Journal of Photochemistry and Photobiology B: Biology 80 (2005) 217–224 www.elsevier.com/locate/jphotobiol

Interaction of pyrene-end-capped poly(ethylene oxide) with bovine serum albumin and human serum albumin in aqueous buffer medium: A fluorometric study Basudeb Haldar, Arabinda Mallick, Nitin Chattopadhyay

*

Department of Chemistry, Jadavpur University, Calcutta 700 032, India Received 9 February 2005; received in revised form 2 May 2005; accepted 2 May 2005 Available online 24 June 2005

Abstract The photophysical behavior of a hydrophobically tailored water-soluble polymer, pyrene-end-capped poly(ethylene oxide) (PYPY), has been studied in aqueous buffered bovine serum albumin (BSA) and human serum albumin (HSA) media. In buffered aqueous solution the polymer shows dual emission corresponding to the monomer and the excimer of pyrene moiety. The relative intensity of the monomer to the excimer emission shows interesting variation with the addition of BSA and HSA and is indicative of significant interaction of these albumin proteins with the polymer. The binding interaction has been shown to have a prominent role on the steady state fluorescence anisotropy of the two emission bands. Attempt has been made to determine the micropolarities of the protein microenvironments from a comparison of the variation of the monomer to excimer relative fluorescence intensities of the probe in water–dioxane mixtures with varying composition.
2005 Elsevier B.V. All rights reserved. Keywords: Serum albumin; Fluorescence; Excimer; Anisotropy; Micropolarity

1. Introduction Associative polymers, especially hydrophobically modified ones, sometimes referred to as telechelic polymers in aqueous solutions have received considerable interest both experimentally and theoretically due to their unusual thickening properties and therefore their various important technological applications [1–4]. These hydrophobically modified water-soluble polymers are finding applications in areas as diverse as thickeners for rheology modifications, water based coating fluid such as paints, cosmetics, drug delivery system, detergents, food stuff, adhesives, sealant, enhanced oil recovery and microencapsulation of active molecules [5,6]. Particular importance has been focused on poly(ethyl*

Corresponding author. Tel.: +9133483 4133; fax: +9133414 6266. E-mail address: [email protected] (N. Chattopadhyay).

1011-1344/$ - see front matter
2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2005.05.003

ene oxide) (PEO)-end capped by various hydrophobic groups, including alkanes [3,7–9], adamantane [10], cholesterol [11], pyrene [12–14] and fullerene C60 [15]. Because of their associative properties, telechelic polymers have been incorporated in colloidal dispersions including emulsions, micelles and lyotropic lamellar phases [16–19]. As biological polymers, the hydrophobic modification of proteins has also been well developed yielding a marked modification of their surface properties [6]. Macromolecule, viz., serum albumin, a multifunctional transport protein should display analogous behavior to micelles or emulsion droplets in terms of association as a function of concentration [20]. Recently, we have shown that a representative telechelic polymer, pyrene-end-capped poly(ethylene oxide) (abbreviated as PYPY, Scheme 1) interacts with micelles of different ionic and non-ionic head groups [21]. In another study,

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CH O(CH CH O) OCH 2 2 2 n 2

Scheme 1. Structure of PYPY.

we have also established the different modes of encapsulation of this polymer in different aqueous cyclodextrin (CD) environments [22]. In the present study, we have extended the work to observe the interaction behavior of PYPY to model proteins bovine serum albumin (BSA) and human serum albumin (HSA) in buffered aqueous environments. The polymer PYPY is relatively monodispersed water-soluble polymer containing one pyrene unit at both the ends of the long PEO chain. We have chosen attachment of the hydrophobic end-caps through the less hydrolyzable ether linkage [23]. Duhamel et al. [23] have shown that in aqueous solution a similar polymer to PYPY (mol. wt. a bit smaller) forms an associated pair in the ground state using the two pyrene moieties at the chain ends. Char et al. [14] have also assumed from their experimental findings that the two pyrenes attached to the chain ends are sufficiently close, within twice the capture radius, to form a dimer, yielding static excimer emission. Upon excitation in aqueous solution, PYPY gives a structured fluorescence between 350 and 450 nm assigned to the pyrene monomer (IM) and an unstructured broad excimer emission IE at around 480 nm [23]. Scheme 2 depicts the equilibria existing between different species. Time-resolved measurements indicate that much of the excimer form very rapidly, within the time resolution of the instrument and leads to the suggestion that hydrophobic association between the pyrene groups takes place prior to

