Interaction of cinnamic acid derivatives with serum albumins: A fluorescence spectroscopic study

Spectrochimica Acta Part A 78 (2011) 942–948

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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa

Interaction of cinnamic acid derivatives with serum albumins: A fluorescence spectroscopic study T. Sanjoy Singh, Sivaprasad Mitra ∗ Department of Chemistry, North-Eastern Hill University, Shillong 793022, India

a r t i c l e

i n f o

Article history: Received 23 June 2010 Received in revised form 18 November 2010 Accepted 30 November 2010 Keywords: Cinnamic acid Fluorescence quenching Serum albumin Ligand binding Energy transfer

a b s t r a c t Cinnamic acid (CA) derivatives are known to possess broad therapeutic applications including anti-tumor activity. The present study was designed to determine the underlying mechanism and thermodynamic parameters for the binding of two CA based intramolecular charge transfer (ICT) fluorescent probes, namely, 4-(dimethylamino) cinnamic acid (DMACA) and trans-ethyl p-(dimethylamino) cinnamate (EDAC), with albumins by fluorescence spectroscopy. Stern–Volmer analysis of the tryptophan fluorescence quenching data in presence of the added ligand reveals fluorescence quenching constant (q ), Stern–Volmer constant (KSV ) and also the ligand–protein association constant (Ka ). The thermodynamic parameters like enthalpy (H) and entropy (S) change corresponding to the ligand binding process were also estimated. The results show that the ligands bind into the sub-domain IIA of the proteins in 1:1 stoichiometry with an apparent binding constant value in the range of 104 dm3 mol−1 . In both the cases, the spontaneous ligand binding to the proteins occur through entropy driven mechanism, although the interaction of DMACA is relatively stronger in comparison with EDAC. The temperature dependence of the binding constant indicates the induced change in protein secondary structure. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Cinnamic acid (CA) and its derivatives are used as important components in flavors, perfumes, synthetic indigo and pharmaceuticals. The corresponding ester derivatives of CA can also act as optical filters or deactivate substrate molecules which have been excited by light for the protection of polymers and organic substances. In cosmetic industry, these are used as sunscreen agents to reduce skin damage by blocking UV-A and UV-B. In biological chemistry, CA is a key intermediate in shikimate and phenylpropanoid pathways. Shikimic acid is a precursor of many alkaloids, aromatic amino acids, and indole derivatives; whereas, phenylpropanoid is a class of plant metabolite based on phenylalanine. They are widely distributed in plants fulfilling several functions, such as plant defense mechanism, pigmentation and external signaling system [1–3]. Furthermore, CA and its esters are also known to show a broad range of therapeutic activities. For example, 3,4-dihydroxycinnamic acid phenyl ester (CAPE) is reported to be a potent antitumor drug [4,5]. It also suppresses TPA-induced tumor promotion and COX-2 expression [6,7]. CA also acts as an antitumor agent; although, the response is somewhat weaker than CAPE [8]. Cinnamic acid and

∗ Corresponding author. Tel.: +91 364 272 2634; fax: +91 364 255 0076. E-mail addresses: [email protected], [email protected] (S. Mitra). 1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2010.11.046

its derivatives were also found to be effective in insulin secretive activity [9]. Serum albumins are the major soluble protein constituents of the circulatory system contributing significantly in the physiological functions as carrier proteins [10–12]. The most widely studied serum albumins, probably because of their well characterized structures and known functions, are either obtained from bovine (BSA) or human (HSA) sources. BSA is a single polypeptide chain consisting of about 583 amino acid residues and no carbohydrates. At the pH range of 5–7, it contains seventeen interchain disulfide bridges and one sulfhydril group [13–14]. The BSA molecule is made up of three homologous domains (I, II, III). Each domain, in turn, is the product of two sub-domains (IA, IB, etc.). Two tryptophan (trp) residues are embedded in two different domains; one of them is located in the proximity of the protein surface, but buried in a hydrophobic pocket of domain I (trp-134), whereas the other is located in an internal part of domain II (trp-214). The structural motif of HSA is similar to that of BSA; however, the primary exception is that it contains only one tryptophan residue in domain II (trp-214) [15]. Many drugs and other bio-active small molecules bind reversibly to albumins. Protein–drug binding greatly influences the absorption, distribution, metabolism and excretion properties of typical drugs [16,17]. Fluorescence studies on protein–ligand interaction with characteristic fluorescence properties of the drug (ligand) have gained enormous interest in recent times [18–24]. Also, serum albumins are known to increase the apparent solubil-

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(SRL), India. The analytical grade type-II water, used as solvent in the measurements, was obtained from Elix 10 water purification system (Millipore India Pvt. Ltd.).

