Langmuir 1996,11, 2410-2413
2410
Fluorescence Monitoring of the Hydrophobic Surface of Dextrin Using p-Toluidinonaphthalenesulfonate Kaustuv Das, Nilmoni Sarkar, Swati Das, and Kankan Bhattacharyya" Physical Chemistry Department, Indian Association for the Cultivation of Science, Jadavpur, Calcutta 700 032, India
D Balasubramanian" Centre for Cellular & Molecular Biology, Hyderabad 500 007, India Received December 14, 1994. I n Final Form: March 10, 1995@ The effect of the linear oligosaccharides dextrin (a-la,4e-~-glucopyranose) and dextran (a-1,6-~(TNS)has been studied glucopyranose)on the emission properties of 2-~-toluidino)-6-naphthalenesulfonate in aqueous solution. TNS exhibits a 30-fold fluorescence enhancement on binding to dextrin, while with dextran there is only a marginal increase in the emission intensity. The polarity values of the binding sites are estimated by comparing the nonradiative rates of TNS with those in aqueous dioxane-water mixtures. The results indicate that the binding surface of dextrin is markedly less polar than that of dextran. This difference is attributed to the differing stereochemical constraints imposed on the two chains. The binding constants of TNS with the two polysaccharides in pure water have been determined in the presence and absence of several salting-in denaturants and salting-out reagents. Salting-in agents cause a decrease in the number of TNS molecules bound to the dextrin surface and an increase in the local polarity, while the salting-out agent LiCl increases the number of TNS molecules bound to dextrin. The interaction of TNS with dextran, however, is found to be very weak even in the presence of LiCl. Energy minimization studies reveal a hydrophobic surface for dextrin while no such amphiphilicity is observed in dextran.
1. Introduction The hydrophobic aggregation of organic molecules in aqueous medium is responsible for many important phenomena such as micellar aggregation, membrane structure, globular protein folding, enzyme-substrate binding, antibody-antigen binding, cell surface recognition, and so 0n.l Sugar molecules are generally considered to be hydrophilic in nature, because of their high water solubility, preponderance of hydroxyl groups, and lack of any nonpolar groups. Yet, as the cyclic oligosaccharides called c y c l ~ d e x t r i n s ~ illustrate, -~ it is possible for sugar chains to display hydrophobic faces. Factors such as the monomer ring conformation (the chair form), the epimeric structure (a-glucopyranose), the glucosidic bond stereochemistry (a-1,4 linkage) and the chirality (D-conformation) dictate the amphiphilic properties of the sugar molecules. The idea of hydrophobicity in sugars, while surprising, has been invoked in the process of molecular recognition and intermolecular interactions. Johnson et
* To whom correspondence should be addressed. Phone: (91)(40)-673487. Fax: (91)-(40)-671195. E-mail:
[email protected]. Abstract published in Advance A C S Abstracts, J u n e 1, 1995. (1)(a) Breslow, R. Acc. Chem. Res. 1991,24,159.(b) Blokzijl, W.; Engberts, J. B. F. N. Angew. Chem., Int. Ed. Engl. 1993,32,1545.(c) Pindur, U.; Lutz, G.; Oho, C. Chem. Reu. 1993,93,741. (2)Clarkey R. J.; Coates, J. H.; Lincoln, S. F. Adu. Carbohydr. Biochem. 1988,467,205. (3)(a)Bender, M.L.; Komiyama,M. Cyclodextrin Chemistry;Springer Verlag: Berlin, 1978.(b) DSouza, V. T.; Bender, M. L. Acc. Chem. Res. 1987,20,146. (c) Wenz, G.Angew. Chem., Int. Ed. Engl. 1994,33,803. (d) Ramamurthy, V.; Eaton, D. F. Acc. Chem. Res. 1988,21,300. (4) (a) Ayoma, Y.; Nagai, Y.; Otsuki, J.; Koayashi, K.; Toi, H.Angew. Chem., Int. Ed. Engl. 1992,31,745.(b) Kitamura, S.;Matsumori, S.; Kuge, T. J. Incl. Phenom. 1984,2,25. (c) Nakatani, H.; Shibata, K.; (d) Bright, F.V.; Catena, Kondo M.; Hiromi, K. Biopolym. 1977,16,2367. G. C.; Huang, J. J. Am. Chem. SOC.1990,112,4023. (5)(a) Sarkar, N.; Das, K.; Nath, D.; Bhattacharyya,K. Chem. Phys. Lett. 1994,218,492. (b) Sarkar, N.; Das, K.; Nath, D.; Bhattacharyya, K. Chem. Phys. Lett. 1992,196,491.(c) Das, K.; Sarkar, N.; Bhattacharyya, K. J. Chem. SOC.,Faraday Trans. 1993, 83,1959. @
