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Spectral studies on the binding behavior of cationic dyes and surfactants

with bacterial polysaccharide of Klebsiella K43

Abstract

The Binding behavior of acidic capsular polysaccharide (SPS), isolated from

Klebsiella serotype K43 with oppositely charged dyes and surfactants have been studied by

absorbance and emission spectroscopic studies. Each repeating unit of the SPS consists of

three D-mannose, one D-galactose and one D-glucuronic acid residue. The anionic polymer

exhibited chromtropic character and induced strong metachromasy in the cationic dye,

pinacyanol chloride (PCYN) through the formation of a 1:1 polymer/dye complex. Evaluation

of thermodynamic parameters, viz., standard changes in free energy (∆G0), enthalpy (∆H

0)

and entropy (∆S0), for the formation of dye-polymer complex and studies on the effect of

different co-solvents were also evaluated to shed light on the binding nature as well as the

extent of stability of the dye-polymer complex. Fluorescence of the cationic dye acridine

orange (AO) was quenched with the progressive addition of SPS, which was found to be of

Stern-Volmer type. Cationic surfactants in their pure form as well as in mixed state with

nonionic surfactant (Tween 20), replaced the dye bound to the polymer matrices; thus the

original band intensities of the dyes could be reverted. Such the studies revealed that both

electrostatics as well as hydrophobic forces is operative within the dye-polymer as well as

surfactant-polymer aggregates.

Published in J. Dispersion Sci. Technol. 31 (2010) 1447-1455

70

1. INTRODUCTION

Klebsiella is a gram negative bacterium which belongs to Enterobacteriaceae family.

They are the causative factors for several human diseases [1-3] like liver abscess [4], septic

endophthalamitis [5, 6], infection in urinary tract, pneumonia and meningitis [1, 7], etc.

Most of these gram negative bacteria form capsular polysaccharides. Till now about

82 serologically distinct K-serotypes [8, 9] have been found in the gram-negative bacteria

Klebsiella which can produce acidic hetero polysaccharides with wide structural variations.

These bacterial polysaccharides also have antigenic properties [1]. Presence of definite

repeating units, ranging from tri- to hepta-saccharides, containing glucuronic acid and/or

galacturonic acid, along with other neutral sugars, contribute to their unique nature, than other

conventional polysaccharides. The existence of potential anionic site in the biopolymers is

due to the presence of acidic sugars in every repeating unit which, in turn, also provide

polyelectrolytic nature. The primary structures of most of the capsular polysaccharides are

now known through chemical analyses [10].

The primary structures of capsular polysaccharides and conformational factors are

responsible for their serological specificity [11]. The biophysical properties of these

polysaccharides are due to their secondary structures. Extra cellular polysaccharides are

produced by Klebsiella bacteria which surround the bacterium as an additional outer layer as

capsule. Since Klebsiella capsular antigens have been found to be safe in human so these

antigenic polysaccharides are also being used as human vaccines [12]. Due to potential uses

of the bacterial polysaccharides in immunological and vaccine preparations, primary

structural studies and conformational analysis, as well as studies on various physico-chemical

properties of these biopolymers are gaining more and more importance with the discovery of

newer serotypes. Thus the physicochemical characterizations of these bacterial

71

polysaccharides are also becoming necessary. Although the present research group is

associated in such studies since last three decades still the results are considered to be

fragmentary in nature [13].

Klebsiella K43 consists of D-mannose, D-galactose and D-glucuronic acid residues in

the ratio 3:1:1. The term ‘SPS’ arises from sauer polysaccharides; (sauer means sour in

German). Presence of one glucuronic acid in each repeating unit enables it to interact with

positively charged dyes and surfactants [14, 15]. The authors have been carrying out dye-SPS

interaction studies through spectroscopic measurements in characterizing different SPS

isolated from various serotypes of Klebsiella [13, 15-18]. Spectral studies on the dye-polymer

interaction could shed light on the detailed structural aspects of the polysaccharides [13].

Metachromasy is a well known phenomenon in case of dye-polymer interaction and is

generally applied to the aggregation of cationic dye on anionic polymers and colloids [19].

The phenomena have also been observed in few cases of cationic polyelectrolyte and anionic

dye systems. Reports dealing with studies on dye-polymer interaction inducing metachromasy

in different cationic dyes by different synthetic polyanions, DNA and naturally occurring

plant polyelectrolytes are also available. A clear idea about the mechanism of polyion induced

metachromasy and the correlations between the dye structures and metachromatic behaviors

could be obtained through such studies, e.g., interaction of heparins with azure dyes [20-22].

