Interactions of alginates with univalent cations

CarbohydrateResearch, 110(1982)101-112 ElsevierscientificPublishingCompany,Amsterdam- Printed in The Netherlands INTERACTIONS

OF ALGINATES WITH UNIVALENT CATIONS

ROBERTSBALE, Dobbie Chemical Laboratories, University College of North Wales, Bangor (Great Britain) EDWIN R. MORRIS,AND DAVID A. REES

Unilever Research, Colworth Laboratory, Sharnbrook, Be&x-d MK44 ILQ (Great Britain)

(ReceivedFebruary 8tb, 1982;acceptedfor publication,April 6th, 1982)

ABSTRACT

Interactions of alginate with univalent cations in solution have been investigated by circular dichroism (c.d.) and rheological measurements. Poly-L-guhuonate chainsegments show substantial enhancement (-50%) of c.d. elliptic@ in the presence of excess of K+, with smaller changes for other univalent cations: Li+ < Na+ < K+ > Rb+ > Cs+ > NH:. The maximum c.d. change is attained by 0.3~~ with no further increase at higher concentrations of cation. No significant dependence on polymer concentration is observed. Spectral changes for poly-D-mannuronate and heterotypic chain-sequences are much smaller. For intact alginates, the magnitude of c.d. change varies almost linearly with poly+guluronate content. Difference spectra (c.d. with excess of univalent counterion minus c.d. in distilled water) can be fitted accurately to two Gaussian bands at 211 and 198 nm, assigned to carboxyl n+z* and X+X* transitions, respectively. The perturbations induced by Li+, K+, Rbf, Cs+, and NHf show a clear family relationship, and are mainly in the z+lt* spectral region. With Na+, by contrast, c.d. change is largely confined to the n+z* transition, and is similar to that previously reported for intermolecular (“egg-box”) binding of divalent cations, consistent with results of rheological studies which indicate Na+-induced association of poly+guluronate chain-sequences. These associations are further enhanced on freezing and thawing. This combined evidence is interpreted in terms of three modes of interaction between univalent cations and alginate chains in solution: (a) ion-pair formation with carboxyl groups of mannuronate and isolated guluronate residues; (b) specific site-binding to contiguous guluronate residues; and (c) co-operative “egg-box” binding, particularly of Na+, between poly-r_-guluronate chain-sequences. INTRODUCTION

Alginate occurs as the major structural polysaccharide of brown seaweed’ (Phaeophyceue), and is of considerable technological importance both for its solution properties and as a gelling agent ‘. It is a linear, (1+4)-linked copolymer of a-~guluronate and /3-D-mannuronate’, the relative proportions of which, and their @ 1982- ElsevierScientificPublishingCompany 0008-621 S/82/OMKMOOO/$O2.75,