aL6 have reviewed the occurrence of apolar contacts between sugar chains and some enzymes. Surolia and co-workers have analyzed7s8the thermodynamics of such interactions between sugars and proteins in aqueous solutions, while Lemieux et al.9J0have indicated that an amphiphilic surface is presented by some sugar chains to the interacting proteins. We have analyzed and interpreted these results on the basis of our finding11-13that the dextrin chain (la,4e-a-n-glucopyranose, or Glcpal4Glc...) is amphiphilic in character, while dextran (a-1,6linked D-glucopyranose units or Glcpal-6Glc ...) and cellulose (B-le,4e-D-glucosechain or GlcpBl-4Glc ...) are not able to project any apolar face and can only be hydrophilic. Our earlier approach to detect and analyze the hydrophobicity of sugar chains involved studying their ability to solubilize lipophiles,ll modulate chemical reaction between organic molecules in water, destabilize globular proteins and micellar aggretates, and offer a competitive surface for the binding of fluorescent dyes.12J3 Of these, the last method is the most direct one to monitor a hydrophobic surface since the emission parameters of dyes such as l-(anilino)-8-naphthalenesulfonate ( A N S ) or 24ptoluidino)-6-naphthalenesulfonate (TNS) are fine-tuned ~
(6) Johnson, L. N.; Cheetham, J.; McLaughlin, P. J.;Acharya, K. R.;
Barford, D.; Phillips, D. C. Curr. Top. Microbiol. Immunol. 1988,139, 81. (7) Sastry, M. V. K.; Banajee, P.; Patanjali, S. R.; Swamy, M. J.; Swamalatha, G. V.; Surolia, A. J. Biol. Chem. 1986,261,11726. (8) Acharya, S.;Patanjali, S. R.; Sajian, S. U.; Gopalakrishanan, B.; Surolia, A. J. Biol. Chem. 1990,265,11586. (9)Lemieux, R. U.;Hindsgaul, 0.;Bird, P.; Narasimhan, S.;Young, W. W., Jr. Carbohydr. Res. 1988,178,293. (IO)Delbare, L. T. J.; Vandonselaar, M.; Prasad, L.; Quail, J . W.; Pearlstone, J. R.; Carpenter, M. R.; Smillie, L. B.; Nikrad, P. V.; Spohr, U.; Lemieux, R. U. Can J . Chem. 1990,68,1116. (11)Sivakama Sundari, C.; Raman, B.; Balasubramanian, D. Biochim. Biophys. Acta 1991,1065,35. (12)Gopala Krishna, A,; Balasubramanian, D.; Ganesh, K. N. Biochem. Biophys. Res. Commun. 1994,202,204. (13)Balasubramanian, D.; Raman, B.; Sivakama Sundari, C. J.Am. Chem. SOC.1993,115,74.
0743-7463/95/2411-2410$09.00/00 1995 American Chemical Society
Hydrophobic Surface of Dextrin to the polarity, microviscosity and size of the substrate to which they bind.14J5 In this paper we exploit these properties of one of them, namely TNS, in order to explore the amphiphilic surface in dextrin chains. TNS displays negligible fluorescence in water. Its quantum yield offluorescence, &,is about 0.001 in aqueous solution, with a very short fluorescence life time (tf= 60 ps). Upon binding to cyclodextrins or linear amylose its q5f and tf increase nearly 50-f0ld.~,~ The extraordinary sensitivity of the emission properties of TNS arises from a n efficient nonradiative process called the twisted intramolecular charge transfer (TICT)process, whose rate increases rapidly with the polarity of the medium.15J6In water, where the polarity is high, the TICT rates of TNS are very high and hence its &and tfvalues are very small. When TNS binds to the nonpolar, hydrophobic surfaces of micelles, proteins or the apolar cavities cyclodextrins, the TICT process is inhibited and this causes a steep rise in $y and tf. The fluorescence enhancement thus gives direct information about the polarity of the binding site of the fluorophore.16,17 We have therefore studied the interaction of TNS with dextrin and dextran, with a view to estimate the polarity of the binding surface of these two polysaccharides. We have also studied how this binding of TNS with dextrin and dextran is affected by urea and different salting-in and salting-out agents. Finally we present a picture of the hydrophobic surface of the dextrin chain obtained from energy minimization calculations. 2. Experimental and Energy Minimization
Methods TNS (Aldrich) was purified by repeated recrystallization. Dextrin (Serval (dextrin-10, mean molecular weight 1800 Da, degree of polymerization ca. 10) and Dextran (Serval (mean molecular weight 4000-6000 Da, degree of polymerization ca. 22-33) were used as received. Absorption and fluorescence spectra were recorded in JASCO 7850 spectrometer and PerkinElmer MPF 44B fluorimeter respectively. The fluorescence quantum yield were measured with respect to that of quinine sulphate in 1N sulfuric acid as 0.55. The emission life times were determined at magic angle polarization using a picosecond single photon counting apparatus described el~ewhere.~ The fluorescencedecays were deconvoluted using the global lifetime analysis soRware.1S The energyminimization studies were donell using the b o approach with the crystal structure data on glucose.
