Mechanistic and conformational studies on the interaction of anti-inflammatory drugs, isoxicam and tenoxicam with bovine serum albumin

Journal of Luminescence 130 (2010) 2052–2058

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Mechanistic and conformational studies on the interaction of anti-inflammatory drugs, isoxicam and tenoxicam with bovine serum albumin Reeta Punith, Umesha Katrahalli, Shankara S. Kalanur, Seetharamappa Jaldappagari n Department of Chemistry, Karnatak University, Dharwad 580 003, India

a r t i c l e in f o

a b s t r a c t

Article history: Received 5 October 2009 Received in revised form 6 March 2010 Accepted 21 May 2010 Available online 1 June 2010

The mechanism of interaction of the non-steroidal anti-inflammatory drugs, isoxicam (IXM) and tenoxicam (TXM) with bovine serum albumin (BSA) has been studied using spectroscopic techniques, viz., spectrofluorescence, circular dichroism (CD), UV–visible absorption and FT-IR under simulative physiological conditions. Stern–Volmer analysis of fluorescence quenching data shows the presence of the static quenching mechanism. Thermodynamic parameters (negative DH0 and positive DS0 values obtained in the present study) revealed that the hydrophobic interactions played a major role in the interaction of these drugs with BSA. The distance, r between the donor (BSA) and acceptor (IXM/TXM) was calculated based on the Forster’s theory of non-radiation energy transfer and the values were observed to be 3.85 nm and 2.60 nm in IXM–BSA and TXM–BSA system, respectively. CD and FT-IR studies indicated that the binding of IXM/TXM to BSA induced conformational changes in BSA. The effect of common ions on the binding of IXM/TXM to BSA has been investigated. & 2010 Elsevier B.V. All rights reserved.

Keywords: Bovine serum albumin Tenoxicam Isoxicam Spectroscopic techniques Interactions

1. Introduction Serum albumins are the most abundant proteins in the circulatory system of a wide variety of organisms. Bovine serum albumin (BSA) being the major macromolecule in blood plasma of animals accounting to about 60% of the total protein corresponding to a concentration of 42 g dm  3 [1]. It consists of a single chain of 582 amino acids, globular nonglycoprotein cross-linked with 17 cysteine residues (eight disulfide bonds and one free thiol). It is divided into three linearly arranged, structurally distinct and evolutionarily related domains (I–III); each domain is composed of two subdomains (A and B) [2]. BSA has two tryptophans, embedded in two different domains: Trp-134, located on the surface of domain I and Trp-214, located within the hydrophobic pocket of domain II. The binding cavities associated with subdomains IIA and IIIA are also referred to as site I and site II according to the terminology proposed by Sudlow et al. [3]. The most exceptional property of a serum albumin is that it serves as a depot protein and a transport protein for numerous endogenous and exogenous compounds [4]. The exogenous substances that bound to protein with a high affinity are drugs. The interaction between albumin and drugs has attracted several researchers since several decades as the drug–protein interaction may result in the

n

Corresponding author. Tel.: + 918362215286; fax: + 918362747884. E-mail addresses: [email protected], [email protected] (S. Jaldappagari). 0022-2313/$ - see front matter & 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2010.05.025

formation of a protein–drug complex, which has important effect on the distribution, free concentration and the metabolism of drugs in the blood stream [4]. Thus, the drug–albumin complex may be considered as a model for gaining fundamental insights into drug– protein interactions. In this regard, BSA has been studied extensively, partly because of its structural homology with human serum albumin (76%) [5]. TXM and IXM (Fig. 1) belong to oxicam series. These belong to the class of medications known as non-steroidal antiinflammatory drugs (NSAIDs). They are employed to relieve inflammation, swelling, stiffness and pain associated with rheumatoid arthritis. The molecular interactions are often monitored by spectroscopic techniques because these methods are sensitive and relatively easy to use. Because of its outstanding sensitivity, selectivity, reproducibility, easy implication and vast theoretical foundation; fluorescence spectroscopy is an appropriate method to investigate the interaction between the drug and protein [6], which can reveal the accessibility of drugs to fluorophore group of the protein and provide an understanding of binding mechanism to drug, and yield clues to the chemistry of the binding phenomenon. Conventional approaches viz., affinity or size exclusion chromatography, equilibrium dialysis, ultrafiltration and ultracentrifugation suffer either low sensitivity or lengthy operation time or both. Spectroscopic techniques have advantages over these conventional approaches [2]. Critical literature survey reveals that attempts have not been made so far to investigate the mechanism of interaction of TXM

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2.3. Procedure 2.3.1. IXM/TXM–BSA interactions On the basis of preliminary experiments, fluorescence spectra were recorded in the range 300–500 nm. BSA concentration was kept fixed at 1.237 mM and drug concentration varied from 1.237 to 10.321 mM.