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sequence within the primary structure, show substantial variations between species, and between different tissues within the same plant4*5. To a reasonable first approximation, alginate may be regarded as a block copolymer, with residues arranged in homopolymeric sequences of both types, and in heterotypic alternating-sequences6*‘, although more detailed studies by enzymic digestions and ‘3C-n.m.r. spectroscopy’ have revealed some deviation from this idealised picture. Conformational-energy calculations of chain flexibility’ o predict a higher characteristic ratio (i.e., greater coil dimensions in solution) for polyguluronate than for polymannuronate, with heterotypic sequences being more flexible (lower characteristic ratio) than either. and these predictions are verified by light-scattering and intrinsic-viscosity measurements’ ‘J 2. In the crystalline state, poly-(o-mannuronic acid) exists m an extended 2, conformation”, analogous to that of (l-+4)-linked /?-o-mannan’4, or of cellulosei which shares the same diequatorial-linkage pattern. By contrast, poly-o-mannuronate (Li+, Na+, K+, and Cazf salt forms) adopts a three-fold helical structure to accommodate the bound counterions I6 . Poly-(L-guluronic acid) also shows 2, symmetry in the crystalline statei’, but, in this case, the chain profile is highly buckled due to the (l-4) diaxial linkage-geometry. Molecular model building18 shows that large cavities or interstices are formed when polyguluronate chains are packed together in this conformation. In the solid state, and in solutions” and gels20*2’, these cavities may be occupied by site-bound cations packed between the chains like eggs in an egg box’s, with retention of 2, chain-symmetry’“. The primary event in the Cazf-induced gelation of alginate is association of poly+guluronate chain-sequences into dimeric “egg-box” junction zones’ 9, although, in the presence of excess of Ca2 + , these may associate further into larger aggregates. Present evidence’ 9~22suggests that poly-o-mannuronate and mixed chain-sequences function predominantly as solubilising, interconnecting stretches between polyguluronate junctions. Other divalent cations may also be incorporated within junctions in an analogous fashion 2’*22, but with marked differences in the strength of binding. The resulting selectivities have been investigated extensively23-25, principally by potentiometric titration and ion-exchange equilibrium experiments. Specificity of interactions between alginate and univalent cations, however, has been far less widely explored. Selective interactions with different univalent cations are very important for the solution and gel properties of another family of seaweed polysaccharides, the carrageenans from certain species of Rkodophyceae. Gelation is promoted specifically by Kf, Rb+, or (to a lesser degree) Cs+ or NHf. with little or no evidence of network formation in the presence of larger (Me,N+) or smaller (Li+ or Na+) univalent cations. It has been suggested2” that the origin of this behaviour lies in cationmediated aggregation of carrageenan double-helices as a necessary step in formation of a cohesive, long-range gel network. Very recently, specific cation binding to carrageenan has been demonstrated2’ by ‘33Cs-n.m.r. spectroscopy. One technique which has proved particularly valuable in previous studies of

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the structure and interactions of polyuronates is circular dichroism (c.d.), since the electronic transitions of the carboxyl chromophore are extremely sensitive to relatively small changes in their local environment. For example, r,-guluronate and D-mannuronate show completely different c.d. behaviour”, which provides a simple method for determination of the overall composition of alginate. More-subtle c.d. changes with residue sequence within the alginate chain may also be utilised to estimate block composition’ 9. The method has been of greatest value, however, in monitoring and quantifying site binding of cations in the vicinity of the carboxyl group. In particular, “egg-box” binding of divalent cations is accompanied’ 8-21 by dramatic changes in c.d. In the present work, we have used cd. to investigate the interaction of univalent cations with alginate in aqueous solution. We also report some preliminary studies of the associated changes in solution rheology. EXPERIMENTAL

The following commercial alginates from Alginate Industries Ltd. were used: F347 (from Laminaria hyperborea); SS/DJ (from L. hyperborea stipes); F387 and SS/LK (both from Ascophyllum no&sum). These are identical to samples VI, IV, VII, and IX in the paper by Penman and Sanderson3’, and we have used the block compositions determined by these workers. Chain segments approximating to each structural type were prepared by partial hydrolysis with acid’, and characterised by n.m.r. spectroscopy 3o. All samples were dialysed extensively against deionised water, accurately neutralised, filtered, and freeze-dried before use. Absolute concentrations were determined by elemental analysis. C.d. spectra were recorded at 25” with a Cary 61 CD Spectropolarimeter, using an integration period of 10 s, at two different sample concentrations (1.5 and 0.1 w/v), using, respectively, l-mm and l-cm pathlengths. Curve fitting was effected by an iterative, least-square computer method, using a standard, Powell minimisation technique. High-resolution, ‘H-n.m.r. spectra were recorded at 100 MHz with a Varian XL-100 spectrometer operating in the Fourier-transform mode. “Zero shear” viscosity of dilute solutions was measured with an OgstonStarrier, low-shear, concentric cylinder viscometer31; at higher concentrations, a Rheometrics Mechanical Spectrometer (Model RMS-605; with a 50-mm cone and plate of cone angle 0.04 rad) was used. In each case, the most concentrated alginate solution used was equilibrated against the appropriate salt solution, and the dialysate used for subsequent dilutions to lower concentrations of polymer. Solutions were heated to 80” and re-cooled, immediately prior to measurement. Thixotropy studies were performed with a Deer Rheometer (50-mm perspex cone and plate; cone angle, 1.5 deg). Shear stress was increased linearly from 0 to 67.5 Pa over a period of 300 s (1 Pa = 10 dyne.cmm2).

