53

Interactions of bacterial polysaccharides with cationic dyes:

Physicochemical studies

Abstract

Capsular polysaccharides (SPS) are an integral component of gram-negative bacteria

and also have potential use as vaccine. In this paper, interactions of SPS isolated from

Klebsiella strains K20 and K51 with cationic dyes pinacyanol chloride (PCYN) and acridine

orange(AO) were studied by absorbance and fluorescence measurements. Both the

polysaccharides having glucuronic acid as the potential anionic site induced strong

metachromasy (blue shift~ 100 nm) in the PCYN. The Spectral changes were studied at

different polymer/dye molar ratios (P/D=0-40). A complete reversal of metachromasy was

observed upon addition of co-solvents suggesting the breakway of dye molecules from the

biopolymer matrix. Binding constant, changes in free energy,enthalpy and entropy of the dye-

polymer complex were also computed from the spectral data at different temperatures to

reveal the nature of the interaction. Quenching of fluorescence of AO by the polymers and the

incorporated mechanisms were also explored.

Published in Indian J. Biochem. Biophys. 46(2009)192-197.

54

1. Introduction

Klebsiella, the gram-negative bacteria of Enterobacteriacea family, having 82

serologically classified strains are causative factor of several diseases like pyrogenic hepatic

abscess[1-3], pneumonia[4-8]. However, till date the relationship between the clinical

presentation and bacterial factors remain unclear[3,5]. Capsular polysaccharides are the

integral components of the outer membrane of gram-negative bacteria. These polysaccharides

are acidic in nature, thus are also known as Sauer (German: means sour) polysaccharides

(SPS). Some of the Klebsiella SPS are used as vaccines[1,2,4,6]. However, knowledge on the

functionality-composition relationships in bacterial strains is still considered to be

fragmentary [9-12]. Therefore, compositional and structural studies on Klebsiella SPS are

considered important.

SPS, isolated from different Klebsiella strains comprise definite repeating units

ranging from tri- to hepta-saccharaides. The primary structure of Klebsiella K20 SPS

Klebsiella K20

Klebsiella K51

Figure 1. Primary structure of Klebsiella K20 and K 51 SPS

3) − β − D-Galp (1 2) - α-D-Manp (1-

β-D-GlcpA- (1 3)-α-D-Galp

3

1

3)− α − D-Galp (1 3) - α-D-Galp (1-

α-D-GlcpA- (1 6)-α-D-Glcp

4

1

55

(Figure.1) reveals the presence of D-galactose, D-mannose, D-glucuronic acid in 2:1:1 molar

ratio[7], while K51 has D-galactose, D-glucose, D-glucuronic acid in the same molar ratio[8].

Glucuronic acid acts as potential anionic site in both the polymers, for which they behave like

polyelectrolytes. It induces metachromasy[9] in the dye molecules by the formation of blue-

shifted spectral band in the visible range[10].

Interaction of different cationic dyes with synthetic/natural polyanions has been well-

studied using techniques, such as calorimetric, surface tension, spectroscopic, conductometric

and light scattering measurements, etc[11,12]. Similarly, interactions of plasmid DNA[13]

and polyelectrolyte like polystyrene sulphonate with cationic surfactants[14], and β-carbonyl

based quinolizine with different cyclodextrins using steady-state and time-resolved

fluorescence have also been reported[15]. Spectral studies on interaction of different dyes

assume importance for their multi-faceted uses, such as evaluation of different physico-

chemical parameters of SPS viz., molar mass per repeating units, conformation in aqueous

media under various environments, and extent of interactions with the dye/drug molecules,

and evaluation of thermodynamic parameters, such as changes in free energy, enthalpy and

entropy for SPS-dye complex formation[10,16,17]. However, such studies are not plenty in

literature.

Both the Klebsiella strains K20 and K51 are known for their ability to synthesize

large amounts of SPS. K20 strain is causative factor for pulmonary, intraperitoneal and burn

infections[18], while the K51 strain causes nosocomical disease[19]. So far, no physico-

chemical studies on both the strains have been reported. Thus, in the present paper, physico-

chemical studies on the interaction of SPS isolated from K20 and K51 strains with cationic

dyes pinacyanol chloride (PCYN) and acridine orange (AO) using absorbance and emission

spectral measurements have been undertaken.

