize the acting forces between CLP and BSA. As stated earlier, the working forces between a small molecule and macromolecule mainly let in hydrogen bonds, van der Waals forces, electrostatic forces and hydrophobic interaction forces. The thermodynamic parameters, free energy change (ΔG), enthalpy change (ΔH), and entropy change (ΔS), are the main evidences to determine the binding mode which were evaluated using the following equations: ln K A ¼ 

ΔH ΔS þ RT R

ð5Þ

and

0.4

ð6Þ

Absorbance

ΔG ¼ ΔH  TΔS ¼  2:303RT log K A

where R and T are the gas constant and temperature in Kelvin scale, respectively. The results obtained are shown in Table 1. The negative values of free energy (ΔG) show that the interaction process is spontaneous. Free energy (ΔG) is more at 295 nm with respect to 280 nm (Table 1). It means that BSA–CLP binding is more spontaneous at 295 nm as compared to 280 nm. Ross and Subramanian [29] have quantified the sign and magnitude of the thermodynamic parameters related with various individual kinds of interactions that may take place in the protein association process, which can be easily resolved as: (i) ΔH4 0 and ΔS 40, hydrophobic force; (ii) ΔHo0 and ΔS o0, van der Waals’ force and hydrogen bonding; (iii) ΔH o0 and ΔS 40, electrostatic interactions. Therefore, from the thermodynamic characteristics summarized above, the positive ΔH and ΔS values suggest that hydrophobic force plays the major role in the BSA–CLP binding interaction. The positive values of enthalpy (ΔH) and entropy (ΔS)

0.3

0.2

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

0.1

0.0 250

260

270

280

290

300

310

λ /nm Fig. 2. Ultraviolet absorbance spectra of native BSA and BSA–CLP complexes. 1.5

1.5

280 nm Temperature /K 298.15 310.15 318.15

1.4

295 nm

Temperature /K 298.15 310.15 318.15

1.4

1.3

F0/F

F0/F

1.3

1.2

1.2

1.1

1.1

1.0 1.0 0.0

0.5

1.0

1.5

2.0

0.0

0.5

1.0

1.5

2.0

-5

-5

[CLP] x 10 mol/L

[CLP] x 10 mol/L

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

Table 1 Binding and thermodynamic parameters of BSA–CLP system at different temperatures. T (K)

KSV  10  4 (L/mol)

kq  10  12 (L/mol/s)

298.15 311.15 318.15

2.75 1.58 1.27

2.75 1.58 1.27

298.15 311.15 318.15

2.33 1.26 0.90

2.33 1.26 0.90

R2 280 nm 0.9965 0.9945 0.9993 295 nm 0.9963 0.9921 0.9911

N

KD  105 (mol/L)

KA  10  5 (L/mol)

 ΔG (kJ/mol)

ΔH (kJ/mol)

ΔS (J/mol/K)

0.89 0.96 1.21

17.97 12.55 0.35

0.05 0.08 2.83

8.77 11.44 13.22

57.52

222.35

1.01 1.10 1.24

1.08 0.36 0.13

0.93 2.75 7.32

12.26 14.15 15.41

34.67

157.46

44

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

160

295 nm

280 nm

120

Temperature /K 298.15 310.15 318.15

500

Temperature /K 298.15 310.15 318.15

140

400

F0/(F0-F)

F0 /(F0-F)

100 80 60

300

200

40 100 20 0

0 0

2

4

6

8

10

12

14

16

0

2

4

5

6

8

10

12

14

16

5

1/[CLP] x 10 L/mol

1/[CLP] x 10 L/mol

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

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

0.05 1000

(a)

900 0.04

800

3.5. Energy transfer distance between CLP and BSA

E¼

R60 6 R0 þ r 60

ð7Þ

where E is the energy transfer efficiency between the donor and the acceptor, r0 is the distance between the acceptor and the donor and the R0 is the critical distance when the transfer efficiency is 50%, which can be calculated by R60