3. Results and Discussion A. Effect of Dextrin and Dextran o n the Emission Properties of TNS. In pure water 4f of TNS is very small (0.001)and tfveryshort (60 P S ) . ~Upon the addition of dextrin, the emission spectrum of TNS exhibits a marked blue shift and enhancement in both the quantum yield (Figure 1)and lifetime which indicates that dextrin indeed possesses a n apolar or hydrophobic surface to which TNS can bind. In 10mM dextrin the &and tfof TNS are nearly 30-fold greater than those in water, while the emission maximum shifts blue to about 450 nm from the value of 490 nm in water (Table 1). The fluorescence excitation spectrum of TNS, however, remains unchanged and identical to the absorption spectrum on addition of dextrin, (14) (a)Kosower, E. M.; Dodiuk, H.; Kanety, H. J. Am. Chem. SOC. 1978,100,4179. (b)Detona, R. P.; Brand, L. Chem. Phys. Lett. 1977, 47, 231. (15)Cardamone, M.; Pun,N. K. Biochem. J. 1992,282, 589. (16) (a) Hicks, J. M.; Vandersall, M.; Babarogic, Z.; Eisenthal, K. B. Chem. Phys. Lett. 1986,116, 18. (b) Chang, L.; Cheung, H. C. Chem. Phys. Lett. 1990,173, 343. (c) Das, K.; Sarkar, N.; Nath, D.; Bhattacharyya, K. Spectrochim. Acta 1992,48A, 1701. (17) Bhattacharyya, IC;Chowdhury, M. Chem. Rev. 1993,93,507. (18)Knutson, J. R.; Beecham, J. M.; Brand, L. Chem. Phys. Lett. 1983,102,501.
Langmuir, Vol. 11, No. 7, 1995 2411
X (nm)
Figure 1. Emission spectra of 10pMTNS in aqueous solution: (a) in water alone; (b) in 30 mM dextran; (c)in 10 mM dextrin and 3 M LiC104;(d)in 10mM dextrin; (e)in 10mMdextrin and 5 M LiC1. and on subsequent addition of urea and salts. Assuming the simplest 1:lbinding equilibrium, T D = T-D, where T, D, and T-D represents TNS, dextrin, and TNS-dextrin complex, respectively, the binding constant ( k b ) and the quantum yield a t infinite dextrin concentration (&J are obtained from the double reciprocal plots of emission quantum yield against dextrin concentration (Figure 2). The values of 4- and k b so obtained are summarized in Table 2. From the value of kb, using the known total concentration of TNS (10 pM) and dextrin (10 mM), one can easily calculate the concentration of TNS present in the bound form, i.e., in the T-D complex. In 10 mM dextrin the concentration of T-D is 4.7pM which implied that 47%of the total TNS molecules are bound to dextrin. The lifetime data (Figure 3 and Table 1)are consistent with the quantum yield measurements. The observed fluorescence decay curves were fitted to a biexponential decay, namely:
+
a, exp(-th,)
+ a2exp(-th2)
In 10 mM dextrin the fluorescence decay of TNS is overwhelmingly dominated (a1 = 0.95) by a long-lived component with lifetime 3 ns, with a minor component (a2 = 0.05) with a lifetime of 240 ps. From the observed & and tf, the rate constants for the radiative (k,) and the nonradiative decay (k,,) are obtained using the equations
4f = 4 Z f k,,
= Zf - 1 -k,
The value ofk, and k,, are given in Table 1, which shows that compared to the the case in water, k , of TNS in 10 mM dextrin is 50 times smaller while k , remains more or less unchanged. As we have shown elsewhere,16J7such dramatic changes in k,, TNS arise because of the nonradiative TICT process, which is highly sensitive to the polarity of the medium. The rate of the TICT process (kT) increases exponentially with the polarity parameterlgET(19) Reichardt, C. Chem. Rev. 1992, 92, 147.