56

2. Materials and methods

2.1. Materials

The K20 and K51 strains of Klebsiella were supplied by Dr. S Schlecht of Max

Planck Institute for Immunobiology, Frieburg, Germany. Bacterial cells were grown on

nutrient agar-agar medium and the polysaccharides were isolated and purified by phenol-

water-cetavlon method[20]. Dyes pinacyanol chloride (PCYN) and acridine orange (AO)

were purchased from Sigma Chemicals Co., USA and were >99% pure and used as received.

Purity was also checked from their reported spectral data. The stock solutions of the dyes (10-

3 M) were prepared in double-distilled water and kept in dark at 4

oC and used within 1 week,

prior to preparation of their aqueous solutions. Absorption spectra were recorded at

400-700nm by a Lambda-25, UV-VIS spectro-photometer (Perkin-Elmer), while fluorescence

spectra were recorded using a Shimadzu RF-5000 spectrofluorimeter.

2.2. Methods

The effects of co-solvents like ethanol, methanol etc. on the dye polymer complex

were investigated by measuring the absorbance of pure dye solution as well the dye polymer

complexes (at P/D = 5) at its monomeric band (J band = 600nm) and also at their

metachromatic bands (H- band = 490 nm). Stoichiometry of the dye-polymer complexes was

determined by MacIntosh method[21]. Increasing amount of K20 and K51 polymers were

added to fixed quantity of PCYN (10-5

M), taken in different test tubes. After homogenization,

petroleum benzene was added to each tube, and was thoroughly vortexed, till the dye-polymer

complex got distributed into the organic phase. The concentration of uncomplexed dye in the

aqueous layer was measured (with the help of a known standard graph) and the complexed

dye was quantified there from. Finally, the stoichiometry was determined from the

breakpoints of the plot of complexed dye concentration vs. concentration of the polymer.

57

Equivalent weights of polysaccharides were determined by spectrophoto-/ fluori-

metric titrations[22]. Absorbance/fluorescence intensities at the original band of pure dye

were noted upon progressive addition of varied amounts of ~10-4

mg mL-1

polymer solution.

From the break points of the plot of absorbance/fluorescence intensity vs. amount of added

polymers, equivalent weight per repeating unit was determined.

Thermodynamic parameters for PCYN-polymer interaction were determined using

absorbance values of the dye at its metachromatic band (H-band, 490nm) and at different

temperatures (303-323) ± 0.1K. Binding constant of dye-polymer aggregate was calculated at

each temperature using the Rose and Drago equation[23]:

D S S

0 C DS D DS D

(C .C ) C1

(A-A ) [ .L (ε ε )] [(ε ε )L]K= +

− − (1)

Where, CD = initial concentration of the dye; CS = initial concentration of the polymer; Ao =

absorbance of the pure dye solution at 600nm; A = absorbance of the dye-polymer solution at

600nm; KC = binding constant between the dye and polymer; εD = molar absorption

coefficient of the dye; εDS = molar absorption coefficient of the dye-polymer complex; and L

= length of the light path.

From the ratio of slope and intercept of the plots of (CD.CS)/(A–A0) vs. CS, KC was

determined. Free energy change (∆G0) was obtained from the expression: ∆G

0 = -RT ln KC.

Changes in enthalpy (∆H0) and entropy (∆S

0) for the complex formation were obtained from

the linear plot of ∆G vs. T, according to the equation ∆G0 = ∆H

0 - T∆S

0.

58

3. Results and Discussion

Primary structures of the SPS isolated from Klebsiella K20 and K51 strains, as shown

in Figure 1 have been previously established[7,8]. Presence of glucouronic acid in the

terminal sugar of repeating units results in the acidic nature of the polysaccharides.

Changes in the absorption spectra of PCYN (1.0 × 10-5

M) upon addition of SPS at

different polymer ratio (P/D = 0~50) are shown in (Figure 2).

Figure 2. Effect of K20 (A) and K51 (B) polysaccharides on the absorption spectra of aqueous

10-5

M PCYN at 298 K [P]/[D]: 1,0 ; 2, 1; 3,2 ; 4, 5; 5, 10; 6, 15; and 7,20

Pure dye exhibited two peaks: one at 600nm and the other at 550nm, corresponding to

its monomeric (J-band) and dimeric (D-band) forms, respectively. Intensities of both the J-

and D-bands of PCYN decreased and a new metachromatic band (H-band) appeared at

59

~490nm with the increase in polymer concentration. At higher P/D ratio, spectral

characteristic changed significantly; a strong metachromatic (H-band) band appeared at

490nm with almost complete removal of J- and D-bands. A metachromatic blue shift of

~100nm indicated strong metachromatic interactions among the dye and SPS. Dye molecules

could form a charge transfer complex with the SPS; hence due to the formation of a charge

transfer complex a blue shift in the original band of the dye molecule occurred. Overcrowding

of dye on the chromotrope lead to the formation of multiple banded structures in the spectra

of PCYN, due to random coil conformation of the polymer at lower P/D as reported

earlier[10,16,17].