Fig. 1. Effect of univalent cations on the c.d. of poly-L-guluronate. In order of increasing (negative) maximum molar ellipticity, the spectra shown are for solutions (1 .S?A w/v) in 0.5~ LiCl; 0.5M NH4Cl; distilled water (alginate in sodium salt form, i.e., N 75mM w.r.t. Na+); 0.5~ NaCI; 0.5~ CsCI; 0.5~ RbCl; and 0.5~ KCI. Closely similar results were obtained for 0.1~~ polyguluronate solutions. Fig. 2. Effect of univalent cations on the c.d. of algiaate heterotypic sequences. In order of increasing (negative) maximum molar ellipticity, the spectra shown are for solutions (1.5 or 0.10/b w/v) in distilled water; 0.5M NH&I; 0.5~ NaCl; 0.5~ CsCl; and 0.5M KCI. Results obtained for 0.5~ LiCl were virtually identical to those for distilled water, and the results for RbCl were closely similar to those for KCI. Fig. 3. Effect of univalent cations on the c.d. of poly-o-mannuronate. Spectra areshown for solutions ), 0.5~ NaCl (.-.-.-), and 0.5~ CsCl(----). Results for (1.5 or 0.1% w/v) m distilled water (----other univalent cations showed smaller deviations from the behaviour in distilled water. Fig. 4. Enhancement in maximum c.d. ellipticity of poly+guluronate (1.5:; w/v) with increasing mnn-ntratinn

nf KCI

I 0

,

40

I

20

I

60

66

I 100

I

I

205

v

1

2m

I

215

I

XOw

220

I

225

230

235

Fig. 6. C.d. changes on addition of univalent cations (0.5~; chloride salt form) to solutions (1.5% w/v) of poly-~guluronate. Experimental values are showu for Li+ (&; Na+ (A); K+ (m); Rb+ (0); Cs+ (0); and NH& (0); and compared with computed spectra () fitted to tWo Gaussian bands at 211 and 198 nm, of width 13.4 and 13.1 run, respectively.

Fig. 5. Degree of cd. enhancement (AI0]) on addition of K+ (0.3~) to samples having different contents of poly+guluronate. Results are shown for chain segments approximating to poly-~guluronate (-O-) and poly-o-mannuronate (-0-), and for alginates from Ascop~yllum nodosum (-Aand -A--), Luminuria hyperhea (-•-), and L. hyperborea stipes (+-). Measurements were made at the lowest wavelength (ZOOnm) at which accurate c.d. readings could be recorded.