Dye-protein interaction study could also reveal the conformational aspects of protein [23].

Different techniques have been reported for the isolation and stability determination of

metachromatic complexes [13]. The concept of reversal of metachromasy can be used to

determine the stability of the metachromatic complex. Reversal of metachromasy may be

attained with the addition of urea, alcohol, electrolytes and also with increasing the

temperature of the dye-polymer aggregate [24].

72

Evaluation of different thermodynamic parameters like changes in free energy (∆G),

enthalpy (∆H) and entropy (∆S) could explain the nature of interaction in the formation of

metachromatic complex and can also predict about the optimum conditions for the interaction

between the oppositely charged dye and macromolecules.

Fluorescence quenching is a process of deactivation of the excited state which results

in a decrease in fluorescence intensity. Acridine orange (AO) could be used as a

photophysical probe under the influence of different polymer matrices [13, 15, 24].

Polymer-surfactant interaction is a very convincing field of research. Several studies

dealing with polyelectrolyte and oppositely charged surfactants [25-27] have been done for its

importance in both fundamental and applied aspects [28]. Surfactant molecules can bind to

the oppositely charged polyelectrolytes, forming the so called polymer-/surfactant complexes.

Additionally hydrophobic interaction may also promote/favor surfactant binding to the

polymer matrix [29, 30]. A comprehensive study using a series of cationic surfactants with

anionic polyelectrolytes such as carboxymethylcellulose (CMC), dextran sulfate (DX),

polyacrylic acid (PPA), poly(methacrylic acid) (PMAA), etc., have been done using various

techniques [25, 27, 30-35]. Surfactant-polymer interaction studies also include biologically

associated systems like polypeptides [33], proteins [30], DNA [31, 35] carbohydrate based

polymer [36]. Different methods to study polymer-surfactant interaction include turbidity

measurement, viscosity measurements, light scattering techniques, calorimetry, conductivity

measurement, e.m.f. measurements, and dye-incorporation techniques, etc. Such experimental

methods allow in gathering lot of information on the nature of interaction in different

polymer-surfactant aggregates. A quantitative understanding on the interaction in surfactant-

biopolymer systems could be achieved by such studies.

73

The present investigation deals with the studies on interaction between anionic

polysaccharide isolated from Klebsiella K43 and cationic dye pinacyanol chloride and

acridine orange in aqueous medium and also associated with oppositely charged surfactant

system. Spectrophoto-/fluori-metric techniques were used for detailed study of dye-polymer

interaction. Thermodynamic parameters of interaction and effects of different co-solvents

were evaluated. Interaction of Klebsiella K43 with different cationic surfactants, viz., benzyl

dimethyl-(-n-) hexadecyl ammonium chloride (BDHAC), cetyltrimethylammonium bromide

(CTAB), cetylpyridinium chloride (CPC) and dodecylpyridinium chloride (DPC)in their pure

form, as well as in their mixed states with a nonionic surfactant Tween 20 were explored

through spectrophoto-/fluori-metric measurements using dye probing techniques. Binding

constant of biopolymer-surfactant aggregates were evaluated by suitably analyzing the

spectral data.

2. Materials and methods

2.1. Materials

The serological strain of bacterial polysaccharide Klebsiella K43 was kindly supplied

by Dr. S. Schlecht of Max Plank Institute for Immunolobiology, Freiburg, Germany. Cationic

dyes pinacyanol chloride (PCYN) and acridine orange (AO) from Sigma Chemicals, USA

were >99% pure and were used as such. HPLC grade alcohols, from E. Merck, Germany were

used. Cationic surfactants benzyldimethyl-n-hexadecyl ammonium chloride (BDHAC),

cetyltrimethyl amminium bromide (CTAB), cetylpyridinium chloride (CPC),

dodecylpyridinium chloride (DPC) and nonionic surfactant polyoxyethylenesorbitan

monolaurate (Tween 20) were the products from E. Merck, Germany. They were used after

purification [17] using standard procedure. Purities were checked through their

74

conductance/surface tension values [37]. Freshly prepared double distilled water with a

resistivity of 18 mΩ cm-1

was used for all the measurements.