OR

2.2. Equipments

H3C

N

CH3

R=H : DMACA = -C2H5 : EDAC Scheme 1. Structure of the cinnamic acid based ICT probes used to study the interaction with albumins.

ity of hydrophobic drugs in plasma and modulate their delivery to cells both in vivo and in vitro; they can play a dominant role in drug disposition and efficacy [25]. Further, considering the potential application of CA type of compounds for antitumor activity against human malignant tumors, such as melanoma, glioblastoma and adenocarcinoma of prostate and lung [8], a thorough study on binding of these drugs to proteins is relevant. This is because the binding of a ligand to the ligand binding domain (LBD) of the protein constitutes the initial step for its transport and activity. The binding pattern of CA and its derivatives also involve demonstrated example to characterize the interactions among multi-component biological systems [26]. In the present work, our aim is to determine the affinity of two CA derivatives, namely, 4-(dimethylamino) cinnamic acid (DMACA) and its derivative trans-ethyl p-(dimethylamino) cinnamate (EDAC) (see, Scheme 1, for structures) to BSA and HSA, and to investigate the thermodynamics as well as stoichiometry of their interaction. Fluorescence spectroscopy was the obvious choice as experimental tool to achieve this goal since it allows non-intrusive measurements of substances at low concentration under physiological conditions. Furthermore, the fluorescence property of both DMACA and EDAC is well characterized and known to give quite clean and unstructured broad emission spectra on excitation. The origin of this polarity sensitive fluorescence is assigned to an intramolecular charge transfer (ICT) state, which is formed within few hundreds of femtosecond from the precursor Franck-Condon excited state [27,28]. It is observed that resonance energy transfer (RET) from protein trp residue to the bound ligands produces this characteristic ICT emission; the corresponding parameters were calculated based on Förster theory [29]. 2. Experimental details 2.1. Materials Essentially fatty acid and globulin free, ≥ 99% (agarose gel electrophoresis), lyophilized powder form of BSA and HSA were obtained from Sigma (cat. no. B4287) and USB Corp. USA (cat. no. 10878), respectively. (trans)-Ethyl p-(dimethylamino) cinnamate (EDAC) was synthesized using standard procedure based on Reformatsky reaction, whereas, 4-(dimethylamino)-cinnamic acid (DMACA, 99%) was received from Alfa Aesar. Both the compounds were further purified by the method described elsewhere [27,28]. The samples were dissolved in Tris–HCl buffer solution (0.05 mol dm−3 Tris and 0.15 mol dm−3 NaCl, pH 7.4 ± 0.1). The buffer Tris had a purity of no less than 99.5% whereas, analytical grade NaCl and HCl were obtained from Sisco Research Laboratory

Steady-state absorption spectra were recorded on a PerkinElmer model Lambda25 absorption spectrophotometer. Fluorescence spectra were taken in a Hitachi model FL4500 spectrofluorimeter and all the spectra were corrected for the instrument response function. The temperature variation experiments were carried out by attaching a circulatory thermostat bath (MLW, Germany, type – U2C) to the cell holder. The solution pH was checked with Systronics ␮-pH system 361. Sample masses were accurately weighted using a microbalance (Sartorius CP-64) of 0.1 mg resolution. 2.3. Spectroscopic measurements The concentration of albumins was kept fixed at 7.5 × 10−6 mol dm−3 and the ligand concentration was varied from 0 to 9.2 × 10−5 mol dm−3 with an increment of 9.2 × 10−6 mol dm−3 in each step. The absorption spectra were measured at room temperature; whereas, the fluorescence emission spectra were recorded at four different temperatures (298, 308, 318 and 328 K) by exciting the sample at 278 nm. Quartz cuvettes of 10 mm optical path length, received from Perkin-Elmer, USA (part no. B0831009) and Hellma, Germany (type 111-QS), were used for measuring absorption and fluorescence spectra, respectively. For the fluorescence measurements, 5 nm band pass was used both in the excitation and emission side. Although the ligands have insignificant absorption at the trp excitation wavelength used in this study (278 nm), each fluorescence spectrum of the protein in presence of different ligand concentration was corrected for any possible inner filter effect using the following equation [29,30]: IFcorr (E , F ) = IF (E , F )