Equivalent weights of the K20 and K51 polysaccharides determined by metachromatic

and fluorometric titrations as shown in (Figure 3) were found to be 646 and 594, respectively.

Figure 3. Determination of equivalent weight of K20 and K51 SPS by metachromatic (A)and

fluorimetric (B) titration [(O), K51; (□), K20]

The results were in agreement with the primary structure of the polysaccharides.

Equivalent weight of polysaccharide was considered to be the molar mass of one repeating

unit[10,16,17].

60

The stoichiometry of the SPS-dye complex was determined by isolation technique of

MacIntosh[21]. Increasing amounts of SPS solution (0.1-1.0 mL,10-4

M) were added to 10 mL

1.0 × 10-5

M PCYN. The metachromatic solutions were isolated with petroleum ether and

uncomplexed dye concentration was measured spectrophotometrically. Concentrations of

complexed dye were plotted against the concentration of added polymer. From the point of

intersection of two linear curves, the stoichiometry of dye-polymer complex was determined.

Stoichiometry was also determined by centrifugation method (results not shown)[24]. In this

method, the dye-polymer complex was centrifuged at a speed of 12,000 rpm, and from the

supernatant, uncomplexed dye concentration was estimated colorimetrically. Stoichiometry

was determined in the same manner as in the earlier experiment and results are shown in

(Figure 4). Identical stoichiometries were found in both

Figure 4. Determination of stoichiometry of K20 and K51 polysaccharide-PCYN complex by

McIntosh method at 298 K [(O), K51; (□), K20]

the cases (P: D = 1:1). Results were in accordance with earlier observations[10,16,17]. 1:1

Stoichiometry of the metachromatic compounds indicated that every potential anionic site of

repeating unit of the polymer was associated with the dye cation and poly-anion. Multiple-

61

banded structures in absorption spectra were possibly due to the stacking conformations of

aqueous polymeric solutions in the presence of dye. It was evident from the structures of K20

and K51 SPS that there was one anionic charge site per repeating unit. Hence, it was quite

expected to observe a 1:1 stoichiometric complex formation among the dye and

polysaccharides.

Effects of different co-solvents on the dye-polymer complexes were studied by measuring

absorbance at 600nm (J-band) and 490nm (H-band). Absorbance at 600nm increased with the

increasing concentration of co-solvents added. After a certain amount of added cosolvents

there was no further change in the abosorbance, once it attained the abosorbance of the pure

dye. Reversal of metachormasy also occurred with the progressive addition of alcohols to the

dye-polymer aggregate. Alcohols have the capability to break down the dye-polymer

aggregate for which the original band of the dye molecule reappeared; simultaneously, the

metachormatic band disappeared. Representative plot of effect of ethanol on the dye-K51

SPS complex is shown in (Figure 5). Similar effect was observed with the K20-PCYN

complex (results not shown). As the K20 SPS formed a weak aggregated species with PCYN,

hence the effect of alcohol addition was higher than in the other case. In reversal of

metachromasy, the order was: n-butanol>n- propanol>ethanol (data not shown). The longer

alcohols were more effective in inducing the metachromasy reversal, and supported the

involvement of hydrophobic interactions. This order of reversal of metachromasy revealed

that the organization/stacking of dye-polymer complex could be alternately visualized through

the thermodynamic parameters[10,16,17].

Thermodynamic parameters of the dye-polymer interaction KC, (∆H0), (∆G

0) and

(∆S0) at different temperatures (303, 308, 313, 318 and 323 K) were determined by putting

the absorbance values (at 490 nm) in Rose and Drago equation[23]. (CD.CS)/(A–A0) values

62

Figure 5. Effect of ethanol on K51 polymer-PCYN dye (10-5

M) complex [Curves: 1, pure dye; 2, dye-

polymer complex at [P]/[D] = 50; 3, dye-polymer complex in presence of 40% (v/v) ethanol; and 4, dye

in presence of 40% (v/v) ethanol]

were plotted against CS as depicted in (Figure 6) . Value of KC was determined from the ratio

of the slope and intercept of straight line. The results were summarized in Table 1.

