Interaction of group I cations with iota, kappa and lambda carrageenans studied by multinuclear n.m.r.

Interaction of group I cations with iota, kappa and lambda carrageenans studied by multinuclear n.m.r. P. S. Belton, V. J. Morris and S. F. Tanner A F R C Food Research Institute, Colney Lane, Norwich NR4 7UA, UK

(Received 16 May 1984; revised 21 August 1984) Results are reported for ZaNa, a9K, S7Rb and 133Cs n.m.r, on kappa and iota carrageenans over a range of temperatures. Results for ZaNa and 39K n.m.r, for lambda carrageenan are also given over the same range of temperatures. A variety o f behaviour is observed which, in general, does not correlate with the theological behaviour in these systems. It is concluded that non-specific ion interactions are of importance in determining rheological behaviour. Keywords: Polysaccharides; ca rrageenan; gels; gelation; multinuclear n.m.r.

Introduction Carrageenans are gel-forming polysaccharides of considerable technological and scientific interest. However, the mechanism of gelation is still a subject of controversy1-6. Considerable attention has, in the past, been centred on the conformation of the polysaccharide chains and on the changes that occur in their conformations on gelation. It has become clear more recently that the counterions associated with those polysaccharides have an important role to play in the gelling process 1' 2,5. Nuclear magnetic resonance (n.m.r.) can be of great value in the study of ion interactions with polymers 7 and we have previously reported on an n.m.r, study of the interactions of alkali metal ions with iota carrageenan using 23Na, 39K, S7Rb and ~33Cs n.m.r, s. In this paper we report a comparative study of kappa, iota and lambda carrageenans. These three species differ from each other in their chemical repeat units (Figure 1) and the number of sulphate residues in each repeating unit 9. They can have one, two and three sulphate residues per repeat, respectively. In addition, under normal conditions lambda carrageenans do not gel x°. For iota and kappa carrageenans results are reported for the sodium, potassium, rubidium and caesium salts, whereas for the lambda form only results for the sodium and potassium forms are reported.

digestion vessel The hot mobile liquid was poured into the n.m.r, tube and allowed to cool to room temperature ovemight. N.m.r. experiments were always started at the lowest temperature to be studied. Measurements were made at 5 or l0 degree intervals. Repeat experiments showed the spectra to be highly reproducible using samples made on different occasions. In all experiments identical polysaccharide concentrations were used, i.e. the molar concentration of repeating units was maintained at 4.7 x 10 -2 mol dm -3. No excess salt was added to any sample. N.m.r. measurements were carried out using a Bruker CXP-300 spectrometer operating at a magnetic field strength of 7.05 T, under conditions described elsewhere s. In all cases spectra were obtained following a 90 ° pulse and care was taken to avoid saturating conditions. Dwell

oso o

0

OR,=

a

Experimental The preparation of the pure ion forms of carrageenan samples has been described previously ~1't2. The assessment of ion purity was made using methods described elsewhere s'~1'~2. Particular attention was paid to the possible contamination of the pure ion forms with calcium and magnesium and, in the case of kappa carrageenan, with potassium. In all appropriate cases such contaminants were found to be absent or present in insignificant amounts. Aqueous preparations were made by adding double distilled deionized water to the dry carrageenan and heating this mixture at 70°C for 2 h in a PTFE-lined 0141-8130/85/010053-04503.00 © 1985 Butterworth & Co. (Publishers) Ltd

oH

o

/o b

NO

Figure 1 Structure of the repeat units of iota, kappa and lambda carrageenans. (a) Iota carragecnan Ra = SO 3 -, kappa carrageenan Ra=H, (b) lambda carragoenan Rb=0.3 H, 0.7

SO3 -

Int, J. Biol. Macromol., 1985, Vol 7, F e b r u a r y

53

Interaction of group I cations with carrageenans : P. S. Belton et al. times for digitizing the data were selected to optimize the acquisition of narrow components of the n.m.r, lines. No attempt was made to characterize the broad components. Details of the acquisition conditions are given in Table 1.