A(E ) Atot (E )

(1)

where A represents the absorbance of the free protein, and Atot is the total absorbance of the solution at the excitation wavelength (E ). The corrected spectrum can be taken as the contribution only from the protein trp residue and was used for further analysis. Fluorescence quantum yields () were calculated by comparing the total fluorescence intensity under the whole resultant spectra with that of a standard as described before [27]. The relative experimental error of the measured quantum yield was estimated within ±5%. 3. Results and discussion 3.1. Effect of EDAC and DMACA on albumin fluorescence Gradual addition of ICT probes into the fixed concentration of both BSA and HSA results in decrease in trp fluorescence intensity. Fig. 1 shows some typical spectral profiles for the BSA/EDAC and HSA/DMACA systems. The quenching of tryptophan fluorescence in presence of CA derivatives was analyzed by Stern–Volmer (SV) equation [30]: F0 = 1 + KSV × [Q ] = 1 + kq × 0 × [Q ] F

(2)

where F0 and F denotes the steady state tryptophan fluorescence intensities from the albumin in absence and presence of quenchers (ICT probes), respectively. KSV denotes Stern–Volmer quenching constant, which basically indicates the efficiency of the quenching mechanism, and [Q] indicates the quencher concentration. The magnitude of KSV can be evaluated by linear regression of F0 /F data

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Fig. 1. Tryptophan fluorescence quenching of the albumins [BSA (a) and HSA (b)] in presence of EDAC and DMACA, respectively. The concentrations (mol dm−3 ) of [albumin] = 7.5 × 10−6 ; [EDAC] = 0 (i), 9.2 × 10−6 (ii), 1.84 × 10−5 (iii), 3.68 × 10−5 (iv), 4.6 × 10−5 (v), 5.52 × 10−5 (vi) and 6.44 × 10−5 (vii); [DMACA] = 0 (i), 8.8 × 10−6 (ii), 1.76 × 10−5 (iii), 3.52 × 10−5 (iv), 5.28 × 10−5 (v) and 6.24 × 10−5 (vi). Inset shows Stern–Volmer analysis of the quenching data in linear region.

points against [Q]. The bimolecular quenching constant (q ) can also be determined, provided the fluorophore life time in absence of any quencher ( 0 ) is known. A variety of processes may be responsible for the decrease in fluorescence intensity in presence of quenchers. These include excited state reaction, molecular rearrangement, collisional quenching and also the ground state complex formation [30,31]. Broadly, the mechanism of fluorescence quenching can be classified into two categories, such as dynamic and/or static quenching. These two mechanisms can be distinguished by their temperature and viscosity dependence. Dynamic quenching is a diffusion controlled phenomenon. Since the diffusion coefficient increases with increase in temperature, the magnitude of q is expected to increase with temperature increase in case of dynamic quenching. On the other hand, increased temperature apparently decreases the stability of the ground state complex responsible for static quenching. As a result, the bimolecular quenching constant is expected to decrease with increasing temperature in this case. As shown in the inset of Fig. 1, the plot of F0 /F versus [Q] gives a good linear relationship until the probe concentration reaches