Scheme 2. Photophysics of PYPY.

the photoexcitation. With pure pyrene, the blue monomer fluorescence shows vibrational fine structure with I3/I1 ratio (the ratio between the third and the first vibronic bands of the monomer emission) sensitive to the local polarity of the medium [24]. Whilst this ratio is strictly valid for pyrene itself, where the (0, 0) transition is symmetry forbidden, in practice, similar ratios of vibronic bands have been observed with pyrene derivatives linked by ether groups [25], and they give qualitative information on the polarity around the pyrene groups. However, because of the complexity of the environments studied here, we have avoided considering I3/I1 ratio of the monomer emission for monitoring the polarity of the medium. Rather we have preferred to monitor IM/IE for the purpose. Bovine serum albumin (BSA) and human serum albumin (HSA) are frequently used in biophysical and biochemical studies [26–28]. In spite of having different folding nature, known from their structural studies, they somehow resemble in their activity. Bovine serum albumin (BSA) (583 aa) and human serum albumin (HSA) (585 aa) are characterized by a high homology in the sequence (80%) and similar conformation. The molecular weights of the two proteins are also quite close being 66 kDa for BSA and 66.5 kDa for HSA. According to Peters, albumin protein contains 17 disulfide bridges and a series of nine loops, assembled in three domains (I, II, and III) each formed by two subdomains, A and B [29]. The principal binding regions are located in subdomains IIA and IIIA and it is generally assumed that in BSA and HSA these sites are homologous, although they may differ in affinity [29]. The three-dimensional structure of human serum albumin (HSA) is believed to be resolved [30]. However, for BSA, there are conflicting reports so far as structural aspect is concerned. The exact 3D structure of BSA is not yet known clearly due to unavailability of suitable crystals for X-ray diffraction (XRD) studies [31]. Though serum albumins have conformational adaptability towards binding to a variety of biological molecules there are evidences showing that serum albumins interact very weakly with PEO chain of low molecular weight upto 4000 Da. With increasing the molecular weight of PEO chain the interaction decreases [32,33]. Due to its low capacity to interact with proteins, it is very difficult to determine the protein–PEG affinity constant. Farruggia et al. [34] found a value of about 102 M
1 for serum albumin – low molecular PEG complex formation by using an indirect method, supporting a very weak interaction. Thus, PEO is known for its protein resistant behavior and is widely used as stabilizing surface coating in biological environments. In the clinic, ethyleneoxide surface grafts are used to reduce protein adsorption onto the surfaces of biomedical polymers [35–37]. Though the interaction behavior of various hydrophobic molecules including pyrene [38–40] and hydrophilic polymers like PEO [32–34] with serum

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2. Experimental Pyrene-end-capped poly(ethylene oxide) (PYPY) was a kind gift from Professor Mats Almgren (University of Upsala, Sweden). It is relatively monodisperse (Mw/ Mn 6 1.10) and has a molecular weight of 9500 determined from dynamic light scattering. BSA (fraction V, >96%) was obtained from SRL, India. The HSA protein (essentially fatty acid free, >96%) and the HEPES buffer were Sigma products. Fifty millimolar HEPES buffer solution was prepared in water and the pH was adjusted to 7.05 and used in all studies of PYPY in protein. The albumin protein concentrations were varied by addition of respective protein stock solution (3 mM) to a 3 ml of PYPY solution in 50 mM HEPES buffer in small aliquots through a microsyringe. The solvent, 1,4-dioxane, used was of UV spectroscopic grade (Spectrochem India). Triply distilled water was used for making the experimental solutions. All the solutions were air-equilibrated at ambient temperature. The absorption and steady state fluorescence measurements were performed using a Shimadzu MPS 2000 spectrophotometer and a Spex Fluorolog-2 spectrofluorimeter, respectively. The steady state fluorescence anisotropy measurements were performed with a Spex Fluromax-3 spectrofluorimeter. For the anisotropy measurements the excitation and emission bandwidths were both 5 nm. Steady state anisotropy, r, is defined by r ¼ ðI VV
G  I VH Þ=ðI VV þ 2G  I VH Þ; where IVV and IVH are the intensities obtained with the excitation polarizer oriented vertically and the emission polarizer oriented in vertical and horizontal orientation, respectively. The G factor is defined as G ¼ I HV =I HH . I terms referring to the similar parameters as mentioned above for the horizontal position of the excitation polarizer [41]. Throughout the experiment the concentration of PYPY was kept at 1.6 · 10
6 M. For the fluorescence and anisotropy experiments the samples were excited at 340 nm.