2.2. Methods

In absorbance study, a 10 µM aqueous PCYN was used. Absorption spectra were

recorded in the wavelength range of 400-700nm with a LS-55 spectrophotometer (Perkin

Elmer, USA). The average molar mass of one repeating unit of the polymer containing one

anionic charge site was referred as one mole of polymer. The absorption spectra at different

polymer/dye ([P]/[D]) molar ratio, by addition of the capsular polysaccharides to the aqueous

solution of 10 µM dye were recorded. Spectrofluorimetric measurements were done with the

help of a RF 5000 spectrofluorimeter (Shimadzu, Japan) by the excitation at 475nm of the AO

dye in aqueous solution, using quartz cell of 1 cm path.

Stoichiometry of the dye-polymer complex was determined by isolating the dye-

polymer complex according to McIntosh method [14, 17], which was modified by Kornors et

al. [38] Briefly, dye, bound to polymer (herein dye-polymer complex), was separated out

from the aqueous layer by vortexing the mixture with benzine. The un-complexed dye in

water, was quantified by measuring the absorbance at 600nm, thus the concentration of the

complexed dye could be calculated. Finally, from the point of intersection obtained by

plotting the values of complexed dye concentration against polymer concentration, the

stoichiometry of the dye-polymer complex was determined. Centrifugation method [15] was

also used to determine the stoichiometry. In this method, the metachromatic complex was

centrifuged at 11,000 rpm (23,000 g) for 20 min whereby the dye-polymer complex got

sedimented. The supernatant was analyzed colorimetrically for the free dye and stoichiometry

was calculated as in the previous method.

75

Metachromatic titration was performed by measuring the absorbance of the complexed

dye at 600nm, from which the volume of the polymer solution required for the equivalent

consumption of the dye was estimated. The description of the procedure of metachromatic

titration is available in literature [13, 15, 24, 39-43].

Effects of different co-solvents like methanol, ethanol, n-propanol, on the reversal of

metachromasy was studied by measuring absorbance of metachromatic complex at a [P]/[D] =

5. Extent of reversal of metachromasy was also compared by studying the absorbance of pure

dye solution in presence and absence of the co-solvents.

Thermodynamic parameters of the interaction between PCYN and the experimental

polymer were determined using Rose and Drago equation [44]. Charge transfer interaction

theory could represent the interaction between the cationic dye and polyanions. The following

Rose and Drago equation was then employed for determining the interaction constant between

the dye and polymer.

O

SD

AA

CC

−

.=

)(.

1

DDSC LK εε − +

)( DDS

S

L

C

εε − [1]

where, CD = Initial molar concentration of PCYN, CS = molar concentration of capsular

polysaccharide Klebsiella K43, єD =molar absorption coefficient of PCYN, єDS= molar

absorption coefficient of the PCYN-polymer complex, KC = interaction constant between the

dye and polymer, AO= absorbance of the pure dye PCYN at 500nm (metachromatic band of

the dye-polymer complex), A=absorbance of the dye- polymer solution at 500nm at a

particular polymer concentration and L = Length of light path.

76

Detailed procedure for determining the thermodynamic parameters for dye-polymer

interaction as per Rose and Drago and other associated equations are available elsewhere [15,

39-43].

In fluorescence quenching experiments 10 µM aqueous solution of AO was used as the

probe. The fluorescence of the dye solution as well as of the dye polymer complex (at

different P/D) was measured. The excitation wavelength was at 475nm and the emission

spectra were recorded in the range 450-650nm.

The quenching of fluorescence in acridine orange dye by K43 polymer were treated

with Stern-Volmer equation [45] as used in our earlier works [15, 39-43], then from the slope

of the linear plot, the Stern-Volmer constant (KSV, herein the binding constant between the

dye and polymer) was evaluated.

The effects of different cationic as well as cationic-Tween 20 mixed surfactant

systems on the dye-polymer complex were studied spectrophoto-/fluori-metrically. In

studying the surfactant effects on bacterial polysaccharide, to a fixed dye-polymer ratio

(P/D=5), increasing amounts surfactant was added and absorbance of each solution was

measured at 600 nm (at J band) of PCYN.

