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Cation-specific vacuum ultraviolet circular dichroism behaviour of alginate solutions, gels and solid films
Cation-specific vacuum ultraviolet circular dichroism behaviour of alginate solutions, gels and solid films J. N. Liang* and E. S. Stevens Department of Chemistry, State University of New York, Binghamton, New York 13901, USA
and S. A. Frangou, E. R. Morris and D. A. Rees Unilever Research, Colworth Laboratory, Sharnbrook, BedJbrd, MK44 1LQ (Received 13 August 1979; revised 8 November 1979) The vacuum ultraviolet circular dichroism o[alginate solutions, gels and solid films is repdrted. Two previously observed bands at ~ 215 and ~ 203 nm are assigned to n-,~z* transitions o] carboxy groups under d([J~,rent conditions tfflocal environment. Three bands not previously observed are at ~ 185 nm, assigned to carbox y ~z~ ~z* transitions, and at ~ 169 and ~ 149 nm, assigned to transitions o[the polymer backbone. In the course o[the sol (Na +) gel (Ca 2+), the sol (Na*) film (Na ~ ) and the gel (CaZ +) film (Ca 2+) transitions, intensity chan qes are observed in both the low energy and high energy hands. The c.d. changes during the three transitions d(ff~,r in magnitude, but are qualitatively the same,.J?om which we conclude that the chain co¢![brmations in the gel and films are similar, and that the principal spectral changes have their origin in perturbation q/ehromophores by sitebound cations.
Introduction In the study of polysaccharide conformation the process of gelation is of particular interest, and in recent years a great deal of attention has been paid to those with high gel formation tendency. Examples include carrageenan and agarose, which undergo heat-induced gel sol transitions 1'2, and pectin and alginates, which form gels by binding with divalent cations 3'4. The sol gel transition in many biopolymers arises from cooperative association and melting of extended regions of ordered tertiary structure 5. Such cooperative interactions in polysaccharide systems have been characterized by optical rotation 1'2. 1H and a3C n.m.r, relaxation 2'5, ultraviolet circular dichroism 2'5, and, more recently, by vacuum ultraviolet circular dichroism (v.u.c.d.)6. In the case of agarose, v.u.c.d, measurements were used to characterize the heat-induced gel sol transition 6. We now report v.u.c.d, measurements on alginate solutions, gels and solid films. Alginate is a (1 ~4)-linked copolymer of Ct-L-guluronate (I) and fl-D-mannuronate (II), with residues arranged v in homopolymeric blocks of each type, and in mixed se0
0
' C02H
0
o (I)
(IT)
quences in which both residues occur in an approximately alternating structure 8. Each block-type shows quite different c.d. behaviour in solution, thus providing a simple index of alginate composition 3. On gelation with Ca 2 ÷ or larger Group II cations pronounced c.d. changes are observed in the carboxy n~rt* spectral region above * Postdoctoral research associate 0!.41 --8130/80/040204 05502.00 © 1980 IPC Business Press
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Int. J. Biol. Macromol., 1980, Vol 2, August
~ 200 nm, which appear to arise principally from cation chelation to polyguluronate chain sequences 4. The spectroscopic origin of these changes, however, is still uncertain. Three possibilities can be suggested. (a) The presence of a cation tightly bound to the chromophore alters the rotational strength of the n--,zr* band, without greatly altering its position. (b) The energy of the transition is raised, moving part of the original band to below the short wavelength cut-off of commercial c.d. equipment. (c) The changes are due to exciton splitting. One objective in extending c.d. measurements into the vacuum ultraviolet is to establish which, if any, of these proposals is correct.