3. Results and discussion 3.1. Absorption and fluorescence study The absorption spectrum of pyrene shows the lowest energy structured band in the range 300 and 370 nm, the band maximum being 334 nm in polar solvents. The absorption spectrum of PYPY is qualitatively similar to that of pyrene. The only difference is that the position of the band maximum is a bit red-shifted in case of the polymer. The band maximum for the polymer system in aqueous buffered solution is observed at 343 nm and the bathochromic shift of this band maximum from that of pyrene itself (334 nm) is non-negligible. This reflects that the pyrene units in PYPY are not totally equivalent to pyrene itself and corroborates the earlier proposition [23] that the pyrene units remain as associated pair in PYPY prior to the photoexcitation. Preassociation of pyrene moieties in this polymer is evidenced from the excitation spectra monitoring the two species (at 376 and 485 nm, respectively). Although the excitation spectra of the monomer and the excimer are similar in nature they are not superimposable. First, the excitation spectrum for the excimer emission is 1–2 nm red-shifted, compared to the spectrum of the monomer. Second, the bands in the spectrum monitored for the excimer are broadened. Third, the peak-to-valley ratio for the 0–0 transition is different for the monomer and the excimer excitation spectra. These observations are consistent with the report of Winnik and co-workers [42] for other telechelic polymeric systems, viz., pyrene labeled hydroxypropyl cellulose of different chain length, where they demonstrated a similar red-shift of 1–4 nm for the formation of the ground state preassociation. The normalized excitation spectra corresponding to the monomer and the excimer emissions of PYPY are shown in Fig. 1. Monomer Excimer

Fluorescence Intensity (a.u.)

albumins is already explored in appreciable volume, the interaction nature and/or extent of interaction of hydrophobically modified polymers like PYPY with serum albumins are very rare. Hence, we intended to see whether the hydrophobic end-capping at the two ends of a long, high molecular weight PEO chain can modify the interaction behavior with BSA and HSA. An endeavor has been made to estimate the micropolarity around the probe within the microenvironment. For the entire study, steady state fluorescence spectroscopic techniques have been employed due to their high sensitivity and relative ease to use.

219

80000

0

600000 0

400000 0

0

20000 0

0

270

285

300 315 330 Wavelength (nm)

345

360

Fig. 1. Normalized excitation spectra of PYPY in aqueous buffer solution monitored at 376 nm (monomer, solid line) and at 485 nm (excimer, dotted line).

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In 50 mM HEPES buffer medium (pH 7.05), PYPY gives its characteristic emission spectrum as in pure aqueous solution of PYPY [21–23]. The spectrum consists of a typical structured monomer emission and a broad and unstructured excimer emission. Addition of BSA and HSA to a 1.6 · 10
6 M solution of PYPY in the aqueous buffer medium shows remarkable modification in the emission spectrum of the fluorophore. The nature of variation observed in the two cases is however very similar. As the albumin concentration increases, the emission intensity of the monomer band increases at the cost of the excimer band resulting in an isoemissive point at 438 nm for BSA and at 449 nm for HSA. The variation in the emission spectrum of PYPY as a function of BSA and HSA concentration has been shown in Fig. 2.