O NH O

N

S O

O

N

2.3.2. Thermodynamics of drug–protein interactions Thermodynamic parameters for the binding of drugs to BSA were determined by carrying out the binding studies at three different temperatures, 294, 299 and 303 K in the range of 300– 500 nm upon excitation at 296 nm in each case by spectrofluorimetric method.

O H

OH

N

O

S

N H HN S O

O

Fig. 1. Chemical structure of (a) IXM and (b) TXM.

and that of IXM with BSA. This is the first attempt made to probe the mode of association between TXM/IXM and BSA. In this paper, the quenching of the intrinsic tryptophan fluorescence of BSA has been used as a tool to study the interaction of TXM/IXM with the transport protein, BSA.

2. Experimental details 2.1. Apparatus Fluorescence measurements were performed on a spectrofluorimeter Model F-2000 (Hitachi, Japan) equipped with a 150 W Xenon lamp and a slit width of 5 nm. A 1.00 cm quartz cell was used for measurements. The CD measurements were made on a JASCO-J-715 spectropolarimeter (Tokyo, Japan) using a 0.1 cm cell at 0.2 nm intervals, with 3 scans averaged for each CD spectrum in the range of 200–250 nm. The absorption spectra were recorded on a double beam CARY 50-BIO UV–vis spectrophotometer (Varian, Australia) equipped with a 150 W Xenon lamp and a slit width of 5 nm. A quartz cell of 1.00 cm was used for measurements. FT-IR spectra were recorded on Nicolet Nexus 670 FT-IR spectrometer (USA) equipped with a Germanium attenuated total reflection (ATR) accessory, a DTGS KBr detector and a KBr beam splitter.

2.3.3. The displacement experiment The displacement experiments were performed using the site probes keeping the concentration of BSA and the probe constant (each of 1.237 mM). The fluorescence quenching titration was used as before to determine the binding constants of IXM/TXM– BSA system in presence of the site probes, warfarin, ibuprofen and digitoxin for sites I, II and III, respectively. 2.3.4. Circular dichroism (CD) measurements The CD measurements of BSA in the presence and absence of IXM/TXM were made in the range of 202–246 nm. A stock solution of 125 mM BSA was prepared in 0.1 M phosphate buffer of pH 7.4 containing 0.15 M NaCl. The BSA to drug concentration was varied (1:2, 1:4, 1:6 and 1:8) and the CD spectrum was recorded. 2.3.5. Fourier transform infrared spectroscopy All FT-IR spectra were taken via the attenuated total reflection (ATR) method with a resolution of 4 cm  1 and using 60 scans. The spectra processing procedure involved collecting spectra of the buffer solution under the same conditions. Next, the absorbance of the buffer solution was subtracted from the spectra of the sample solution to obtain the FT-IR spectra of the proteins. The subtraction criterion was that the original spectrum of the protein solution between 2200 and 1800 cm  1 was featureless. 2.3.6. UV–visible absorption studies The absorption spectra of BSA were recorded in the presence and absence of IXM/TXM. 2.3.7. Effects of some common ions The fluorescence spectra of IXM/TXM–BSA were recorded in presence and absence of various common ions, viz., K + , Ca2 + , Co2 + , Ni2 + , Cu2 + and Zn2 + upon excitation at 296 nm. The overall concentration of BSA was fixed at 1.237 mM and that of common ion was maintained at 1.237 mM.

3. Results and discussion

2.2. Reagents

3.1. Fluorescence quenching mechanism

Bovine serum albumin (BSA, Fraction V) was obtained from Sigma Chemical Company, St. Louis, USA. Tenoxicam and isoxicam were obtained as gift sample from Torrent Drugs and Chemicals, India. The solutions of IXM/TXM and BSA were prepared in 0.1 M phosphate buffer of pH 7.4 containing 0.15 M NaCl. BSA solution was prepared based on its molecular weight of 65,000. All other materials were of analytical reagent grade and double distilled water was used throughout.