Finally, the binding constant between the SPS and oppositely charged cationic as well

as cationic-nonionic mixed surfactants were evaluated by Rose and Drago equation [44] by

suitably processing the absorption spectral data. The terms involved in Rose and Drago

equation as used in dye-polymer interaction study, bear separate meaning. In the present case,

the term Cp indicates the concentration of the polymer, AO is the absorbance of the dye-

polymer solution at 600 nm, A is the absorbance of the dye-Polymer solution at 600 nm when

CS concentration of surfactant added, Ka is the binding constant between the polymer and

77

surfactant, and ε PS is the molar absorption coefficient of surfactant-polymer complex,ε S is

the molar absorption coefficient of surfactant and L is the length of light path.

O

SP

AA

CC

−

.=

)(.

1

SPSa LK εε −+

)( SPS

P

L

C

εε − [2]

3. Results and Discussions

The primary structure of Klebsiella K43 consists of pentasaccharide repeating unit of

D-mannose, D-galactose and D-glucuronic acid in the ratio 3:1:1 [14]. The proposed structure

may be represented through Figure1.

Figure1. Structure of Klebsiella K43 SPS.

The equivalent weight of the polymer obtained through metachromatic titration (as to

be shown later) was found to be 820, which was very close to the value calculated from its

structure. The presence of glucuronic acid in the repeating unit provides potential anionic site

for its interaction with the oppositely charged dyes and surfactants.

The cationic dye PCYN, which has been used, belongs to cyanine group of dyes [26]

and because of the presence of aromatic moiety, it is amphipathic in nature and has a tendency

to aggregate in aqueous media, specially at higher concentration (even in the micromolar

range). Two sharp bands at 600nm (J-band) and at 550nm (D-band) were observed for the

aqueous solution of the dye. These two peaks correspond to the absorption peaks of the

3) − α − D-Manp (1 2) - α-D-Manp (1 3)-α-D-Galp (1-

2

1

β-D-Manp- (1 4)-β-D-GlcpA

78

monomeric and dimeric form which is due to the vibration less electronic transition and

vibrational electronic transition respectively [17]. With increase in polymer/dye molar ratio

(P/D) values the J-and D-band intensities decreased and a new band with a maximum at 490

nm (H-band) appeared. This was an evidence of blue shift of ~110nm and induction of strong

metachromasy in PCYN induced by Klebsiella K43 polymer. Occurrence of initial

broadening at the J- and D- bands alongwith newly formed H-band was due to multiple

conformational structures of the polymer. The absorption spectra obtained by addition of K43

polymer at different P/D ratios (P/D=0 to 30) with PCYN dye (10 µM) has been shown in

Figure 2. The nature of the spectra depends upon the conformation of polyanions as well as

Figure 2. Absorption spectra of 10µM PCYN at different [SPS]/[Dye]:1,0; 2,1; 3,2 ;4,5; 5,10;

6,20; 7,30 and 8,50

dye ion in aqueous solution. Conformational influence on the spectra of PCYN by other

synthetic polyanions has been reported [46]. Occurrence of multiple banded broad spectra

suggested random coiled structure of the polymer in the solution whereas at higher

concentration of polymer almost a single banded spectrum was observed, which suggested

conformational change of the polymer from random coil to helical structure.

79

Metachromatic titration was carried out spectrophotometrically at lower P/D values (0

~ 2) and yielded identical stoichiometry. The absorbance values at 600nm were plotted

against volume of the polymer solution added as shown in Figure 3. The molecular weight of

Figure 3. Spectrophotometric () and fluorimetric (∆) titration of Klebsiella K43 at 303K, 10µM PCYN

and AO were used for metachromatic and fluorimetric titrations, respectively, λex for AO: 475nm.

the polymer per repeating unit was calculated from the intersecting point. Spectrofluorimetric

titration also yielded identical result.

The stoichiometry of the dye-polymer complex was determined by McIntosh and

centrifugation methods which were found to be 1:1 as in Figure 4. This suggested that the

glucuronic acid was the only potential anionic sites for interaction with the cationic dye

PCYN and thus suggested a stair case stacking arrangement of the dye molecules to polymer

matrices. Appearance of isosbestic point at ~535 nm in the absorption spectra of PCYN also

confirms the formation of 1:1 stoichiometric complex between dye and polymer.