Fluorescence Intensity (arb. unit)

8000000 7000000

xi

6000000 5000000

i

4000000

i

3000000 2000000

xi

1000000 0

350

400

450

500

550

600

Wavelength (nm)

Fluorescence Intensity (arb. unit)

(a)

7000000

The plot of the relative emission intensity of excimer to monomer (IE/IM) against albumin concentrations is presented in Fig. 3. The figure shows a gradual decrease in IE/IM, suggesting that the intramolecular association of the pyrene chain ends get somehow hindered in the protein environments. The smaller variation in IE/IM value for HSA suggests that the intramolecular association is less hindered in HSA compared to BSA. Since the proteins have hardly any interaction with the PEO chains (vide infra) the variation in the emission intensity and existence of the isoemissive point indicate that the polymer PYPY binds with the albumin proteins through the hydrophobic pyrene units. A higher value of IE/IM value at the plateau in case of HSA compared to BSA (Fig. 3) is a reflection of the fact that the polymer is adsorbed mainly on the surface of HSA while it goes inside the BSA environment. From the view point of protein resistant behavior of high molecular weight PEOs, it should not be irrational to assume that the unbound excimers resulted from the preassociated pyrene moieties are responsible for the higher value of IE/IM in case of HSA. In addition to the emission spectra, another interesting difference is observed in the excitation spectra of PYPY in two albumin protein media. A distinct 2 and 1 nm red-shift is observed in the excitation spectra of monomer and excimer, respectively, between zero and high (274 lM) BSA concentrations. But in the case of HSA, though the same shift (2 nm) in the excitation spectrum is found for the monomer, even lesser shift is observed for the excimer. The observation led us to conclude that both the monomer and the associated pair get accommodated in the hydrophobic grooves of BSA whereas for HSA only the monomer is bound in the hydrophobic pockets. From the above findings it appears that BSA has bigger grooves in its structure compared to those within HSA that allows binding of the

6000000

xi

5000000

HSA BSA

4000000

0.5

i

3000000

i 0.4

2000000

IE/IM

xi

1000000

0.3

0

350 (b)

400

450

500

550

600

Wavelength (nm)

Fig. 2. Fluorescence spectra of aqueous buffered PYPY solution in varying albumin concentrations. In (a) the concentrations of BSA from (i) to (xi) are 0.0, 8.0, 25.9, 37.7, 57.1, 97.1, 120.4, 143.3, 170.4, 231.5, 273.6 lM, respectively, and in (b) the concentrations of HSA from (i) to (xi) are 0.0, 4.2, 12.4, 24.7, 49.0, 80.6, 103.8, 130.3, 156.3, 192.3, 261.2 lM, respectively. In both the cases PYPY concentration is 1.6 · 10
6 M.

0.2

0.1 0

50

100

150

200

250

300

6

[Albumin] x 10 (M) Fig. 3. Variation in IE/IM with increasing albumin concentration.

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preformed pyrene associated pair with the former protein. 3.2. Fluorescence anisotropy study Measurement of fluorescence anisotropy is claiming the role for its tremendous potential in biochemical research owing to the fact that any factor affecting size, shape or segmental flexibility of a molecule will affect this parameter [41]. Since polarization measurements can give details about any association or binding phenomenon, the technique has been employed to gather additional evidence in support of interaction of the hydrophobic pyrene units to the native albumin protein. The anisotropy values (r) have been measured for both the monomer and the associated species monitoring at 376 and 485 nm, respectively. The variation in the anisotropy value as a function of albumin concentration has been plotted in Figs. 4(a) and (b) for the monomer and the excimer species, respectively.

0.040 0.035

Anisotropy (r)

0.030 0.025 0.020 0.015 0.010

HSA BSA

0.005 0

50

100

150

200

250

300

6

[Albumin] x 10 (M)

(a) 0.16 0.14

Anisotropy (r)