Fluorescence measurements were carried out to investigate the binding of IXM and TXM with BSA. For this, the fluorescence intensity of BSA was recorded upon excitation at 296 nm in presence of increasing amounts of the drug. As shown in Fig. 2a and 2b BSA exhibited strong fluorescence emission while IXM/TXM did not show intrinsic fluorescence under the experimental conditions. Further, the fluorescence intensity of protein decreased in presence of the drug. This indicated that the

R. Punith et al. / Journal of Luminescence 130 (2010) 2052–2058

IXM/TXM interacted with BSA. Further, the quenching was also accompanied by blue shift (from 340 to 332 nm with IXM and from 340 to 331 nm with TXM) of the respective maximum emission wavelength. The blue shift signified that the binding of IXM/TXM was associated with changes in the local environment in BSA. This suggested that the chromophore was placed in an increased hydrophobic environment after the addition of IXM/ TXM [7]. Moreover, the occurrence of an isobestic point at 384 nm in IXM–BSA indicated the complex formation between IXM and BSA [8]. The fluorescence quenching data was analyzed using the Stern–Volmer equation [9] shown below: F0 =F ¼ 1 þKq t0 ½Q  ¼ 1 þ Ksv ½Q 

ð1Þ

where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively, Kq is the bimolecular quenching constant, t0 is the lifetime of the fluorophore in the absence of quencher, [Q] is the concentration of quencher and Ksv is the Stern–Volmer quenching constant. The plot (Fig. 3a and 3b) of F0/F versus [Q] yielded the values of Ksv and in turn Kq (Table 1). The probable quenching mechanism of fluorescence of BSA by the drug was proposed based on the dependence of Ksv with temperature. In the present study, the Ksv values decreased with increase in temperature thereby indicating the presence of static quenching [10]. Moreover, the values of Kq (which are in the order of 1012 for IXM–BSA and TXM–BSA) are far larger than

2.0  1010 l mol  1 s  1, the maximum value reported for diffusion quenching rate constant of various quenchers with the biopolymer [11]. So we propose the presence of static quenching mechanism in the binding of IXM and TXM to BSA. 3.2. Binding constants and binding sites For static quenching, the relationship between fluorescence intensity and concentration of a quencher can be described by the equation shown below[12]: logðF0 -FÞ=F ¼ log K þ n log½Q 

ð2Þ

where K is the binding constant and n is the number of binding sites per BSA. The values of K and n (Table 2) are evaluated from

2 1.8 1.6 1.4

Fo/F

2054

1.2 1 0.8

500

0.6 0.4 0.00E+00

fluorescence [a.u.]

400

4.00E-06

1.00E-05

1.20E-05

8.00E-06

1.00E-05

1.20E-05

2

10

1.5

Fo/F

100

1

350

400

450

500

wavelength (nm)

0.5

0 0.00E+00

500

1

2.00E-06

4.00E-06

6.00E-06

[Q]

400

luorescence [a.u.]

8.00E-06

2.5

200

Fig. 3. The Stern–Volmer curves for quenching of BSA with (a) IXM and (b) TXM at (~) 289 K, (m) 294 K, and (K) 299 K.

300

10

200

Table 1 Stern–Volmer quenching constants for the interaction of IXM/TXM with BSA at 289, 294 and 299 K.

100 0 300

6.00E-06

[Q]

300

0 300

2.00E-06

1

350

400

450

Kq  1012 (l mol  1S  1)

T (K)

IXM

289 294 299

7.08 6.56 4.78

7.08 6.56 4.78

TXM

289 294 299

12.81 10.61 09.49

12.81 10.61 09.49

500

wavelength (nm) Fig. 2. Fluorescence spectra of BSA in presence of (a) IXM and (b) TXM. Concentration of BSA was fixed at 1.237 mM (1) and that of IXM/TXM was maintained in the range of 1.237–10.321 mM (2–10).

Ksv  104 (M  1)

Drug

R. Punith et al. / Journal of Luminescence 130 (2010) 2052–2058

3.4. CD spectra In order to obtain an insight into the structure of BSA, the CD spectra were recorded in presence and absence of drug. The CD spectra of BSA in presence of a representative drug, TXM are shown in Fig. 5. The CD spectrum of BSA exhibited two negative bands in UV region at 208 and 220 nm, characteristic of a-helix