∼65 ␮M, for both the albumins. Further increase in probe concentration induces curvature in SV plots. The calculated KSV values from the SV plots for EDAC/BSA and DMACA/HSA systems are (1.81 ± 0.1) × 104 and (2.36 ± 0.1) × 104 dm3 mol−1 , respectively. Although the tryptophan fluorescence largely depends on the environment, as a first approximation, the intrinsic fluorescence decay time of tryptophan ( 0 ) can be approximated as ∼10 ns under the present experimental condition [32]. The apparent values of q , estimated from Eq. (2), are of the order of ∼1012 dm3 mol−1 s−1 ; which is about two orders of magnitude higher than the diffusion limited quenching rate constant. Therefore, it is obvious that the quenching of albumin fluorescence both by EDAC and DMACA is mainly governed by static quenching mechanism rather than a dynamic process. Further, the magnitude of KSV indicates a relatively strong ground state protein–ligand complex formation. To further verify the mechanism of quenching, the effect of temperature on the quenching process was monitored. Some representative plots are shown in Fig. 2. Interestingly, the SV plots tend to show upward curvature as the temperature is raised even when the ligand concentration is below 65 ␮M. Positive deviation

Fig. 2. Temperature dependent Stern–Volmer (SV) plot for HSA/EDAC (a) and BSA/DMACA (b) systems. The curved lines are only to guide the eye along the non-linear variation at higher temperatures. Inset: the percentage of deviation from linearity at higher temperatures for a particular ligand concentration (∼5.4 × 10−5 mol dm−3 ) relative to that at 298 K (see text for details).

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Table 1 Stern–Volmer quenching constants (KSV ) at different temperatures.a Temperature (K)

BSA

HSA

EDAC

298 308 318 328 a

DMACA

EDAC

DMACA

KSV

S.D.

R

KSV

S.D.

R

KSV

S.D.

R

KSV

S.D.

R

1.81 1.53 1.40 1.30

0.15 0.14 0.13 0.10

0.95 0.94 0.94 0.95

5.42 3.84 3.67 2.83

0.09 0.03 0.09 0.07

0.99 0.99 0.99 0.99

2.91 2.25 1.94 1.66

0.18 0.20 0.19 0.18

0.98 0.96 0.95 0.94

2.36 1.43 1.42 1.40

0.05 0.02 0.02 0.02

0.99 0.99 0.99 0.99

KSV values are represented in the unit of 104 dm3 mol−1 ; standard deviation (S.D.) and correlation coefficient (R) is also given in each case.

in SV plots is an indication of contribution from both the static and dynamic mechanism in the quenching process [30]. The values of KSV are calculated from the linear region of the plots using Eq. (2) and given in Table 1. The magnitude of KSV decreases with increase in temperature in all the cases, which further confirms the static quenching mechanism proposed earlier. It is also important to note the temperature dependence of the deviation from linear SV region at a certain ligand concentration. For example, quantitative estimation of this deviation at ∼55 ␮M ligand concentrations for some representative cases at three different temperatures, viz. 308, 318 and 328 K, relative to 298 K, are shown in the inset of Fig. 2. Interestingly, at 308 K, the deviation was calculated to be only 26% for EDAC, in comparison with 31% in case of DMACA. This indicates that DMACA binds more preferentially to the ligand binding domain of the albumins in comparison with EDAC (see below). The calculated KSV value at 298 K for BSA/EDAC and HSA/DMACA systems imply that ligand concentrations of ∼55 ␮M and 42 ␮M, respectively, are needed to quench half of the intrinsic protein fluorescence. This indicates that DMACA interacts more strongly with the albumins when compared with EDAC. Furthermore, the quenching of fluorescence intensity is associated with a shift in tryptophan fluorescence maxima in presence of both the ligands. Interestingly, titration of albumins with EDAC results a slight blue-shift in tryptophan fluorescence (Fig. 1a), whereas, similar treatment with DMACA results a red shift (Fig. 1b). The blue-shift in emission spectrum suggests an increased hydrophobicity of the region surrounding the albumin tryptophan site [33,34] in presence of EDAC. Although, the reason for red shift in case of DMACA interaction is not clear, it may be due to the following. Albumins are known to transport anions around the body and the carboxylic acid group of DMACA may get deprotonated at physiological pH [28]. It is, therefore, more likely that DMACA would bind more preferentially to a positively charged region of albumins causing the stabilization of the fluorescing state of tryptophan residue. As a result, a red shift in the trp fluorescence peak position is observed when bound to DMACA. 3.2. Apparent binding constant and number of binding sites For the static quenching process in drug (quencher)–protein (fluorophore) interaction, under the assumption that there are n number of same and independent binding sites in the protein

and single binding site of the drug, the binding process can be described as P + nD  Dn P