looo-

,..a T

_

E; WI

_

R. SEAL&

106 RESULTS

E. R. MORRIS,

D. A. REES

AND DISCUSSION

Fig. 1 shows the c.d. behaviour of poly+guluronate segments (Na+ salt form) in distilled water, and in the presence of a large excess (0.5~) of various univalent cations (chloride salt form). In the presence of Kf, the maximum molar elliptic@ is enhanced by - 50 7: relative to that in water, with lower elliptic@ values for larger or smaller cations, as follows: Lif < Nat < K+ > Rb” > Cs+ > NHf. This order parallels the effectiveness of the same ions in promoting gelation of carrageenan’ 6. Heteropolymeric chain-sequences show far smaller c.d. changes with univalent cations (Fig. 2), but essentially the same order IS observed. In the case of poly-o-mannuronate, the corresponding spectral changes (Fig. 3) are virtually undetectable. Comparison of spectra recorded at 1.5 and 0.1 y; polyuronate (w/v) shows that residue molar ellipticity is essentially independent of polymer concentration. However, the magnitude of c.d. enhancement varies with cation concentration, reaching a final maximum value at - 0.3~. This is illustrated in Fig. 4 for the addition of KC1 to polyguluronate, where the largest and most accurately quantifiable c.d. changes were observed. As shown in Fig. 5, the magnitude of c.d. enhancement in the presence of excess of K+ over that in water for intact alginate varies almost linearly with poly-Lguluronate content. For this series of measurements, a KC1 concentration of 0.3M was used to optimise transmission, since the results presented in Fig. 4 show no further change at higher concentrations. The values shown are for the c.d. change between water and salt solution at the lowest wavelength at which accurate c.d. measurements could be made (200 nm). It is evident from Frg. 1 that the polyguluronate spectra recorded in the presence of excess of Li+, K+, Rb+, Cs+, and NHf show a clear family relationship, whereas the spectrum in NaCl solution is shifted appreciably to longer wavelength. To investigate this further, the c.d. changes induced by each cation (c.d. in the presence of 0.5~ univalent cation minus c.d. in distilled water) were calculated. In all cases, these “difference spectra” can be fitted accurately (Fig. 6) in terms of two Gaussian bands, one centred at 211 nm and of width (half-width at l/e of maximum height) 13.4 nm, and the other at 198 nm and of width 13.1 nm. These are in spectroscopically reasonable positions for the carboxyl n-+x* and rr--trc* transitions, respectively2*. Again the exceptional behavrour of Na+ is evident. The c.d. changes induced by K+ are centred predominantly in the Z-W* spectral region, and the principal differences between the other ions are in the steady decrease in the magnitude of this band through the series K+, Rb+, Cs+, NH:, and Li+. In the case of Na+, however, the c.d. changes are largely confined to the n-+x* spectral region, as observed for “egg-box” binding of Ca2+ and related divalent cations’8-21. Solution rheology. Alginate solutions containing high concentrations of NaCl also appeared to be of appreciably higher viscosity than comparable solutions in the presence of other univalent cations. To explore and quantify this behaviour, Circular dichroism.

-

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Fig. 7. Concentration &L-l) dependence of “zero-shear” specific viscosity (qsp) for alginate from Ascophyllumn&sum in 0.5~ (-O-) and 0.05~ (-O-) KCl.

Fig. 8. Concentrationdependence of “zero-shear” specific viscosity for alginate from Ascophyllum twdosum in 0.5~ (-O-)

and 0.05~ (-0-)

NaCl.

the concentration dependence of “zero shear” viscosity for alginate solutions at high (0.5~) and low (0.05~) concentrations of Naf and K+ was examined. In common with almost every polymer, alginate solutions exhibit “shear thinning” behaviour; i.e., doubling the applied stress (r) more than doubles the resulting rate of shear flow (j), and thus the apparent viscosity (q) is decreased. At very low rates of shear, however, T is proportional to 9, and viscosity remains constant at a maximum “zero shear” value of Q,. The fractional enhancement of solution “zero shear” viscosity over that of the solvent is then known as the “zero-shear” specific viscosity (q,J. The concentration (c) dependence of qSPundergoes an abrupt change at a critical value (c*) at which isolated polymer coils are forced to interpenetrate, and chain “entanglement” begins3’. Fig. 7 shows a double logarithmic plot of qSPagainst c for alginate from Asco&llum nodosum (21% polyguluronate) equilibrated against 0.5 and 0.05M KCl. In general, the viscosity of polyelectrolyte solutions would be expected to decrease with increasing ionic strength as intramolecular electrostatic repulsions between different chain-segments of the polymer are progressively screened, thus reducing overall coil dimensions. Alginate, however, is known to be comparatively stiff33 and, within the precision of the present measurements, no significant differences were observed between results obtained for high and low concentrations of KCl. In both cases, the slope of log qSPVS.log c above c* is -3.2 (Le., q. -c~.~). This is