Experimental The alginate used was the same material as sample IV in the paper by Penman and Sanderson 9, who determined its block composition as 58.6~,, polyguluronate, 18.7'J'~(,polymannuronate, and 22.7°~, mixed sequences. Solutions were prepared by dispersing the alginate in water, adjusting the pH to 7, and filtering to optical clarity (1.2 /~m Millipore). Final solution concentrations were determined by total carbon analysis (Galbraith Laboratories, Knoxville, Tennessee). Solution c.d. measurements were made in 0.1 mm path length quartz cells. Gels were prepared by dialysis against 5 mM CaC1 z. Films were cast on calcium fluoride discs by evaporating to dryness, either in the atmosphere or in a dessicator. The absence of molecular orientation was confirmed by observing no change in signal when the film was rotated in the light beam. The v.u.c.d, spectrometer was operated as described elsewhere 6' l o.11, using a 3.2 nm spectral width, 10 or 30 s time constant, and 1.0 nm min- 1 scan speed. Molar ellipticities were calculated on the basis of a residue molecular weight of 198.
Alginate Circular dichroism." J. h¢. Liang et al.
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blue shift of several nanometers is observed. Previous computer curve-fitting studies ~4, however, suggest that this may be due to overlap with the growing positive band at ~ 203 nm, leading to displacement of the apparent peak maxima from their true spectral positions. Alginate solutions also form gels at low pH on dialysis against acid solution, but the c.d. spectrum of the resulting gel (Figure 3) is quite different from that of the calcium gel, presumably largely due to partial replacement of the C O 0 - chromophore by COOH. The negative n~rt* band is enhanced rather than diminished, and there is no evidence of the appearance of a positive band at lower wavelength. On drying down such acid gels to solid films, band shape and position remain unchanged (Figure 3), and although exact quantitative comparison of film and gel spectra is complicated by uncertainties in film thickness, there is no evidence of any substantial change in intensity. Similarly films cast from Ca 2 + gels (Figure 4) show essentially the same c.d. behaviour as the parent gel. By contrast, on drying down sodium alginate solutions to solid films, appreciable c.d. changes are observed,
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Figure 1 Circular dichroism of alginate solution (Na +), and of
gels formed in the presence of different Group II cations: A, Ba2~-; B, Sr2+; C, CaZ+: D, Mg2+; E, Na + Im O
Results
As shown in Figure 1, sodium alginate solution displays two negative c.d. bands, one in the region 210-215 nm, and the other near "185 nm. The first has been assigned 3'4 to the carboxy n--*lt* transition. The second has not previously been observed, although its presence has been inferred 3 from the asymmetry of the n ~ * band above 200 nm. It is close in energy to the 182 nm band of glucuronic acid, which has been assigned 1° to the carboxy 7z~Tz* transition, and we therefore propose the same spectroscopic origin. Cation-induced gelation is accompanied by major c.d. changes (Figure 1). Ca z +, Sr 2 + and Ba 2 + produce spectral changes of comparable form and magnitude, while Mg z + shows a substantially smaller effect, consistent with previous studies of the relative binding affinities of Group II cations to polyguluronate chain sequences 12,13. During the course of gelation (Figure 2) a progressive decrease is observed in the intensity of the negative n-,rt* band at 215 nm, which is accompanied by the development of a positive band centred at ~ 203 nm. Little change in c.d. intensity is observed in the rt~Tt* spectral region around 185 nm, although at C a 2 + concentrations approaching the stoichiometric equivalent of the alginate, an apparent
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Figure 2 Spectral changes during the Ca" ~ gelation of alginate. Calcium ion concentrations as a percentage of the total stoichiometric cation requirement of the alginate were: AO , oo: -tL 2 8 ,o ,, , C, 41%; D, _%°'o, E, 83 °o," F, 103'~o
Int. Ji Biol. Macromol., 1980, Vol 2, August
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AI.qinate circular dichroism: J. N. Liang et al. Computer matching of the film spectra in Figure 4 gives a measure of the variation in intensity of these bands with Ca 2 ~ concentration, as shown in Figure 5. 4
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Competitive cation binding studies 12'14 indicate the following hierarchy of Ca z + chelation to alginate chains. ( 1) The primary mechanism of interchain association is by dimerization of polyguluronate sequences, with specific site binding of Ca/+ or larger G r o u p II cations between chains of regular two-fold symmetry 4'14. This process reaches completion at a Ca 2 + level equivalent to half the stoichiometric requirement of the polyguluronate sequences, since only the interior faces of the chains are involved in chelation. In the particular case of the alginate used in this study (~60~o polyguluronate) this will correspond to ~ 3 0 % of the total cation requirement of the alginate, and offers a direct explanation of the observed discontinuity in the Ca 2 + dependence of the ~ * transition (184 nm) at this calcium level (Figure 5).