(3)

where P, D indicates the free protein and drug, respectively; Dn P indicates the bound complex. The apparent association constant (Ka ) is defined as Ka =

[Dn P]

(4)

[P], [D]n

From the stoichiometric coefficient of Eq. (3), the total protein concentration can be written as [P]0 = [Dn P] + [P]

(5)

If under the experimental condition, protein is the only fluorescing species, the relationship of the protein concentration and the fluorescence peak area can be represented as [P] F = F0 [P]0

(6)

Substituting this relationship in Eq. (4), one can deduce [35,36]: log

F − F  0 F

= log Ka + n log[D]

(7)

From the slope and intercept of the simulated linear plot using the above equation, the binding constant, Ka and number of binding sites, n can be obtained for different albumin/ligand combination at various temperatures. The corresponding values are displayed in Tables 2 and 3, respectively. Some representative plots are also shown in Fig. 3. Interestingly, the apparent association constant increases with increase in temperature. In the albumins, trp-214 is located in subdomain IIA, which is a well-characterized binding cavity for small organic molecules [11,15]. Similar magnitudes of Ka and n values for both BSA and HSA (Tables 2 and 3) indicate identical nature of binding for these ligands in both these albumins. Also, it indicates that the ligands interact with trp-214 only. In case of BSA, which contains two tryptophan molecules, the other tryptophan residue (trp-134, located in domain-I) remains inaccessible to the water soluble ligands. Resonance energy transfer studies from the tryptophan donor site to EDAC and/or DMACA as acceptor (discussed below) also points to the similar conclusion.

Table 2 Association constant and number of binding sites obtained from the double-log plot of interaction of EDAC and DMACA with BSA at different temperatures.a Temperature (K)

BSA EDAC

DMACA

log Ka 298 308 318 328 a

3.87 4.94 4.92 5.13

± ± ± ±

n 0.57 0.53 0.61 0.52

0.92 1.2 1.2 1.2

± ± ± ±

0.12 0.10 0.13 0.11

S.D.

R

log Ka

0.09 0.08 0.09 0.08

0.96 0.97 0.97 0.98

4.62 5.14 6.15 7.71

Standard deviation (S.D.) and correlation coefficient (R) is given in each case.

± ± ± ±

n 0.29 0.26 0.29 0.50

0.98 1.12 1.18 1.16

± ± ± ±

0.06 0.05 0.06 0.11

S.D.

R

0.04 0.04 0.04 0.07

0.99 0.99 1.01 0.99

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Table 3 Association constant and number of binding sites obtained from the double-log plot of interaction of EDAC and DMACA with HSA at different temperaturesa . Temperature (K)

HSA EDAC

DMACA

log Ka 298 308 318 328 a

4.08 4.98 5.40 6.66

± ± ± ±

n 0.47 0.57 0.66 0.65

0.92 1.17 1.29 1.22

± ± ± ±

0.10 0.12 0.15 0.15

S.D.

R

log Ka

0.09 0.10 0.12 0.12

0.96 0.97 0.96 0.97

4.07 4.74 5.32 4.93

± ± ± ±

n 0.64 0.19 0.39 0.42

0.96 1.14 1.18 1.19

± ± ± ±

0.13 0.04 0.08 0.09

S.D.

R

0.09 0.03 0.05 0.06

0.96 0.99 0.99 0.98

Standard deviation (S.D.) and correlation coefficient (R) is given in each case.