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in good agreement with results for other polysaccharides34, and is consistent with physical entanglement of polymer chains3’. In the presence of NaCl, by contrast, the concentration dependence of q. For dilute solutions, shows an appreciable variation with Na+ concentration. measured viscosities are somewhat higher at low levels of salt, as expected from simple polyelectrolyte theory, but, with increasing concentration of polymer, the curves cross (Fig. 8), so that, in concentrated solution (c 2 1.3 7; w/v), viscosity is enhanced by the addition of Na+. This behaviour cannot be explained in terms of simple entanglement, but suggests specific, Na+-induced intermolecular association. At concentrations above c*, the “zero shear” viscosity in the presence of a high level of Na+ (0.5~) increases far more steeply than normal with increasing concentration of polymer (q. h c4.1). Similar behaviour has been reported for other polysaccharides (notably the plant galactomannans)34*3s which show evidence of specific, intermolecular, segment-segment association3 6. In these systems, intermolecular association can be enhanced by freezing and thawing. The rationale of this approach is that the growth of ice crystals during the freezing process progressively raises the polymer concentration in the residual, unfrozen solution, and that associations induced in this way may persist on thawing. After freezing and thawing, solutions of alginate in the presence of high concentrations of Na+ (0.5~) show clear evidence of interchain association. Some slight enhancement of viscosity is also apparent in the presence of high levels of K+, but the effects are most pronounced for Na+, and for alginates having a high content of polyguluronate, where a weak, gel-like structure is developed. In contrast to true gels (such as those formed with Ca*+), however, these can be spread, gradually recover from mechanical damage, and, given long enough, will flow. For alginates rich in polyguluronate, progressive development of network character in concentrated Naf solution is evident even without freezing. It is for this reason that our studies of the concentration dependence of ‘lo (Figs. 7 and 8) have been confined to alginate having a relatively low content of polyguluronate, since no meaningful viscosity measurements can be made in the presence of a continuous network. To give a quantitative indication of the extent and time-scale of interchain association in these systems, measurements obtained with a Deer Rheometer are shown in Fig. 9. In this instrument, applied shear stress may be controlled as an independent variable, and the resulting rate of shear measured, in contrast to conventional rotational viscometers in which shear rate is varied and the resistance to flow is measured. Results are shown for alginate from Latninaria Igprrborea stipes (59 y/, polyguluronate; 1.5 “/;, w/v) in water, in 0.5~ NaCl (freshly prepared solution), and in 0.5~ KC1 and 0.5~ NaCl after freezing and thawing. The visual impression of a gel-like structure in the last of these samples is confirmed by the requirement for a relatively large shear-stress (-20 Pa) to be applied before any detectable shear-rate is generated (i.e., the sample exhibits a yield stress). On decreasing the applied stress, the network character is not restored within

ALGINATE INTERACTION

WITH UNIVALENT

109

CATIONS

2oc

16C

80

0

IO

20

30 40 7 (Pa)

50

60

Fig. 9. Shear rate (9) generated in response to applied stress (7) for alginate (1.5% w/v) from Luminaria hyperborea stipes in distilled water ( -.-.-), 0.5~ NaCl (---), 0.5~ NaCl after freezing and thawing (), and 0.5~ KCl after freezing and thawing (. . . . . . .).

the time-scale of the measurement (300 s). Similar, but less pronounced, thixotropic behaviour is observed for freshly prepared solutions in the presence of high levels of Na+. In distilled water, the sample shows normal “shear thinning” behaviour, with no evidence of thixotropy. On heating to 80” and re-cooling, samples which have been frozen and thawed largely revert to the same flow properties as those of freshly prepared solutions, indicating that the Naf-induced, interchain associations are thermally labile.

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In the presence of high concentrations of K+ (OSM), viscosity is somewhat enhanced after freezing and thawing, but again no thixotropy is evident. This might either suggest a much shorter time-scale of interchain association, or (more probably) indicate that no cohesive network structure is formed and that the predominant response to applied stress is by disentanglement, with limited, specific associations serving only to increase slightly the effective molecular weight. CONCLUSIONS

Although the most familiar and extensively investigated alginate-cation interactions are those involved in gel formation, solution properties are also sensitive to ionic environment. Indeed, evidence from light-scattering and viscosity studies indicates3’ that the solubility of alginate arises not from favourable polymer-water interactions, but from electrostatic stabilisation, and may therefore be substantially altered by binding of cations to the polyelectrolyte chain. In the present work, cd. has been used to investigate these interactions at a local level, rather than from their effect on overall hydrodynamic behaviour. The results indicate considerable specificity in the interactions of alginates with univalent cations in solution, in agreement with previous evidence from the saltdependence of intrinsic viscosity 33. The interactions are most pronounced for K+, which has been used as a selective precipitant in the fractionation of alginatej’, and for alginates rich in polyguluronate. A possible interpretation of this latter selectivity is that adjacent guluronate residues may offer a suitable site for specific chelation of cations, which is absent in other component-disaccharide groupings within the alginate chain (see Fig. 10). This proposal explains the similarity in form (but not in magnitude) of c.d. changes