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Figure 3 Comparison of the c.d. behaviour of alginic acid gels and films with that of sodium alginate at neutral pH. A, Sol(Na ÷); B, gel(H +); C, film(Na +); D, film(H +)
which are qualitatively similar to the changes observed on Ca 2+ gelation, but smaller in magnitude. Indeed, comparison of the sodium alginate film c.d. in Figure 4 with the spectrum obtained under hydrated conditions in the presence of 41'Y;, of the total stoichiometric Ca 2 + requirement [Figure 2) shows close similarity in position, widths and relative intensities of the bands at ~215, ~203 and 185 nm. In addition to these low energy transitions, two high energy bands, which are inaccessible in solutions and gels, are observed at ~ 170 and 150 nm in the c.d. spectra of both sodium and calcium alginate solid films (Figure 4). The relative magnitudes of these bands vary substantially with Ca 2 + level, as shown in Figure 4. Similar bands have been observed for iota carrageenan 11, agarose 6 and galactomannans 15, and are attributedto transitions of the polymer backbone. They are always observed to be of opposite sign, and have not yet been definitely assigned. It has been suggested, however, that they have t h e s a m e spectroscopic origin as the band seen in the vapour phase c.d. of tetrahydropyran ~''1i. i Quantitative analysis of alginate c.d. behaviour has previously been confined to the spectral region above 200 nm ~4. Similar computer curve-fitting in the vacuum ultraviolet region below 200 nm confirms that good quantitative agreement with the observed c.d. behaviour of both sodium and calcium alginate films in this spectral region can be obtained in terms of three Gaussian c.d. bands. These are all of width 10.2_+0.5 nm, and are centred at 149_+1, 169-t-1 and 184_+1 nm, respectively.
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Figure 4 Cation-dependent spectral changes in the vacuum ultraviolet circular dichroism of algi-nate in the solid state. The agreement between observed ( ) and fitted (e) c.d. spectra is shown for Na + and Ca 2 + films. A similar quality of fit was obtained at intermediate Ca 2 + levels, which are shown as a percentage of the total cation requirement of the alginate: A, 0%; B, 14",,: C, 28",,: D, 56",,; E, 83%; F, 103%
Alginate circular dichroism: J. N. Liang et al.