Reversal of metachromasy is a phenomenon of destruction of charge-transfer complex

formation (i.e., formation of metachromatic complex). The reversal of metachromasy was

80

Figure4. Determination of stoichiometry ofKlebsiella K43 SPS-PCYN complex at 303K, = McIntosh

method and O= Centrifugation method

studied by measuring absorbance both at J- and H- band upon addition of different co-

solvents like methanol, ethanol and n-propanol. The destruction of metachromatic compound

with the progressive addition of alcohols has been attributed to the involvement of

hydrophobic bonds in the induction of metachromasy. The reversal of metachromasy taking

n-propanol as representative case is shown in Figure 5. With gradual addition of ethanol, the

Figure 5. Reversal of metachromasy induced by propanol in Klebsiella K43 SPS-PCYN complex: 1=

dye-polymer complex; 2= pure dye; 3= dye-polymer complex in 30% propanol, and 4=dye in 30%

propanol.

intensity of the J-band gradually increased with the decrease in intensity of D-band. Finally,

in presence of 30% propanol at [P]/[D] = 5 the metachromatic band (H-band) completely

81

disappeared and the spectrua became identical with that of the pure dye solution (in 30% n-

propanol). Experimental results suggested that the dye only interacts with SPS through

predominant electrostatic binding in the anionic charge centers of the polymer.

Rose and Drago equation was used to evaluate the thermodynamic parameters [44]. The

parameters Kc, ∆H0, ∆G

0 and ∆S

0 of the dye-polymer interaction were determined at different

temperatures (303, 308, 313, 318 and 323 K) by computing the absorbance values (at 490 nm)

in Rose and Drago equation. (CD.CS)/(A-AO) values were plotted against CS as depicted

graphically in Figure 6. Kc value was determined from the ratio of the slope and intercept of

the graph. The results are summarized in Table 1. The values of free energy change was

Figure 6. Plot of CD.CS/(A-Ao) vs. CS to determine the PCYN. Klebsiella K43 SPS binding constant at

different temperatures. [PCYN]= 10µM. Temperature (K): =303 ; O =308; ∆= 313; ∇=318 and ◊=323.

determined at all temperatures with the help of the equation ∆G0= - RT ln KC. The value of

standard enthalpy change, ∆H0 was obtained from the plot of ln Kc versus 1/T and ∆S

0

standard entropy change was obtained from the plot of ∆G0 versus T according to the

equation ∆G0 = ∆H

0 – T∆S

0. The negative values of ∆G

0 were within the range of a reversible

biological process involving any non-chemical type of interaction. Binding constant values

decreased with the increase in temperature which supports the exothermic nature of the

interaction process .The negative value of ∆G0 shows the spontaneity of the process [17].

82

Table 1. Thermodynamic parameters for the interaction of Pinacyanol chloride (PCYN) with Klebsiella

K43 capsular polysaccharides.

Temp./K 10-4KC (M

-1)a G

0(kcal mol

-1)b H

0(kcal mol

-1)c S

0(cal mol

-1 K

-1)c

303 0.12 -4.29

308 0.10 -4.26

313 0.08 -4.19 -6.54 0.98 7.45 0.51

318 0.07 -4.18

323 0.06 -4.14

PCYN]=10µM. a Calculated from Figure 6,according to Rose and Drago equation[43].

b Calculated from the thermodynamic relation ∆G

0= - RT ln KC.

cCalculated from the graphical plot of ∆G

0 vs T according to the relation ∆G

0 = ∆H

0 -T∆S

0.

The fluorescence quenching was applied to study the interaction of fluorescent dye

acridine orange (AO) with polymer. The emission spectra of the dye and the dye-polymer

mixture at different [P]/[D] values were recorded. The fluorescence intensity of the dye was

progressively quenched with increasing concentration of the polymer, as shown in the

Figure 7.

Figure 7. Emission spectra of 10µM AO at 303K in presence of varying amounts of Klebsiella

K43 SPS. [SPS] (10µM)=1,0; 2,1; 3,2; 4,5; 5,10; 6,20; 7,30 and 8,50. Λex =475nm.

The results were also treated in Stern-Volmer equation to study the interaction between the

dye and the polymer molecules in solution and the plot was shown in Figure 8. The plot was

83

Figure 8. Stern-Volmer plot for determining Ksv for Klebsiella K43 SPS-AO complex at 303K.

Fluorescence were recorded at 525nm , λex = 475nm.

linear in nature with a unit intercept. From the slope, the binding constant, KSV was found to

be 0.74 x 104

M-1

.

The anionic polyelectrolyte can interact with different cationic surfactants. Hence

addition of cationic surfactant or cationic-nonionic mixed combinations would be able to

replace the dye bound to the polymer matrix; thus the original band of pure dye could be

regained. The cationic surfactants used were CTAB, CPC, DPC and BDHAC. The absorbance

of a dye-polymer complex was measured by adding surfactants (to a fixed P/D =5) at 600nm.