The behaviour of the spectra is dependent both on ion type and polysaccharide type. The effect of polysaccharide type is illustrated for 39K in Figure 3. Figure 4 illustrates the effects of temperature on the peak area of the 39K resonance of the three different types of carrageenan. Clearly the lambda form does not show the same dramatic change in area as the kappa and iota forms. In order to interpret the spectra it is necessary to consider the magnetic properties of the nuclei in some detail. All the nuclei studied here are quadrupolar. 23Na, 39K and STRb have a spin of 3/2 and lS3Cs has a spin of 7/2. We shall consider caesium separately from the other nuclei. For the spin 3/2 nuclei three main spectral regions may be distinguished. These are: extreme motional narrowing, where the lines are Lorentzian in shape; motional narrowing, where the spectra consist of two Lorentzian lines centred at about the same frequency13; and a static region, where the spectra are very broad and generally consist of three separate lines. A further subregion may be distinguished between the extreme motional narrowing and motional narrowing regions. In this region, which we will call intermediate motional narrowing, the lineshape remains Lorentzian but its width becomes dependent 14 on the Larmor frequency. In the experiments reported here we cannot distinguish between extreme narrowing and intermediate narrowing. In the motionally narrowed case one line is generally much broader than the other and contains 60% of the total signal intensity~s. The remaining 40% is contained in

Results and discussion Figure 2 shows the 39K, S7Rb and 133Cs spectra of iota and kappa carrageenan at selected temperatures. The 2aNa spectra (not shown) of all three polysaccharide forms show only gradual increases in line width with decreasing temperature and no changes in intensity even at temperatures as low as 5°C. On the other hand the K, Rb and Cs spectra show, with the exception of caesium iota carrageenan, sharp changes in intensity. The apparent intensity loss in the caesium form of iota carrageenan is due to changes in linewidth rather than area loss s.

Table 1 N.m.r.acquisition parameters used for differentnuclei =/2 Spectrometer pulse Sweep frequency l e n g t h width Nucleus (MHz) (~) (Hz) 23Na

agK S7Rb x33Cs

79.3 13.9 98.1 39.3

20 30 17 22

5 000 5 000 30000 15 000

Points No. acquired scans 1 024 1 024 1 024 16 384

512 10 000 10000 200

Kappa

iota

I

~___

I

160 Hz

t

3i K

!

1600 Hz

87Rb

i

133Cs

~60 Hz 303

313

323

303

343

313

Temperature( K )

Figure 2 39K, STRband 13SCsspectra of iota and kappa carrageenans at selected temperatures

54

Int. J. Biol. Macromol., 1985, Vol 7, February

323

343

Interaction of group I cations with carrageenans: P. S. Belton et al.

Lambda

Iota

Kappa 313

303

323

333

343

200 Hz

Temperature ( K )

Figure 3

a9K n.m.r, spectra of iota, kappa and lambda carrageenans at different temperatures

1.0

0.5

293

303

313

323

333

343

Temperature(K)

Figure 4

Peak areas, normalized to the area at maximum

temperature, of the agK spectra of iota, kappa and lambda carrageenans as a function of temperature, rT, lambda carrageenan; A, iota carrageenan; V, kappa carrageenan. (The lines on the diagram are a guide for the eye only)

the narrow fine. The acquisition conditions used here will preferentially select for the signal arising from the narrow component. Thus for Rb and K iota carrageenan the sharp change in intensity observed at about 45°C is due to a transition from the extreme or intermediate motional narrowing situation to the motional narrowing state. The observed K + intensity is somewhat less than that expected but this is probably due to the problems of poor signal to noise ratios and acoustic ringing experienced with 39K resonance. This also explains a slight loss in intensity of the lambda carrageenan signal at low temperatures. In the case of the K + and Rb + kappa carrageenans the