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Figure 5 Cation dependence of alginate v.u.c.d. The film spectra in Figure 4 may be fitted adequately by three bands below 200 nm, each of width 10.2 + 0.5 nm, and centred at 149 +1 m), 169+1 (A) and 184+1 (e) nm, respectively. The variation in intensity of these bands with Ca 2 ÷ level is as shown (Ca 2÷ concentrations expressed as a percentage of the total cation requirement ofthe alginate). Similar quantitative analysis of lower energy c.d. behaviour has already been reported elsewhere 14
data indicate stiff, extended chain geometry for polyguluronate in solution, suggesting only limited conformational mobility about the preferred solid state conformation, in contrast to polymannuronate and mixed chain sequences which show considerably greater flexibility 19. This is perhaps consistent with the observed cation dependence of the high energy transition at ~ 169 nm, which has previously been shown to provide a sensitive index of conformational changes during the sol-gel transition of agarose 6. Little variation in the magnitude of this band with Ca 2÷ level is observed up to the stoichiometric equivalent of the polyguluronate chain sequences (Figure 5), but at higher calcium concentrations there is a marked change in intensity, which may reflect conformational changes associated with Ca 2 ÷ chelation to mannuronate-containing chain sequences. If, as suggested by the above evidence, intermolecular association of polyguluronate involves little change in backbone geometry, then the observed spectral changes which accompany this process must have their origin in some other effect. We suggest that the most likely explanation lies in perturbation of chromophores by sitebound cations. Computer model-building studies, and visual inspection of physical models of polyguluronate chains in the preferred two-fold conformation, show 4 the existence of oxygen-lined pockets or 'nests' formed by O(1), (the glycosidic 'linking' oxygen), 0(2) and 0(3) of one sugar residue, and O(5), (the ring oxygen) and O(6), (one of the carboxy oxygens) of the adjacent residue in the reducing direction (see Figure 6). These 'nests' are periodically spaced along both sides of the chain, and their dimensions satisfy the criteria for Ca 2 ÷ chelation. From this it has been proposed that intermolecular association in alginate gels and in the solid state involves interchain chelation of extended arrays of site-bound cations (the 'eggrbox' model, shown schematically in Figure 6). The involvement of the ring oxygen and glycosidic oxygen of the polymer backbone in cation chelation may explain the sensitivity of the 149 nm band to ionic
dime,
(2) Subsequent association of these dimers into larger aggregates would involve further cation chelation, reaching a theoretical maximum of stoichiometric equivalence (calcium polyguluronate), in the present case at ~ 6 0 ~ of the total cation requirement for the entire chain. As shown in Figure 5, discontinuities in the Ca 2 ÷ dependence of both the high energy backbone transitions (at 149 and 169 nm) are, in fact, observed at this calcium level. (3) At higher Ca 2 ÷ levels some binding to polymannuronate and mixed chain sequences may occur. In solutions and gels this may be weak and non-specific, but in the solid state is likely to involve at least some degree of site binding, on loss of mobility of the participating chain sequences. X-ray fibre diffraction and i.r. dichroism studies ~7'18 have established an extended two-fold conformation for polyguluronic acid, which appears to persist in all salt forms so far investigated. Viscosity and light scattering
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Aggregation of climers o
Figure 6 Schematic representation of cooperative intermolecular cation binding to alginate polyguluronate chain sequences
Int. J. Biol. Macromol., 1980, Vol 2, August
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AI,qinate circular dichrosim: J. N. Lian,q et al. environment (see Fi,qure 5), although differences in the behaviour of this band and of the other backbone transition at 169 nm must remain the subject of further investigation. The positions and widths of these far vacuum u.v. bands are in excellent agreement with the values which we have recently obtained for the corresponding backbone transitions in the galactomannan family of plant polysaccharides 1s. The lower energy c.d. bands of alginate all involve transitions of the carboxy group, which is likely to experience the most severe perturbations from cation binding. Our results show no evidence of displacement of these bands into the vacuum u.v. region on cation binding, nor of exciton splitting, and thus the observed spectral changes must have their origin in changes in rotational strength. As indicated in the following scheme, four states of alginate may be considered, each having its own characteristic c.d. behaviour from the carboxy chromophore.