The result has been shown in Figure 9. With the addition of cationic surfactants, the

absorbance values of the dye-polymer complex increased to a considerable extent. This result

indicated that the surfactant molecules interacted with the capsular polysaccharide, replacing

the cationic dye molecules.

The anionic polyelectrolyte taken here interacted with cationic surfactants and the

association was strong as they were oppositely charged. The interaction was electrostatic in

nature, which was stabilized by hydrophobic interactions.

84

Figure 9. Effect of cationic surfactants on Klebsiella K43 SPS-PCYN complex at 303K. [SPS]/[PCYN]

=5, [PCYN]= 10µM. Surfactants = CTAB; O = CPC, ∆ = DPC and ∇=BDHAC.

Binding constant of these interactions was calculated by using the Rose and Drago

equation from the absorbance values. The results are reported in Figure 10. Results are

summarized in Table 2.

Table 2. Values of binding constants between Klebsiella K43 SPS and different cationic/cationic-

nonionic mixed surfactant systems at 303K

Surfactant CMC /mM 10-4 x binding constant/M

-1

BDHAC 0.042 2.06

CPC 0.90 0.90

DPC 14.70 0.80

CTAB 0.80 1.00

CTAB:T20(4:1) 0.17 0.48

CTAB:T20(1:1) 0.07 0.43

CTAB:T20(1:4) 0.05 0.50

*CMC values were adopted from references

[17,37].

85

Figure 10. Plot of CP.CS/(A-Ao) versus Cs to determine the binding constant of Klebsiella K43

SPS-surfactants at 303K.[SPS]/[PCYN] =5,[PCYN]= 10µM. Surfactants: = CTAB; O = CPC

and ∆ = DPC, Inset: BDHAC.

The effect of cationic-nonionic mixed surfactants, taking CTAB and Tween 20, in

different compositions, on the absorbance of Klebsiella K43 SPS-PCYN complex were also

studied. The result of CTAB-Tween 20 mixture has been shown as representative graph in

Figure 11.

Figure 11. Plot of CP.CS/(A-Ao) versus Cs to determine the binding constant of Klebsiella K43.

[SPS]/PCYN=5, [PCYN]= 10µM , CTAB/Tween-20 mole-ratio : [O= 0; = 0.8; ∆= 1 and ∇=0.2]

The results indicated higher binding capability in case of mixed surfactants in

comparison with pure cationic surfactants [47-51]. Addition of nonionic surfactant could

lower down the critical micelle concentration; hence hydrophobicity would be enhanced.

86

Fluorescence studies of polymer-surfactant interaction obtained by using single as

well as mixed surfactants have been shown in Figures 12 and 13. The results also revealed

identical binding effect between surfactant and SPS by absorbance studies.

Figure 12. Effects of cationic surfactants on the fluorescence of AO-Klebsiella K43 polymer

complex [P/D], [Dye]=5, [PCYN]=10µM, = CPC; ∇= DPC ,O = CTAB , and ∆ =BDHAC.

Figure 13. Effect of CTAB-Tween 20 Surfactant mixture on Klebsiella K43 SPS-PCYN

complex at 303K. [SPS]/[PCYN]=5 ,[PCYN]= 10µM CTAB/Tween-20 molar ratio [= 0; O =0.8;

∆= 1 and ∇ =0.2]

4. Conclusion

Studies on the interaction of K43 SPS with PCYN and AO were performed

spectrophoto-/fluorimetrically. K43 SPS was found to be acidic in nature with definite

repeating sugar unit. Thermodynamic parameters for the dye polymer interactions were found

87

to be comparable with the reversible biological processes. The polymer quenched the

fluorescence of AO. The present set of physicochemical studies thus could shade light on the

tertiary conformation of the SPS in aqueous solutions. The effects of surfactants, pure as well

as mixed, were also studied and the results indicated higher binding capacity in case of mixed

surfactants in comparison with pure cationic surfactants. The present studies strongly revealed

that the two forces, viz., electrostatic and hydrophobic forces are essentially involved during

the aggregation process of polymer-dye and polymer-surfactant. However, further studies like

light scattering, zeta potential measurements are essential to understand the complete solution

behavior of such polymeric materials.

88

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