signal is totally lost implying a sudden very considerable increase in linewidth. This may result from a transition to the static region - with the implication that the ions have become rigidly bound to immobilized polymer in the junction zones of the gel. However. since the line is not observed some caution is necessary in interpretation: it is possible that the motionally narrowed r e , m e obtains but that the linewidth of even the narrow component is considerable. In lambda carrageenan the potassium line shows no change in intensity even at temperatures as low as 10°C. Although the width of the line is greater at high temperatures than corresponding spectra of the iota and kappa forms the system remains throughout in a condition of extreme or intermediate motional narrowing. Caesium has a spin of 7/2 and as a resultitsbehaviour is somewhat more complex than the spin 3/2 nuclei. In general the 13aCs resonance will consist of up to four Lorentzian components ts depending on the Larmor frequency and the stateof motion. However, in extreme or intermediate narrowing the lineshape is that of a single Lorentzian component 14,ts.For iotacarrageenan the line remains single Lorentzian and of constant intensity throughout the temperature range examined; however, with kappa a sharp change in lineshapeisevident at about 45°C. This represents a transition between motional narrowing and extreme or intermediate motional narrowing and is consistent with the previous observations of Grasdalen and Smidsrod I~. These authors also noted that no transitionswere seen in the lineshape of caesium lambda carrageenan.

Int. J. Biol. Macromol., 1985, Vol 7, February

55

Interaction o f group I cations with carraoeenans: P. S. B e l t o n et al.

The motionally narrowed conditions can arise in two ways. The first is associated with ion binding. If some fraction of the ions is bound to the polyelectrolyte with a correlation time in the regime described, and is exchanging with the remaining free ions on a time-scale which is rapid compared with the slowest relaxation rate in the system, then the signal behaves as if all of it were in the motionally narrowed regime. As a consequence there will be two components to the line and a 60% intensity loss may occur. The spectral details will be a function of the relaxation times, bound fraction and exchange rate between the sites 17. A word of caution is required on the use of the word 'bound'; use of this word should not be taken to imply formation of covalent bonds or ion pairing, it merely implies that this fraction is experiencing fluctuations in quadrupole coupling, at a rate significantly slower than the ions in free solution. Thus ions in a state of 'delocalized binding' as described in the Manning model I s would be considered bound in the sense implied here 19. Specific site binding has, however, been invoked to explain the behaviour of l aacs in kappa carrageenans 16. An important and alternative mechanism for generating the motionally narrowed state has been proposed by Berendsen 2°. This arises when anisotropic i:egions are present through which the ions diffuse. These ions can experience anisotropic fluctuations in the quadrupolar coupling and this leads to a situation in which an additional correlation time must be used. This correlation time arises from translational diffusion between regions of different orientation in which rapid anisotropic motion occurs. When a sufficient number of differently oriented regions have been experienced the residual static interaction, arising from the anisotropic motion, is reduced to zero and thus the motional narrowing state obtains. The length of this correlation time is the determining factor in the resulting lineshape. It is suggested that anisotropic domains with dimensions of the order 10 nm or more could give rise to the characteristic 40/60, two-component lineshape. In carrageenan gels the dimensions of the anisotropic junction zones have been estimated to be of the order of 100 nm 21. Thus, such behaviour must be considered to be likely in the gelforming samples studied here. The loss of signal intensity on gelation alone cannot therefore be considered to be direct evidence of long-lived binding at well-defined sites on macromolecules as has been claimed 16. On the other hand there is other evidence to support this contention. Concurrent with the observed lineshape changes large chemical shift changes are observed in caesium kappa carrageenans 16. Removal of potassium from this type of carrageenan is also difficult22, again pointing to some very strong interaction. Comparison of the high temperature 39K spectra of the three carrageenan types may also be considered to lend plausibility to this argument. At high temperatures the 39K linewidth in lambda carrageenan is greater than that for iota or kappa at corresponding temperatures. If ion condensation is occurring this is to be expected since the charge density of lambda is greater than that in iota or kappa. However, the changes in charge density on helix formation and subsequent gelation of iota and kappa will be relatively small and