Thermodynamic parameters, except the entropy change were higher for K51 than K20. In the

present experiment, values of binding constants of both the polymers decreased with the rise

in temperature, suggesting that the binding processes were exothermic in nature, as indicated

by the negative values of enthalpy change. In case of K20-PCYN complex, the binding

parameters were found to be lower than K51-PCYN complex. The results suggested that the

dye molecule could easily get stacked to the coil form of K-51 SPS than in the

other[10,12,16,17,24-26]. The negative values of ∆G0 were within the range of a reversible

63

Figure 6. Plot of (CDCS)/(A-A0) vs CS for K20 (A) and K51 (B) polymers [Temperatures/K: (●), 303;

(○), 308; (▼), 313; (∇), 318; (■), 323]

Table 1. Thermodynamic parameters for the interaction of pinacyanol chloride (PCYN) with Klebsiella K20 and K51 polysaccharides.

Polymer Temp./K 10-4xKc(M

-1)a G

0

(kcal mol-1)b

H0

(kcal mol-1)c

S0

(calmol-1K-1)c

K20

303 0.34 -4.93

-6.82 1.89

6.25 0.61

308 0.32 -4.97

313 0.22 -4.87

318 0.17 -4.72

323 0.19 -4.69

K51

303 5.60 -6.63

-17.26 1.79

3.40 0.57

308 4.90 -6.65

313 7.67 -6.34

318 1.89 -6.24

323 1.03 -5.97

[PCYN]=1.0 x 10-5 M

a Calculated from Fig. 6 according to Rose and Drago equation.

b Calculated from the thermodynamic relation G0 = -RT ln KC

c Calculated from the graphical plot of G

0 vs T according to the relation G

0= H

0 - T

0

biological process involving any non-chemical type interaction. Negative values of entropy

change also indicated formation of ordered structures during the formation of dye-polymer

complexes.

64

The fluorescence studies on the interaction of K20 and K51 polymers with AO were

carried out by adding increasing amounts of SPS to a fixed quantity of dye. Fluorescence

intensity of AO was quenched upon the addition of both the polymers. Aqueous solution of

AO was excited at 440nm (λex) and fluorescence intensity was recorded at 522nm

(λem). Representative fluorescence spectra are shown in (Figure 7).

Figure 7. Effect of K20 (A) and K51 (B) SPS on the emission spectra of aqueous 10-5

M AO

at 298 K [P/D:1,0; 2, 02; 3, 04; 4, 06; 5,1; 6,14; 7,20; 8,24; 9, 28; 10,32; 11, 36, and 12, 40].

The ratio of fluorescence intensity of pure dye (Fo) and dye-SPS mixture (F) was plotted

against the concentration of SPS, where SPS acted as quencher [Q]. The plots are graphically

summarized in Figure 8.

65

Figure 8. Stern-Volmer plots for the interaction of acridine orange with K20 (Ο) and K51(∆) polymers at

298 K [Dye conc 10 × 10-5

M]

Binding constants (KSV) of the dye-polymer complexes were determined using Stern-

Volmer equation [27]:

Fo/F = 1 + KSV [Q] (2)

Stern-Volmer constant value for K20 (10.93 × 10-3

M) was found to be higher than

that of K51 (9.2 × 10-3

M), indicating binding efficiency of the dye with K20. Efficiency of

SPS as quencher to the fluorescent probe AO was an established fact. SPS being anionic in

nature could donate their electrons to the cationic dyes through the formation of charge

transfer complexes. This would eventually reduce the transition probability of electrons of AO

bonding molecular orbital to the anti-bonding/non-bonding molecular orbital. Thus, the

fluorescence of AO got quenched upon the addition of SPS to aqueous AO solution. Linear

plots with an intercept of unity indicated Stern-Volmer type of quenching[16,17].

66

Higher binding constant for K20 than K51 was due to a different tertiary

conformation of the polymer for which the dye molecule could not properly get stacked.