2412 Langmuir, Vol. 11, No. 7, 1995
Das et al. Table 1. Emission properties of TNSa
host
reagent
Aem m",nm
df
Tdal),ns
0.001
urea, 5 M LiC104,3 M LiCl, 5 M
490 450 455 455 460 460 465
0.06 3.0 (0.95) 2.6 (0.90) 2.0 (0.90) 3.1 (0.96) 0.9 (0.50) 2.0 (0.70)
water dextrin, 10 mM dextrin, 10 mM dextrin, 10 mM dextrin, 10 mM dextran, 30 mM dextran, 30 mM a
LiCl, 5 M
0.03 0.009 0.007 0.075 0.003 0.005
TZ( a d , ns 0.24 (0.05) 0.17 (0.10) 0.16 (0.10) 0.20(0.04) 0.12 (0.50) 0.20(0.30)
k,(xlO7)
k,,(xlOg)
XZ
1.6
16.6 0.3 0.38 0.5 0.3
1.15
2.0
0.8 0.75 6.0
1.20 1.33 1.25 1.35 1.30 1.32
8.0
0.4 0.4
8.0
Two exponential fits of the decay curves were adequate, as the goodness of fit parameter x2 values show.
io "0,O
I
I
I
200.0
l/(dextrin)
I
400,O
I
1 600.0
(M-')
Figure 2. Double reciprocal plots of A 4 = [ 4f - d0f1 of TNS (@'f = quantum yield of TNS in water) against concentration of dextrin in the presence of 5 M LiCl. Table 2. Binding Constants k b and Quantum Yields 4- of TNS in Different Mediaa
system kb d. TNS + dextrin 90 f 20 0.06 TNS + dextrin + 5 M urea 50 f 20 0.028 TNS + dextrin + 3 M LiC104 65 f 20 0.009 TNS + dextrin + 5 M LiCl 400 f 50 0.1 TNS + dextran 60 f 30 0.004 [TNSI = 10 wM; [Dextrin] = 10mM, [Dextran]= 30 mM; room temperature (ca. 298 K). (30) of the medium. Thus a t high polarity ( E ~ ( 3 0>) 50) one can write, k,, = k~ so that the plot of In (knr)against E ~ ( 3 0is ) a straight line.16 From the comparison of the values ofk,,, &, and zfofTNSbound to dextrin and dextran with those of TNS in dioxane-water mixtures16 the ET(30) values of binding site of TNS in 10 mM dextrin is estimated to be 53.5 & 0.5 (Table 3) which is in between the E ~ ( 3 0of) ethanol (51.7) and methanol (55). In sharp contrast to the effect displayed by dextrin, the addition of dextran produces only a 2- to %fold increase in the emission quantum yield of TNS and a small blue shift. The fluorescence decay of TNS shows (Figure 3) that the lifetime of the long-lived component (rl= 0.9 ns) in 30 mM dextran is shorter than that of the long-lived component observed in 10 mM dextrin (3 ns). Further, the amplitude of the long-lived component is equal to that of the very short component (za = 120 ps) (Table 1). This indicates that the binding sites in dextran are much more polar than those for dextrin. The values of k,,, &, and tf of TNS in 30 mM dextran corresponds to a E ~ ( 3 0value ) 61 & 1which is only marginally less than that of water (63.5). The binding constant for dextran is also lower than that for dextrin. In summary, though TNS binds strongly with dextrin and experiences a microenvironment with polarity much less than water (perhaps comparable to that of the surface of micelles); in the case of dextran
I
I
I
14
2.8
I
12.