-1.6 -1.2 log (Fo-F)/F

the plot (Fig. 4a and 4b) of log(F0–F)/F versus log[Q] [10]. The larger values of K observed in the present study indicated the presence of strong binding between drug and protein. Further, the binding constant values decreased with increase in temperature suggesting the reduction in stability of drug–BSA complex [13]. The values of n are observed to be close to unity indicating that there is one independent class of binding sites on BSA for IXM/ TXM. It looks most likely that the drug binds to hydrophobic cavities in subdomain IIA, the principal region of the ligand binding site in protein. That is to say, tryptophan-214 is near or within the binding site. In order to determine the specificity of IXM/TXM binding and the location of drug binding site on BSA, the competitive displacement experiments were carried out using different site probes, viz., warfarin (for site I), ibuprofen (for site II) and digitoxin (for site III) [14,15]. For this, varied amounts of the drug were added to a solution containing fixed amounts of BSA and site probe and fluorescence intensities were noted down upon excitation at 296 nm. The binding constant values were evaluated. From the results shown in Table 3, it is evident that the ibuprofen and digitoxin are not significantly displaced by IXM/TXM. However, both drugs exhibited significant displacement of warfarin suggesting that the site for both drugs and warfarin is same. This means that the binding site for drug on BSA is site I located in subdomain IIA near Trp-214 [16].

2055

-0.8 -0.4 0 -4.8

-5

-5.2

-5

-5.2

-5.4 log [Q]

-5.6

-5.8

-6

-1.2

3.3. Binding mode

log K ¼ DH0 =2:303RT-DS0 =2:303R

ð3Þ

where K is the binding constant at the corresponding temperature (289, 294 and 299 K) and R is the gas constant. DH0 and DS0 can be calculated from the slope and ordinate of the plot of log K versus 1/T, respectively. The free energy change (DG0) is calculated from the following Gibbs–Helmholtz relationship:

DG0 ¼ DH0 -T DS0

ð4Þ

The values of DH0, DG0 and DS0 are listed in Table 2. The negative sign of DG0 values supported the assertion that all binding processes are spontaneous. The values of DH0 are negative and large, while the values of DS0 are positive. According to Ross and Subramanian [18], the positive DS0 value was frequently taken as an evidence for hydrophobic interaction, while small negative DH0 values reveal the presence of electrostatic interactions [18,19]. Since DH0 o0 and DS0 40, the acting force between IXM/TXM and BSA is believed to be predominantly hydrophobic force [20].

-0.8 log (Fo-F)/F

Generally, the acting forces contributing to the macromolecules interactions with small ligands include hydrogen bond, van der Waals force, electrostatic interaction and hydrophobic force [17]. The force acting may be predicted by knowing the value of the enthalpy change (DH0) and entropy change (DS0), which can be evaluated using the following van’t Hoff’s equation:

-0.4 0 0.4 -4.8

-5.4 log [Q]

-5.6

-5.8

-6

Fig. 4. Binding equilibria of (a) IXM–BSA and (b) TXM–BSA at (~) 289 K, (m) 294 K, and (K) 299 K.

Table 3 The binding constant of drug–BSA in presence of site probes. System

IXM (M  1)

TXM (M  1)

Drug–BSA Drug–BSA+ warfarin Drug–BSA+ ibuprofen Drug–BSA+ digitoxin

4.82  104 3.26  104 4.72  104 4.64  104

1.30  105 2.56  104 1.21  105 1.26  105

DH0 (kJ mol  1)

DG0 (kJ mol  1)

DS0 (J mol  1 K  1)

 20.2768  20.6199  20.9637  16.5138  16.7829  17.3214

+ 68.69

Table 2 Thermodynamic parameters of IXM–BSA and TXM–BSA system. Drug

T (K)

K (M  1)

n

R2

IXM

289 294 299 289 294 299

9.15  104 4.82  104 2.64  104 8.97  105 1.30  105 0.58  105

1.0191 0.9669 0.9404 1.1760 1.0194 0.9544

0.9940 0.9947 0.9912 0.9951 0.9995 0.9987

TXM

 0.425  0.954

+ 53.84

2056

R. Punith et al. / Journal of Luminescence 130 (2010) 2052–2058

structure of the protein [10]. The binding of TXM to BSA decreased both of these bands, clearly indicating the decrease in a-helicity of protein. The CD results are expressed in terms of mean residue ellipticity (MRE) in deg cm2 dmol  1 according to following equation [21]: MRE ¼

observed CD ðm degÞ Cp nl x 10

ð5Þ

where Cp is the molar concentration of the protein, n is the number of amino acid residues, and l is the path length. The helical content of free and combined BSA is calculated from MRE values at 208 nm using the equation as described by Lu et al. [22].