¼ 8:79x10

 25

2

K n

4

ΦJ

ð8Þ

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

0.03

600 500

0.02

400

(b)

Absorbance (b)

According to Förster’s non-radiation energy transfer theory [30], the rate of energy transfer depends on: (i) the relative orientation of the donor and acceptor dipoles, (ii) the extent of overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor, and (iii) the distance between the donor and the acceptor. Fluorescence resonance energy transfer (FRET) is a distancedependent interaction in which excitation energy is transferred nonradiatively 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 molecule (acceptor) and the distance between the donor and the acceptor is no longer than 7 nm [31,32]. The distance between the donor and acceptor and extent of spectral overlap determine the extent of energy transfer. Fig. 5 shows this overlap for the investigated donor–acceptor pair. The molecular distance r0 was then calculated for the BSA–CLP complex which is actually the average value between the bound CLP and the two tryptophan residues [17]. The energy transfer effect is related not only to the distance between the acceptor and the donor, but also on the critical energy transfer distance R0, i.e.,

Intensity (a)

700

300 0.01

200 100 0 300

0.00 320

340

360

380

400

420

440

λ /nm Fig. 5. Spectral overlap of BSA fluorescence (a) with CLP absorption (b) (CBSA/CCLP ¼1:1 at pH¼ 7.40).

Its value can be estimated by the following equation: J¼

∑FðλÞεðλÞλ4 Δλ ∑FðλÞΔλ

ð9Þ

where F(λ) is the fluorescence intensity of the fluorescent donor in wavelength λ and is dimensionless, and ε(λ) is the molar extinction coefficient of the acceptor in wavelength λ. The energy transfer efficiency is calculated from the relative fluorescence yield in the presence (F) and absence of acceptor (F0): E ¼ 1

F F0

ð10Þ

In the present case, K2 ¼ 2/3 [33], n ¼1.336, and Φ¼ 0.15 [34]. According to Eqs. (7)–(10), the overlap integral J, R0, E, and r0 can be evaluated. The value of J was found to be 2.87  10  14 cm3/L/mol and R0 to be 2.52 nm. The efficiency of energy transfer E was 0.02129. The actual distance r0 was 4.47 nm. It can be seen that the distance r0 is less than 7 nm, which indicates that energy transfer from BSA to CLP occurs with high probability. At the same time, it is proved again that the fluorescence quenching of BSA is caused

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

3.6. Circular dichroism (CD) studies Circular dichroism (CD) spectroscopy is a well-established method in biological chemistry and structural biology, with a progressively wide range of applications. It is a technique that plays an important role in the characterization of proteins, particularly for secondary structure (e.g., α-helix, β-sheet) determination. Informations related to the polypeptide backbone conformations of proteins are provided by the spectra in the ultraviolet wavelength range (typically from 200 to 250 nm). The CD measurements for the drugserum albumin complexes were made in the range of 200–250 nm using a 0.1 cm path length cell. The molar ratio for all serum albumin/drug complexes was in between 1:5 and 1:20. The CD results are expressed in terms of mean residual ellipticity (MRE) in deg/cm2/dmol according to the equation: MRE ¼

observed CD ðmdegÞ C P nl  10

ð11Þ

where Cp is the molar concentration of the protein, n the number of amino acid residues and l is the path length of the cell. The secondary structure was estimated from spectra recorded between 200 and 247 nm using K2d CD secondary structure server, which uses an unsupervised neutral network to predict secondary structure [25]. In solution, far-UV CD spectrum of serum albumin is characterized by the presence of two minima at 222 nm and at 208 nm associated with the existence of predominant α-helical structure in the protein molecule. The BSA concentration used in this study was 5 mM. The acquired data indicate that the secondary structure of 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 at 1:5, 1:10, 1:15 and 1:20 M ratios, α-helical structure is induced and β-sheets reduced (Table 2). The values of the negative peaks slightly 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 severe increase of the ellipticity is noted which arises from a folding process underwent by the protein in the presence of excess drug molecules. This process involves an important gain of α-helix content in the secondary structure of the protein in contrast to a little loss in β-sheet and an extensive ordered conformation for the drug, as seen in Table 2 after deconvolution of CD spectra. This corroborates that, as the concentration of drug increases, the protein folding occurs, which can be facilitated by attractive electrostatic interactions between the drug molecule bound to the exposed binding sites. Furthermore, the CD spectra of BSA in the presence and absence of CLP molecules were observed to be similar in shape indicating that the structure of bovine serum albumin was also predominantly α-helical [37].