0.12 0.10 0.08 0.06 0.04

HSA BSA

0.02

The anisotropy variation in the presence of albumin reveals a restriction in the rotational diffusion of both the pyrene and the associated pair supporting a hydrophobic binding interaction between PYPY and the albumins. A greater anisotropy value in BSA compared to that in the same concentration of HSA suggests a greater motional restriction around the fluorophoric moiety in BSA compared to HSA. This observation suggests, in conformity with the discussions in the previous section, that the polymer is mainly adsorbed on the surface of HSA while it goes more inside in the case of BSA. A noticeable difference between the two albumin proteins is the overall increase in the anisotropy values for the monomer and the excimer. The enhancement in the anisotropy of the excimer emission is remarkably large compared to that for the monomer. It is also interesting to note the pattern of increment in the excimer anisotropy. These two observations suggest that the mode of interaction of albumins with the associated pair is different from that with the monomer pyrene units. It is also not expected that the excimer possessing charge transfer character should respond to a hydrophobic interaction in the same way as that of a pyrene unit. It is therefore, probable that the monomer should sit deeper inside the hydrophobic cavity whereas the associated pair should be exposed to a less hydrophobic and comparatively open site. It is pertinent to mention here that when a macromolecule like high molecular weight PEO is labeled with a fluorophore, the motions that the fluorophore can ÔreportÕ are not only dependent on the size of the fluorescing unit but also on: (i) the nature of the attachment of the fluorophore (e.g., rotation around a single bond that links the fluorophore with the macromolecule), (ii) the three-dimensional shape and motions of the entire macromolecule and (iii) the global Brownian tumbling of the protein–macromolecule aggregate. The overall dimension of the protein-bound associated pair is markedly larger compared to that of the unbound species. This leads to a marked reduction in the tumbling motion for the former resulting in an increase in the anisotropy value. A higher increase in the anisotropy value for the excimer emission in BSA compared to that in HSA is consistent with our proposition of greater binding of the associated pair with the former and lesser with the latter. The larger size of the associated pair along with the tumbling motion of the protein molecules may significantly contribute to favor the higher anisotropy value and the different pattern in the anisotropy variation for the excimer emission. 3.3. PYPY–albumin binding interaction

0.00 0

(b)

221

50

100

150

200

250

300

6

[Albumin] x 10 (M)

Fig. 4. Variation in steady state fluorescence anisotropy (r) value as a function of albumin concentration for monomer (a) and excimer (b).

To find the extent of binding between the two species PYPY and serum albumins, the binding constant values have been determined from the fluorescence intensity

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considering the following rearranged Eq. [43] of the original one developed by Benesi and Hildebrand [44]: 1=DF ¼ 1=DF max þ ð1=K  ½LT Þð1=DF max Þ;

ð1Þ

where DF = Fx
F0 and DF max ¼ F 1
F 0 where F0, Fx and F1 are the fluorescence intensities of the monitoring species of PYPY in the absence of serum albumin, at an intermediate concentration of serum albumin and at the saturation level of interaction, respectively; K being the binding constant and [LT] the protein concentration. Rearranging the above equation we have the following form DF max =DF ¼ 1 þ 1=ðK½LT Þ.

ð2Þ

The linearity in the plot of DFmax/DF against 1/[LT] confirms a one to one interaction between the probe and the serum albumin molecule. Fig. 5 depicts the corresponding plots for the monomer and the excimer species in two serum albumin environments. 5

3

8 7 6

max

/∆F

2

∆F

∆Fmax/∆F

4

5 4 3 2

1

1 0 0.00

0 0.00

0.01

0.02

0.03 -6

0.04 -1

0.05

0.06

1/LT x 10 M

0.01

0.02

0.03

0.04 -6

0.05

0.06

-1

The binding constant (K) values thus obtained from the slopes of the plots for the monomer and the excimer with BSA are 1.33 · 104 and 8.4 · 103 M
1, respectively. The changes in free energy (DG) for the binding process of these two species with BSA have also been calculated from the equation DG =
RT ln K at 25 C and they are found to be
5.6 and
5.3 kcal mol
1 for the monomer and the excimer, respectively. The fluorescence intensity data for the monomer of PYPY in HSA protein give appreciably good linear fit and the binding constant value thus obtained is 2.7 · 104 M
1 giving the DG value of interaction as 6.0 kcal mol
1. It is pertinent to discuss here about the apparent discrepancy between the relative binding constant values with the IE/IM plot (Fig. 3). While Fig. 3 displays that the plateau is attained with HSA at a higher IE/IM value compared to BSA environment, the binding constant between the PYPY monomer is more with HSA. Actually the value of IE/IM depends not only on the binding parameter but also on the polarity of the microenvironment. Since the polymer binds with HSA at the surface (vide infra), the polarity is higher compared to that in BSA. Thus a higher polarity leads to a higher value of IE/IM at the HSA plateau than the BSA plateau. However, monitoring the excimer emission a non-linear plot was obtained reflecting that there is inappropriate or non-stoichiometric association between the HSA and PYPY associated pair. This can perhaps be ascribed to the preference of the excimer/associated pair to stay in the peripheral zone of the HSA rather than getting encaged within the hydrophobic core. The weak non-stoichiometric interaction between the associated pair of PYPY and HSA is reflected in the smaller increase in the fluorescence anisotropy of the excimer band compared to the same in BSA environment.