M-M

M-G

HO

Fig. 10. Disaccharide sequences present in alginate: O-~-~-ManpA-(l+4)-O-~-~-ManpA (M-M), O-p-D-ManpA-(144)-0-a-L-GulpA (M-G), 0-a+GulpA-(1+4)-O-/?-D-ManpA (G-M), O-a-LGulpA-(l-*4)-0-a-L-GulpA (G-G). The proposed site for inter-residue binding of univalent cations (M+) to G-G is indicated.

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observed for polyguluronate and heteropolymeric chain-sequences, since the latter are also known to contain an appreciable proportion of contiguous guluronate residues*39. Binding of univalent cations to other residues would then be confined to weak chelation by isolated residues (which may correspond to the small, positive intercept in the plot of c.d. change against polyguluronate content in Fig. 5), and loose association due to the polyelectrolyte character, which would not be expected to influence cd. behaviour. The c.d. changes induced by Na+ ions are qualitatively different from those observed with the other univalent ions studied. In spectral form, however, they resemble the changes which accompany “egg-box” binding of divalent cations, although they are of considerably lower magnitude and opposite in sign, The atypical bebaviour of Nat extends to its influence on solution properties, where clear evidence of specific, cation-induced interchain association has been observed. It seems possible, therefore, that, under forcing conditions (high concentrations of Na+; high content of polyguluronate; freezing and thawing), sodium ions may be capable of stabilising intermolecular “egg-box” junctions analogous to those formed with divalent cations, but of considerably shorter time-scale and lower binding-energy. In summary, therefore, we propose three modes of binding of univalent cations to alginate chains in solution (in addition to general polyelectrolyte effects): (a) weak chelation by mannuronate and isolated guluronate residues (as is found for monomer residues); (b) specific site-binding to adjacent guluronate residues; and (c) cooperative “egg-box” binding, particularly of Na+, between polyguluronate chainsequences. ACKNOWLEDGMENTS

We thank the SRC for a CASE award (to R.S.), Mr. A. N. Cutler and Mrs. S. A. Frangou for advice and practical assistance, and Dr. J. R. Turvey for advice, discussions, and encouragement throughout this work. REFERENCJiS 1 E. PIUKIVAL AND R. H. MCDOWELL, Chemistry and Enzymology of Marine Algal Polysoecharids, Academic Press, London, 1967. 2 R. L. WHISTLER, Zndustrial Gums, Academic Press, New York, 1973. 3 D. A. RBBSAND J. W. B. SAMUJ%, J. Chem. Sot., C, (1967) 2295-2298. 4 A. HAIJO,B. LARSEN, AND 0. fhrixW~, Curb&y&. Res., 32 (1974) 217-225. 5 B. STOCKTON, L. V. EVANS,E. R. MORRIS, D. A. POWELL,AND D. A. REES,Bat. Mar., 23 (1980) 563-567. 6 A. HAUG, B. LARSEN, AND0. SMIDSRBD, Acta Chem. Scud, 20 (1966) 183-190. 7 A. HAUG, B. LARSEN, AND 0. SMIDSR~, Acta Chem. Scud, 21 (1967) 691-704. 8 J. Bon, AND J. R. TURVEY, Curbohy&.Res., 66 (1978) 187-194. 9 H. GRASDALEN, B. LARSEN, AND0. SMIDSIWD, Carbohydr.Res., 89 (1981) 179-191. 10 S. G. WHITIINGTON,Biopolymers, 10 (1971) 1481-1489; 1617-1623. 11 R. F. BRUCKER AND C. M. WORMINGTON, J. Macromol. Sci., Chem., 5 (1971) 116%1185. 12 0. SMIDSR~D, R. N. GUIVER,AND S. G. WHITITNGTON, Car6ohydr. Res., 27 (1973) 107-118.

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