4. Conclusion

Studies on the interaction of SPS isolated from K20 and K51 strains of Klebsiella were

performed using PCYN and AO as probes in absorption and emission spectroscopy. Both SPS

were found to be acidic in nature with definite repeating sugar units. Charge densities on the

polymer were different for which the extent of interaction with the dyes were different. K51

SPS, having a systematic structure with higher change density, resulted stronger interaction

than the K20. Thermodynamic parameters for the dye polymer interactions were found to be

comparable with reversible biological processes. Both the polymers 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. However, further studies like light

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

behavior of such polymeric materials.

67

References

[1] Cryz, S. J.; Mortimer, P.; Cross, A. S.; Fürer, E.; Germanier, R. Vaccine 4 (1986)15-20

[2] Edelman, R.; Taylor, D. N.; Wasserman, S. S.; McClain, J. B.; Cross, A. S.; Sadoff, J.

C.; Que, J. U.; Cryz, S. J. Vaccine 12 (1994) 1288-1294

[3] Fung, C. P.; Chang, F. Y.; Lee, S. C.; Hu, B. S.; Kuo, B. I. T.; Liu, C. Y.; Ho, M.; Siu.

L. K. Gut 50 (2002) 420-424

[4] Cryz, S. J.; Mortimer, P. M.; Mansfield, V.; Germanier, R. J. Clin. Microbiol. 23 (1986)

687-690

[5] Fung, C. P.; Hu, B. S.; Chang, F. Y.; Lee, S. C.; Kuo, B. I. T.; Ho, M.; Siu, L. K.; Liu,

C. Y. J. Infect. Diseases 181 (2000) 2075-2079

[6] Cryz, S. J. Adv. Biotechnol. Process 13 (1990) 87-103

[7] Choy, Y. -M.; Dutton, G. G. S.; Can. J. Chem. 51 (1973) 3015-3020

[8] Chakraborty, A. K.; Dabrowski, U.; Geyer, H.; Geyer, R.; Stirm, S. Carbohydr. Res.

103 (1982) 101-105

[9] Blanchette, E. A.; Rubin, S. J. J. Clin. Microbiol. 11 (1980) 474-478

[10] Mitra, A.; Nath, R. K.; Biswas, S.; Chakraborty, A. K.; Panda, A. K. J. Photochem.

Photobiol. A: Chem. 178 (2006) 98-105

[11] Chakrabarty, A.; Mallick, A.; Haldar, B.; Das, P.; Chattopadhyay, N.

Biomacromolecules 8 (2007) 920-927

[12] Pal, M. K.; Biswas, M. Histochemie 27 (1971) 36-42

[13] Zhu, D. M.; Evans, R. K. Langmuir 22 (2006) 3735-3743

[14] Monteux, C.; Williams, C. E.; Meunier, J.; Anthony, O.; Bergeron, V. Langmuir 20

(2004) 57-63

68

[15] Park, J. W.; Lee, S. Y.; Kim, S. M. J. Photochem. Photobiol. A Chem. 173 (2005) 271-

278

[16] Dasgupta, S.; Nath, R. K.; Biswas, S.; Hossain, J.; Mitra, A.; Panda, A. K . Colloids

Surf. A Physicochem. Eng. Asp. 302 (2007) 17-23

[17] Panda, A. K.; Chakraborty, A. K.; J Photochem. Photobiol. A Chem. 111 (1997) 157-

162

[18] Süsskind, M.; Brade, L, Brade, H.; Holst, O. J. Biol. Chem. 273 (1998) 7006-7017

[19] Sechter, I.; Mestre, F.; Hansen, D. S. Clin. Microbiol. Infect. 6 (2000) 233-238

[20] Westphal, O.; Jann, K. Methods Carbohydr. Chem 5 (1965) 83-91

[21] MacIntosh, F. C. Biochem. J .35 (1941) 776-782

[22] Diakun, G. P.; Edwards, H. E.; Allen, J. C. Anal. Biochem. 94 (1979) 378-382

[23] Rose, N. J.; Drago, R. S. J. Am. Chem. Soc. 81 (1959) 6138-6141

[24] Zhang, S.; Zhao, F.; Li, N.; Li, K.; Tong, S. Spectrochim. Acta A :58 (2002) 2613-2619

[25] Pal, M. K.; Schubert, M. J. Am. Chem. Soc. 84 (1962) 4384-4393

[26] Zhang, S.; Li, N.; Zhao, F.; Li, K.; Tong, S. Spectrochim. Acta A: 58 (2002) 273-280

[27] Carraway, E. R .; Demas, J. N. Anal. Chem. 63 (1991) 332-336