a-Helix ð%Þ ¼

MRE208 4000  100 330004000

ð6Þ

where MRE208 is the observed MRE value at 208 nm; 4000 is the MRE of the b-form and random coil conformation cross at 208 nm. The results indicated the reduction of a-helix structure of protein (13.7% in TXM–BSA and 10.24% in IXM–BSA) upon interaction with the drug. Further, the CD spectra of BSA in presence of TXM were found to be similar in shape, revealing that the structure of BSA is predominantly a-helix even after the addition of TXM. Similar results were noticed with IXM (not shown). From these

10000 5000

MRE

0 -5000

5

-15000 -20000 -25000 200

210

220

230

240

250

3.5. FT-IR spectra The FT-IR spectra of drug free and drug bound protein were recorded. The protein amide I bands are observed in the region, 1600–1700 cm-1 (mainly due to C=O stretch) and amide II bands are noticed  1550 cm-1 (due to C–N stretch coupled with N–H bending mode). These amide bands have a relationship with the secondary structure of protein [23]. The peak positions of amide I (1641 cm  1) and amide II (1564 cm  1) bands of free BSA were observed to be shifted to 1633 cm  1 and 1550 cm  1 in IXM–BSA and to 1637 cm  1 and 1575 cm  1 in TXM–BSA, respectively. This indicated that the secondary structure of protein was changed upon interaction with IXM/TXM [24]. 3.6. UV–vis absorption studies UV–visible absorption measurement is a very simple method to explore the structural change [25] and to know the complex formation [26]. In the present study, UV absorption intensity of BSA is increased with the addition of IXM/TXM (Figs. 6 and 7). Further, the maximum peak positions of IXM/TXM–BSA systems are noticed to be shifted slightly towards shorter wavelength. This indicated the formation of complex between IXM/TXM and BSA [17]. 3.7. Energy transfer between IXM/TXM and BSA

1

-10000

it is apparent that the decrease in alpha helicity caused conformational changes in the protein.

260

wavelength (nm)

The spectral studies suggested that BSA forms complex with IXM/TXM. In order to evaluate the distance, r between fluorophore (donor) of BSA and bound drug (acceptor), the fluorescence resonance energy transfer (FRET) is used. Generally FRET occurs whenever the emission spectrum of a donor overlaps with the absorption spectrum of acceptor. For this, we have recorded the absorption spectrum of drug and emission spectrum of BSA. Good overlap between the fluorescence spectrum of BSA and absorption spectrum of IXM/TXM was noticed and the same is shown in Figs. 8 and 9. As the fluorescence emission of protein was affected by the excitation light around 296 nm, the emission spectrum ranging from l ¼300 to 500 nm was chosen to calculate ¨ the overlapping integral. According to the Forster’s theory [27],

Fig. 5. CD spectra of (1) BSA (1.5 mM) in presence of (2) 3.0 mM, (3) 4.5 mM, (4) 9.0 mM, and (5) 12 mM TXM.

0.25

10

0.05 0.2

8 absorbance

absorbance

0.04 0.03

1

0.02

0.1

1

0.05

0.01 0 290

0.15

x 310

330

350

370

390

wavelength (nm) Fig. 6. Absorption spectra of (1) BSA (1.237 mM) in presence of (2) 1.237 mM, (3) 2.45 mM, (4) 3.64 mM, (5) 4.807 mM, (6) 5.952 mM, (7) 7.075 mM, and (8) 8.177 mM IXM. A concentration of 1.237 mM (x) was used for IXM only.

0 300

x 320

340

360

380

400

Wavelength (nm) Fig. 7. Absorption spectra of (1) BSA (1.237 mM) in presence of (2) 1.237 mM, (3) 2.45 mM, (4) 3.64 mM, (5) 4.807 mM, (6) 5.952 mM, (7) 7.075 mM, (8) 8.177 mM, (9) 9.259 mM, and (10) 10.321 mM IXM. A concentration of 1.237 mM (x) was used for TXM only.