1/LT x 10 M

(a)

3.4. Micropolarity around the pyrene units 3.5 3.0

2.0

25

20

1.5

∆ Fmax / ∆ F

∆Fmax/∆F

2.5

1.0

15

10

5

0.5 0 0.00

0.01

0.02

0.03 -6

0.0 0.00

0.01

0.02

0.03

0.04 -6

(b)

0.04 -1

0.05

0.06

1/LTx10 (M )

0.05

0.06

-1

1/LTx10 (M )

Fig. 5. Plot of DFmax/DF against 1/[LT] for the monomeric emission of pyrene in (a) BSA and (b) HSA. Corresponding plots for the excimer emission are given in the insets.

For a couple of decades, fluorescent probes have been serving an important role in the determination of the microscopic polarity of the biological systems. The polarity determined through different photophysical parameters of fluorophores gives a relative measure of the polarity of different microenvironments. The local polarity of a biological system like protein, having binding interaction with fluorescent molecules can be estimated by comparison of the spectral properties of the fluorophore in the environment with those of the probe in pure solvents or in solvent mixtures of known polarity parameters [45–47]. It is true that the polarity of the homogeneous environment should not be exactly the same as the polarity of the heterogeneous environments, however the method gives a good relative measure of the desired parameter in such environments. Sytnik and Kasha [48] have referred to this local polarity as static polarity.

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In our recent works [49,50], we have monitored the variation in the relative emission intensity of the neutral to cationic species of a bioactive molecule, norharmane, to estimate the micropolarity in BSA and micellar environments. Here, we have followed the same method for PYPY to get a semiquantitative measure of the local polarity around the pyrene units in these two proteinous environments. Fig. 6 depicts the calibration plot of IM/IE against ET (30) for different water–dioxane mixtures [51]. Superposition of the IM/IE value of the PYPY emission at high albumin concentration (when the probe– protein interaction is complete) on the calibration curve gives an approximate measure of the micropolarity around the binding site. These values for BSA and HSA environments have been determined to be 55.7 and 59.9 kcal mol
1, respectively, in terms of ET (30). The determined ET (30) value of BSA agrees well with our earlier report [49]. A lesser polarity in case of BSA compared to that of HSA indicates that the probe goes deeper into the BSA protein compared to the other one. This is consistent with the higher value of fluorescence anisotropy in BSA than HSA (cf. Fig. 4) and a higher value of IE/IM at the plateau in HSA than that in BSA (cf. Fig. 3). Proximity of the fluorophore molecule to the tryptophan (Trp) moiety of the protein is often determined through observation of fluorescence resonance energy transfer (FRET) from Trp to the probe. In spite of the fact that there is a good overlap between the emission spectrum of Trp present in the albumins (donor) and the absorption spectrum of PYPY (acceptor), we could not identify the occurrence of the FRET process by exciting the composite system at the excitation band of Trp. This suggests that the fluorophore PYPY is not located close to the Trp of BSA or HSA. However, the exact location of the pyrene units is yet to be determined.

24 20

IM/IE

16 12

BSA 8

HSA

4 0 46

48

50

52

54

56

58

60

62

64

-1

ET(30) kcal mol

Fig. 6. Variation of IM/IE against ET (30) in varying water–dioxane mixture and the polarities in the albumin environments.

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4. Conclusion The present work reports the study of interaction of a telechelic polymer PYPY with two albumin proteins in aqueous buffer medium. The photophysical behavior of PYPY is modified remarkably in the proteinous environments compared to that in pure aqueous phase. This has been exploited to study the binding efficiency, nature of microenvironment and the micropolarity around the fluorescing units. Acknowledgments Financial supports from CSIR and DST, Government of India, are gratefully acknowledged. B.H. thanks the CSIR for awarding the research fellowship. We are indebted to Prof. S. Basak and Mr. H. Chakraborty of SINP, Calcutta, for their cooperation in the fluorescence anisotropy measurements.

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