56

Int. J. Biol. Macromol., 1985, Vol 7, February

make relatively small changes to the bound fraction. It would not be expected to cause large changes in correlation time. Hence the intensity loss observed in these two samples may be attributed to site binding effects if a binding model is assumed. At this stage, however, anisotropic effects cannot be discounted. Irrespective of the mechanism underlying the origin of the lineshapes it is clear that two general classes of behaviour are exhibited by ions interacting with carrageenans. One type of behaviour shows dramatic changes in the spectra at about the temperature of gelation, the other shows much more gradual transitions. It would be convenient if those species exhibiting sharp changes were only those which gelled. However, as we have shown previously s, all alkali metal ion forms of iota carrageenan may be induced to gel, but the sodium and lithium spectra do not show sharp transitions. Of the species described here only the lambda carrageenans and the sodium form of kappa carrageenan did not gel in the temperature ranges examined. It is true that the potassium forms of the polysaccharide do show a correlation between gelling behaviour and spectral change, but this does not hold generally, however. By contrast there are demonstrable effects of counterions on gel rheology 8 so that it may not be argued that the nature of the counterion is unimportant. The conclusion must therefore be drawn that non-specific ion effects are of great importance and that the nature of the specific ion interactions is not the sole or, even perhaps, the most important factor in the determination of gelling behaviour.

References 1 2 3 4 5 6 7 8

9 10 11 12 13 14 15 16 17 18 19 20 21 22

Morris,E. R., Rees,D. A. and Robinson, G. J. Mol. Biol. 1980, 138, 349 Smidsred,O. and Grasdalen, H. Carbohydr. Polym. 1982,2, 270 Bayley,S. T. Biochim. Biophys. Acta 1955, 17, 194 Pernas,A. J., Smidsrod,O., Larsen,B. and Haug, A. Acta Chem. Scand. 1967,21, 98 Reid,D. S. in 'Proc. 29th Symp.Colston Res.Soc.',Scientechnica, Bristol, 1978 Anderson,N. S., Campbell, J. W., Harding, M. M., Rees, D. A. and Samuel, J. W. B. J. Mol. Biol. 1969,45, 85 Forsen,S. and Lindman,B. Methods Biochem. Anal. 1982,27, 289 Belton, P.S.,Chilvers, G.R.,Morris, V.J. andTanner, S.F. Int.J. Biol. Macromol. 1984,6, 303

Towle,G. A. in 'Industrial Gums' (Ed. R. L. Whistler),Academic Press, New York and London, 1973,p. 83 Rees, D. A. Adv. Carbohydr. Chem. Biochem. 1969,24, 267 Mords, V.J.andChilvers, G.R.J.Sci. FoodAgric. 1981,32,1235 Morris,V. J. and Belton, P. S. Prog. Food Nutr. Sci. 1982,6, 55 Hubbard,P. S. J. Chem. Phys. 1970,53, 985 Halle,B. and Wennerstrom,H. J. Magn. Reson. 198l, 44, 89 Bull, T.E.,Forsen, S. andTurner, D.L.J. Chem. Phys. 1979,70,

3106 Grasdalen,H. and Smidsred, O. Macromolecules 1981, 14, 229 Bull, T. E. J. Magn. Reson. 1972,8, 344 Manning,G. S. Q. Rev. Biophys. 1978, 11, 179 Gustavsson,H., Lindman,B. and Bull,T. J. Am. Chem. Soc. 1978, 100, 4655 Berendsen, H. J. C. and Edzes, H. T. Ann. N.Y. Acad. Sci. 1973, 204, 459 Morris, V. J. Int. J. Biol. Macromol. 1982, 4, 155 Smidsred, O., Andresen, I-L., Grasdalen, H., Larsen, B. and Painter, T. Carbohydr. Res. 1980, 80, C11