56
I
,
I
70 &4 tlME(ns1
I
I
n.2
I 1
ix
UD
Figure 3. Fluorescencedecay of 10pM aqueous TNS solution: (a)in 10 mM dextrin; (b) in 10 mM dextrin and 3M LiC104; (c) in 30 mM dextran. Table 3. E ~ ( 3 0Values ) of Binding Sites of TNS medium 10 mM dextrin 10 mM dextrin 5 M urea 10 mM dextrin + 3 M LiC104 10 mM dextrin 5 M LiCl 30 mM dextran
+ +
E~(30) 53.5 f 0.5
54.5 f 0.5 55.5 f 0.5 53.5 f 0.5 61.0 f 1
the binding is weaker and the binding site of dextran has a polarity closer to that of water. Comparison of results obtained with dextrins and dextrans of different molecular weights (1800 and 40006000, respectively)leads to the possibility of the differences in the viscosity might contributing to the differences in the fluorescence properties in the two sugar solutions. Unfortunately, we have not so far been able to obtain samples of comparable molecular weights so as to do away with this confounding factor. However, many authors have reported that viscosity effects on the TICT process in TNS are rather minor and that this process is controlled almost exclusively by the polarity of the medium.14J6J7 B. Effect of Chaotropic Agents. We have reported earlier that the salting-in and salting-out agents (chaotropes) markedly influence the binding of TNS to cyclodextrin^.^ In the present study, similar large effects are observed for dextrin. The salting-in denaturants urea and LiC104 cause significant reduction in the @f, rf, and KI,values of TNS in 10 mM dextrin (Tables 1and 2). The decrease in W b indicates that the number of TNS bound to dextrin a t high concentration decreases as urea or LiC104 is added. From the value of kb a t 10 mM dextrin, it is estimated that out of 10 pM TNS about 3.3 pM (33%) remains in the bound form in 5 M urea and 3.9 pM (39%) in 3 M LiC104. (In the absence of these additives, 47%of the TNS molecules are estimated to be complexed with dextrin, see above). Along with the removal of TNS from
Langmuir, Vol. 11,No. 7,1995 2413
Hydrophobic Surface of Dextrin
increase the local polarity of the binding sites. In contrast, LiC1, a salting-out agent, increases &,zf,and k b of TNS bound to dextrin. Upon the addition of 5 M LiCl to 10 mM dextrin, the concentration of the T-D complex increases to 8.0 pM,i.e., nearly 80%of TNS remains in the bound form in 5 M LiC1. The addition of salting-in denaturants has little effect in 30 mM dextran since the quantum yield of TNS increases barely from that in pure water. In this case, 5 M LiCl increases the quantum yield by only 1.5 times, with a marginal increase in the lifetime. Thus the interaction of dextran with TNS remains feeble even in the presence of LiC1. C. Energy Minimization Studies. Energy minimization studies that we have performedll reveal a bipolar ribbonlike conformation for the dextrin chain, with one face of the ribbon, comprisingthe methine protons of the glucopyranosidering, being relatively apolar and the other side of the chain backbone, wherein all the hydroxyls are disposed being hydrophilic, as shown in Figure 4. Long dextrin chains, also called a-amylose,adopt the 712 helical conformation, whose inner cavity is relatively apolar. This enables amylose to complex with stilbene derivatives and with molecular i ~ d i n e . ~ OIn - ~contrast, ~ the a-1,6 linked glucopyranosidechain dextran has no hydrophobic surface; neither does the /3-1,4 linked chain cellulose. Amphiphilic surfaces are generated in /3-1,3 and /3-1,4 galactans, a-1,4 and/3-1,3-~-mannans, and/3-1,3and a-1,4D-XyhnS. 4. Conclusion
This work demonstrates that depending on the constraints imposed by the epimeric structure and stereochemistry, linear polysaccharide chains can generate amphiphilic surfaces and display mild hydrophobicity. As a result of the differences in the polarity of their binding surfaces, dextrin and dextran affect the emission properties of the fluorophore TNS in drastically different ways. Salting-in agents such as urea and LiC104 significantly weaken the binding of TNS to dextrin and also increase the local polarity, while the salting-out agent LiCl enhances the binding. In the case of dextran the interactions continue to be very weak even in the presence of LiC1. The role of the hydrophobic effect in recognition and interaction of the cell surface polysaccharides merit further investigation and have important implications in many biological phenomena in aqueous medium.
Acknowledgment. Thanksare due to the Department of Science and Technology, Government of India, for a generous research grant and to the Council of Scientific and Industrial Research, India, for providing fellowships to K.D., N.S., and S.D.D.B. thanks the Jawaharlal Nehru Centre for Advanced ScientificResearch, Bangalore, India, of which he is a honorary Professor. LA941005A Figure 4. Top: (a) Energy minimized picture of the dextrin trimer chain. Bottom: (b) Energy minimized picture of the dextran trimer chain. dextrin, urea and LiC104 reduce the & and zfof the T-D complex and cause a small red shift, indicating that they
(20) Kim, 0.;Choi, L. Langmuir 1994, IO, 2842. (21) Rees, D.A. Polysaccharide Shapes;Chapman and Hall: London, 1977. (22) Wu, H. C.; Sarko, A. Carbohydr. Res. 1978,61, 7,27. (23) Hui, Y.;Russell, J. C.; Whitten, D. G. J.Am. Chem. Soc. 1983, 105, 1374.