3.3. Thermodynamics of ligand binding The interaction forces between drugs and bio-molecules may include electrostatic interactions, formation of multiple hydrogen bonds, van der Waals interaction, hydrophobic and steric contacts within the antibody binding site, etc. [37]. The sign and magnitude of the thermodynamic parameters can account for the main factor(s) responsible towards the stability of drug–protein complex [38]. In order to elucidate the nature of the interaction of cinnamic acid based ICT systems with albumins, the thermodynamic parameters were calculated using van’t Hoff relation [39]. Assuming the change in enthalpy (H) for the protein–ligand binding process to be insignificant over the temperature range studied, the association constant (Ka ) and the change in other thermodynamic parameters with temperature can be written by following relations: log Ka =

H s − 2.303R 2.303R

G = H − TS

(8) (9)

The enthalpy (H) and entropy (S) change was calculated from the slope and intercept of the van’t Hoff plot at four different temperatures, viz. 298, 308, 318 and 328 K; whereas, Gibb’s free energy change (G) can be estimated from Eq. (9). Some of the representative plots are shown in Fig. 4 and all the thermodynamic parameters are collected in Table 4. The negative value of the free energy change (G) is indicative of a spontaneous binding of these ligands to the proteins. Further, this exothermic process is accompanied by positive S values in all the cases. A positive S value is often indicative of a hydrophobic mechanism in drug–protein interaction [38]. Specific electrostatic interaction among ionic species in solution is characterized by positive S and negative H values; whereas, negative entropy and

enthalpy changes indicate the importance of van der Waals force as well as hydrogen bond formation. Therefore, the binding of these ligands with albumins predominantly involves hydrophobic interaction; although, the possibility of specific electrostatic interaction cannot be ruled out completely. Overall, the whole binding process is considered as entropy driven and the increase in entropy due to the ligand binding might be due to the destruction of the protein secondary structure.

3.4. Energy transfer from albumins to bound ligands As discussed before, the change in protein fluorescence intensity in presence of CA based ligands is due to the decrease in fluorescence intensity of the trp residue located in hydrophobic domain IIA of the protein structure. The distance between the trp residue and the bound ligands can be determined using fluorescence resonance energy transfer (FRET). Normally, FRET occurs in the situation where the emission spectrum of the donor overlaps with the absorption spectrum of the acceptor. If the acceptor molecule is itself fluorescent, the characteristic emission profile appears from it even when excited at the donor absorption peak [40]. The UVabsorption spectrum of both DMACA and EDAC has strong overlap with albumin trp fluorescence. A representative spectral overlap for the BSA/EDAC system is shown in Fig. 5(a). Furthermore, the emission spectra of both DMACA and EDAC were checked to be negligible at the excitation wavelength of trp (278 nm). So, appearance of characteristic ICT emission from both EDAC and DMACA with concomitant quenching in donor fluorescence clearly indicates FRET to occur in all the protein–ligand complexes. A representative plot is shown in Fig. 5(b) for HSA/EDAC system. According to Förster’s theory [30], the energy transfer efficiency (E) depends on the distance (r) between the donor–acceptor pair

Fig. 3. Double-log plot to calculate the binding constant and number of binding site for HSA–DMACA (a) and BSA–EDAC (b) systems. Correlation coefficient (R) and standard deviation (S.D.) in the fitting procedure are also given.

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Fig. 4. van’t Hoff plot for HSA–EDAC (a) and BSA–DMACA (b) systems. Correlation coefficient (R) and standard deviation (S.D.) in the fitting procedure are also given.

Table 4 Relative thermodynamic parameters for the interaction of EDAC and DMACA with albumins.a Temp. (K)

BSA

HSA

EDAC

298 308 318 328 a

DMACA

EDAC

DMACA

G

H

S

G

H

S

G

H

S

G

H

S

−22.28 −25.76 −29.24 −32.73

81.42

0.35

−24.85 −32.09 −39.33 −46.57

190.90

0.72

−22.93 −28.82 −34.69 −40.57

152.29

0.59

−23.21 −27.78 −32.36 −30.94

113.28

0.46

Gibb’s free energy (G) and enthalpy (H) changes are given in kJ mol−1 and entropy (S) change is given in kJ mol−1 K−1 .

with the following relation: E =1−

R06

F = 6 F0 R0 + r 6

(10)