R. Punith et al. / Journal of Luminescence 130 (2010) 2052–2058

the energy transfer efficiency, E is calculated using the equation given below [28]: E ¼ R60 =R60 þ r 6 ¼ 1-F=F0

ð7Þ

where F0 and F are the fluorescence intensities in the absence and presence of IXM/TXM, respectively, r is the distance between donor and acceptor and R0 is the distance at 50% transfer efficiency, which can be calculated using following equation [29]: R60 ¼ 8:79  1025 k2 N 4 cJ

ð8Þ

2057

Table 4 Effects of common ions on binding constants of IXM–BSA and TXM–BSA. Metal ion

K for BSA–IXM (M  1)

K for BSA–TXM (M  1)

K+ Ca2 + Co2 + Ni2 + Cu2 + Zn2 +

15.30  106 20.98  106 2.73  106 3.18  106 9.33  103 2.98  104

1.55  103 7.84  103 8.73  103 5.60  103 3.09  105 1.70  104

2

where k is the spatial orientation factor describing the relative orientation in space of the transition dipoles of the donor and acceptor, N is the refractive index of the medium, c is the fluorescence quantum yield of the donor in the absence of the acceptor and J is the overlap integral between the donor fluorescence emission spectra and the acceptor absorption spectra. J is calculated using the equation shown below [30]: P 4 FðlÞeðlÞl Dl P J¼ ð9Þ FðlÞDl F(l) is the fluorescence intensity of the donor at wavelength l,

evaluated using Eq. (7) and were found to be 3.85 nm and 2.6 nm for IXM–BSA and TXM–BSA, respectively. The closeness between TXM and BSA might have resulted in increased interaction in TXM-BSA compared to that in IXM-BSA. Obviously, the donor-toacceptor distances are lower than 8 nm in both cases, which indicated that the energy transfer from BSA to IXM/TXM has occurred with high probability [33]. Larger r values compared to those of R0 revealed the presence of static type quenching mechanism [34–36].

e(l) is the molar absorption coefficient of the acceptor at wavelength (l). So J could be calculated by integrating the overlap spectra for l ¼300–500 nm and was found to be 0.8058  10  15 and 0.5806  10  15 for IXM and TXM, respectively. Under these experimental conditions the values, k2 ¼2/3 [31], N ¼1.36, and c ¼0.15 are reported in the literature [32]. Based on these data, the critical distance, R0 can be calculated by using Eq. (8) and these values were calculated to be 1.93 nm and 1.7 nm for IXM and TXM, respectively. Finally, the values of r were

0.02

500 b

300 0.01 200

a

absorbance

intensity

400

100 0 305

355

0

405

3.8. Influences of common ions on the binding constant Common ions are widely distributed in human blood and they have a definite ability to bind proteins. Hence, they may affect the binding of drugs to albumin. In order to investigate the effect of some metal ions on drug–albumin binding, the binding constants of drug–BSA were determined in presence of various metal ions. These values are listed in Table 4. The increase in the binding constant of drug–BSA complex indicates stronger binding of BSA to IXM/TXM. The higher binding constants may result from the formation of metal ion-drug complexes, which further interact with the protein to form metal ion-drug-protein complexes. This may prolong the time during which IXM/TXM remains in blood and thus enhance its maximum therapeutic effects [37]. However, the observed decreased binding constant of drug-protein in presence of some metal ions indicated the competition between the metal ion and drug for the same site in the protein. This may result in the displacement of drug from the binding site of the protein and in turn increase the free drug concentration in plasma. This may necessitate higher doses of drugs to achieve desired therapeutic effect [38].

wavelength (nm) Fig. 8. The overlap of the UV absorption spectrum of (a) IXM (1.237 mM) with fluorescence spectrum of (b) BSA (1.237 mM).

0.05

500

300

b

a

0.025

200

absorbance

intensity

400

100 0 305

355

405

0

wavelength (nm) Fig. 9. The overlap of the UV absorption spectrum of (a) TXM (1.237 mM) with fluorescence spectrum of (b) BSA (1.237 mM).

4. Conclusions This paper provided an approach for studying the interactions of anti-inflammatory drugs, IXM and TXM with BSA by fluorescence, CD, absorption, and FT-IR methods. The results indicated that the drugs quenched the intrinsic fluorescence of BSA through static quenching mechanism. In addition, the values of standard enthalpy change and the standard entropy change suggested that the hydrophobic forces played a major role in the interaction between IXM/TXM and BSA. 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 a-helices lying at the surface of the protein, which suggested the change in secondary structure of protein. The biological significance of this work is evident since albumin serves as a carrier protein for multiple drugs. Since, the interaction of BSA with IXM and TXM has not been studied so far, the present study assumes importance in pharmacology and clinical medicine.

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Acknowledgements The financial support of the CSIR, New Delhi (No. 01(2279)/08/ EMR-II dated 20–11–2008) is gratefully acknowledged. We are thankful to Prof. M.R.N. Murthy, Indian Institute of Science, Bangalore, for CD measurement facilities. Thanks are also due to the authorities of the Karnatak University, Dharwad, for providing necessary facilities. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]

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