4. Conclusions In present study the mechanism of CLP interacting with BSA was investigated by various spectroscopic methods under physiological

0

-5000

Native BSA 25 μM CLP 50 μM CLP 75 μM CLP 100 μM CLP

-1

-10000

-15000

2

MRE /deg cm dmol

by the static quenching of compound formation because the value of r0 obtained in present study is less than R0 [35,36]. BSA has two tryptophan residues: trp-212 is located in a hydrophobic fold and the additional tryptophan (trp-134) is located on the surface of the molecule. In this study, CLP was probably bound to the trp-212 residue mainly through the hydrophobic interaction according to the thermodynamic results. However, an interaction between trp134 and CLP cannot be ruled out and, therefore, the distance calculated here is actually the average distance between CLP and the two tryptophan residues in BSA.

45

-20000

-25000

-30000

-35000 205

210

215

220

225

230

235

240

245

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

Table 2 Secondary structural (i.e., α-helix, β-sheets, and random coil (RC)) analysis for the native BSA and its complexes with CLP. Molar ratio of drug: BSA Native BSA (5 mM) BSA:CLP 1:5 1:10 1:15 1:20

α (%)

β (%)

RC (%)

0

67

8

25

25 50 75 100

68 70 74 76

7 5 2 1

25 25 24 23

Conc. of drug (mM)

conditions (i.e., pH 7.4). The knowledge of the binding characteristics of biomolecules and drugs plays an important role in understanding the biological process. The biological significance of this work is evident since albumin serves as a carrier molecule for multiple drugs and the interaction of CLP with albumin is not characterized so far. Hence, this report has a great impact in pharmacology and clinical medicine as well as methodology. Experimental results indicated that CLP could bind with the serum albumin and quench the fluorescence of serum albumin. Based on the Stern–Volmer equation, the quenching rate constants were evaluated and their values suggested that the fluorescence quenching was a static process. The negative value of ΔG implies the spontaneity of the complexation. In addition, the values of enthalpy change and the entropy change suggested that the hydrophobic forces played a major role in the interaction between CLP and BSA. Based on the theory of the FRET, the energy transfer efficiency (E) and the molecular distance (r0) between CLP and BSA were calculated. The intensity of negative CD bands at 208 and 220 nm differed in the presence of drug due to changes in the chemical environment of α-helices lying at the surface of the protein, which suggested 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. (130059-D1434). The authors, therefore, acknowledge with thanks DSR technical and financial support.