R0 is the critical Förster distance, defined as the distance where the transfer efficiency is 50%, and can be expressed as R0 (Å ) = 0.211 [k2 n−4 D J()]

1/6

these quantities are usually taken as 1.36 and 0.14, respectively for trp fluorescence in aqueous buffer [41]. The quantity J() is the overlap integral which represents the degree of spectral overlap between the donor and the acceptor molecules, and can be expressed as

∞ J() =

(11)

where n is the refractive index of the medium, D is the quantum yield of the donor in the absence of acceptor. The values of

0

FD ()εA ()4 d

∞ 0

FD ()d

(12)

FD () is the fluorescence intensity of the donor in the wavelength range of  to  + d and is dimensionless, εA () is the extinction

Fig. 5. (a) Spectral overlap (shown by the shaded area) between BSA emission and EDAC absorption. (b) Decrease in trp fluorescence of HSA at 340 nm and simultaneous increase in ICT emission at 460 nm (shown in inset) on gradual addition of EDAC with an isosbestic point at 423 nm (exc = 278 nm).

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Table 5 Calculated values of energy transfer efficiency (E), overlap integral J(), critical Förster’s distance (R0 ) and donor–acceptor distance (r) between the tryptophan (trp-214) donor and CA acceptors at pH 7.4.

References

Donor

Acceptor

E

J(), dm3 mol−1 cm−1 nm4

R0 , Å

r, Å

HSA

EDAC DMACA

0.41 0.26

5.078 × 1011 5.117 × 1011

10.34 10.35

10.98 12.36

BSA

EDAC DMACA

0.29 0.56

4.744 × 1011 5.398 × 1011

10.34 10.56

12.04 10.16

coefficient (in dm3 mol−1 cm−1 ) of acceptor at wavelength . If  is in nm, then J() is in units of dm3 mol−1 cm−1 nm4 . In Eq. (11), 2 represents a factor expressing the relative orientation of the donor to the acceptor molecule and is expressed as k2 = (sin D sin A cos − 2cos D cos A )

2

is gratefully acknowledged. TSS thanks UGC for providing him with a fellowship (RGNFS).

(13)

where is the dihedral angle between the planes containing emission transition dipole of the donor and absorption transition dipole of the acceptor,  D and  A are the angles between these dipoles and the vector joining the donor and acceptor. Depending upon the relative orientation of the donor and acceptor, this factor may vary from 0 to 4. However, for random distribution of donor and acceptor molecules, the value of 2 is generally assumed to 2/3. The small variation in the value of 2 obtained from the rigorous calculation using Eq. (13) does not seem to incorporate a substantial error in the calculated distance. The low concentration of the protein (∼7.5 ␮M) used in these experiments ensures the absence of any intermolecular energy transfer among the protein molecules [42]. The values of J() over the spectral region of donor emission and acceptor absorption, energy transfer efficiency (E), critical Förster’s radius (R0 ) and donor–acceptor separation distance (r) for several systems are displayed in Table 5. It is seen that the average distance between the donor–acceptor pairs in all the cases fall within the range of 0.5R0 < r < 1.5R0 , that indicates a highly efficient energy transfer [43] between Trp-214 donor of the proteins and CA acceptors used in this study. 4. Conclusions The interaction between model water soluble albumins, namely, BSA and HSA with cinnamic acid based intramolecular charge transfer ligands like EDAC and DMACA were studied by monitoring the tryptophan fluorescence quenching of proteins in presence of the ligand at different temperatures. The results show that the binding patterns of both the ligands are similar indicating a preferential interaction with trp-214 at the hydrophobic site of protein residue. The binding interaction is relatively strong with an apparent association constant ∼104 dm3 mol−1 in 1:1 protein–ligand stoichiometry. Estimation of thermodynamic parameters indicates that the spontaneous complexation occurs through an entropy driven pathway. FRET analysis of the experimental data suggests an efficient energy transfer from the trp donor of the proteins to the CA acceptors and characterized by the appearance of ICT fluorescence band. Acknowledgements Financial support through research project 34-299/2008 (SR) from University Grants Commission (UGC), Government of India

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