46

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

References [1] A.B. Khan, J.M. Khan, M.S. Ali, R.H. Khan, Kabir-ud-Din, Spectrochim. Acta, Part A 97 (2012) 119. [2] S. Tabassum, W.M. Al-Asbahy, M. Afzal, F. Arjmand, R.H. Khan, Mol. Biosyst. 8 (2012) 2424. [3] A.B. Khan, J.M. Khan, M.S. Ali, R.H. Khan, Kabir-ud-Din, Colloids Surf., B 87 (2011) 447. [4] A. Varshney, M. Rehan, N. Subbarao, G. Rabbani, R.H. Khan., PLoS One 6 (2011) e17230. [5] A. Varshney, P. Sen, E. Ahmad, M. Rehan, N. Subbarao, R.H. Khan, Chirality 22 (2010) 77. [6] B. Ahmad, S. Parveen, R.H. Khan, Biomacromolecules 7 (2006) 1350. [7] U. Kragh-hansen, Pharmacol. Rev. 33 (1981) 17. [8] K. Sneppen, G. Zocchi, Physics in Molecular Biology, Cambridge University Press, Cambridge, UK, 2006. [9] Z.M. Wen, S.T. Ye, Asian Pac. J. Allergy Immunol. 11 (1993) 13. [10] L.A. Sklar, B.S. Hudson, R.D. Simoni, Biochemistry 16 (1977) 5100. [11] J. Steinhardt, J. Krijn, J.G. Leidy, Biochemistry 10 (1971) 4005. [12] M. Yamasaki, T. Yamashita, H. Yano, K. Tatsumi, K. Aoki, Int. J. Biol. Macromol. 19 (1996) 241. [13] H. Yuan, W.E. Antholine, W.K. Subczynski, M.A. Green, J. Inorg. Biochem. 61 (1996) 251. [14] W. Parker, P.S. Song, Biophys. J. 61 (1992) 1435. [15] D. Carter, J.X. Ho, Advances in Protein Chemistry, vol. 45, Academic Press, New York (1994) 153–203. [16] T. Peters, Advances in Protein Chemistry, vol. 37, Academic Press, New York, 1985. [17] L. Jiaquin, T. Jianniao, J. Zhang, Z. Hu, C. Xingguo, Anal. Bioanal. Chem. 376 (2003) 864.

[18] D. Tang, H.J. Li, P. Li, X.D. Wen, Z.M. Qian, Chem. Pharm. Bull. 56 (2008) 360. [19] A. Sulkowska, J. Mol. Struct. 614 (2002) 227. [20] B. Valeur, J.C. Brochon, New Trends in Fluorescence Spectroscopy, sixth ed., Springer Press, Berlin, 1999. [21] X.W. Zhang, F.L. Zhao, K.A. Li, Chem. J. Chin. Univ. 20 (1999) 1063. [22] J.R. Lakowica, Principles of Fluorescence Spectroscopy, second ed., Plenum Press, New York, 1999. [23] D.M. Togashi, A.G. Ryder, J. Fluorescence 18 (2008) 519. [24] G.Z. Chen, X.Z. Huang, J.G. Xu, Z.Z. Zheng, Z.B. Wang, The Methods of Fluorescence Analysis, second ed., Beijing Science Press, 1990. [25] Z. Cheng, Y. Zhang, J. Mol. Struct. 889 (2008) 20. [26] B. Chakraborty, S. Basu, J. Lumin. 129 (2009) 34. [27] M.M. Yang, P. Yang, L.W. Zhang, Chin. Sci. Bull. 9 (1994) 31. [28] M. Jiang, M.X. Xie, D. Zheng, Y. Liu, X.Y. Li, X. Chen, J. Mol. Struct. 692 (2004) 71. [29] P.D. Ross, S. Subramanian, Biochemistry 20 (1981) 3096. [30] T. Förster, in: O. Sinanoglu (Ed.), Modern Quantum Chemistry, vol. 3, Academic Press, New York, 1996. [31] J.H. Yang, W.D. Zhao, C.L. Tong, N.Q. Jie, G.L. Zhang, Z.Q. Gao, Chem. J. Chin. Univ. 17 (1996) 451. [32] C.N. Yan, H.X. Zhang, P. Mei, Y. Liu, Chin. J. Chem. 23 (2005) 1151. [33] S. Kasai, T. Horie, T. Mizuma, J. Pharm. Sci. 76 (1978) 387. [34] L. Cyril, J.K. Earl, W.M. Sperry, Biochemists-Handbook, E&FN Spon., London, 1961. [35] W.Y. He, Y. Li, C.X. Xue, Z.D. Hu, X.G. Chen, F.L. Sheng, Bioorg. Med. Chem. 13 (2005) 1837. [36] F.-L. Cui, J. Fan, J.-P. Li, Z.-D. Hu, Bioorg. Med. Chem. 12 (2004) 151. [37] Y.-H. Chen, J.T. Yang, H.M. Martinez, Biochemistry 11 (1972) 4120.