Solution (Na+)~Film (Na +) Gel (Ca2 +)+Film (Ca z+) The major change in c.d. is observed on going from solution to gel. Little further change occurs on drying down to the solid state (Ca 2+ film), consistent with previous evidence 2 that association of polyguluronate sequences in alginate gels approaches completion. The principal spectral change is inversion of sign of the n--, rt* band, with a change in position from ~ 215 nm to ~ 203 nm. Similar energy differences are observed in the n--,rt* transitions of uronate monomers, and have been interpreted in terms of rotational isomerism about the C(5)C(6) bond 3. It seems probable, therefore, that the spectral changes associated with Ca 2+ chelation by polyguluronate have their origin in locking of the carboxy group in a fixed orientation relative to the sugar ring, which is different from the preferred orientation in solution. The proximity of the divalent cation is likely to have a substantial effect on the asymmetric environment of the carboxy group, and hence on the rotational strength of the transition. It is perhaps significant, therefore, that the spectral changes on going from sodium alginate solution to the Na + film are closely similar in form to those observed with Ca 2+, but of approximately half the magnitude, particularly since Na + and Ca 2 + are almost isomorphous, and may therefore be capable of occupying the same binding sites, with little change in local geometry. Thus the major differences in their influence on c.d. behaviour (Figures 4 and 5) may arise from differences in ionic charge. In summary, therefore, we conclude that both the high energy transitions of the alginate backbone, and the low
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energy transitions of the carboxy groups show appreciable changes on intermolecular association, and that for polyguluronate sequences these changes arise principally from perturbations of chromophores by site-bound cations. Our results also indicate the possibility of conformation-sensitive c.d. changes for mannuronatecontaining chain sequences, and this is currently the subject of further research. The data described here support a model of alginate gel structure previously proposed, i.e. the work is put forth as confirming evidence for that model. It is possible that our results are compatible with some other model for the gel structure, but the data displayed in Figure 5 must be considered a very strong constraint on alternative models.
Acknowledgements Acknowledgement is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research. Partial support was also received from the US National Institute of Health through Grant G M 24862.
References 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Dea, l . C . M . , M c K i n n o n , A.A. andRees, D . A . J . MoI. Biol. 1972, 68, 153 Bryce, T. A., McKinnon, A. A., Morris, E. R., Rees, D. A. and Thorn, D. Faraday Discuss. Chem. Soc. 1"974, 57, 221 Morris, E. R., Rees, D. A., Sanderson, G. R. and Thorn, D. J. Chem. Sot'. Perkin Trans. 2 1975~ 1418 Grant, G. T., Morris, E. R., Rees, D. A., Smith, P. J. C. and Thorn, D. FEBS Lett. 1973, 32, 195 Rees, D. A. and Welsh, E. J. Anoew. Chem. Int. Ed. Enql. 1977, 16, 214 Liang, J. N., Stevens, E. S., Morris, E. R. and Rees, D. A. Biopolymers 1979, 18, 327 Haug, A., Larsen, B. and SmidsrCd, O. Acta Chem. Stand. 1967, 21,691 Boyd, J. and Turvey, J. R. Carbohydr. Res. 1978, 66, 187 Penman, A. and Sanderson, G. R. Carbohydr. Res. 1972, 25, 273 Buffington, L. A., Pysh, E. S., Chakrabarti, B. and Balazs, E. A. J. Am. Chem. Soc. 1977, 99, 1730 Balcerski, J. S., Pysh, E. S., Chen, G. C. and Yang, J. T. J. Am. Chem. Sot'. 1975, 97, 6274 Smidsr~bd, O. and Haug, A. Acta Chem. Scand. 1968, 22, 1989 Haug, A. and SmidsrCd, O. Acta Chem. Stand. 1970, 24, 843 Morris, E. R., Rees, D. A., Thom, D. and Boyd, J. Carbohydr. Res. 1978, 66, 145 Buffington, L. A., Stevens, E. S., Morris, E. R. and Rees, D. A. Int. d. Biol. Maeromol. 1980, 2, 199 Pickett, L. W., Hoeflich, N. J. and Liu, T. C. J. Am. Chem. Soc. 1951, 73, 4865 Atkins, E. D. T., Mackie, W. and Smolko, E. E. Nature (London) 1970, 225, 626 Atkins, E. D T., Mackie, W., Parker, K. D and Smolko, E E J. Polym. Sei. (B) 1971, 9, 311 SmidsrOd, O., Glover, R. M. and Whittington, S. G. Carbohydr. Res. 1973, 27, 107
and S. A. Frangou, E. R. Morris and D. A. Rees Unilever Research, Colworth Laboratory, Sharnbrook, BedJbrd, MK44 1LQ (Received 13 August 1979; revised 8 November 1979) The vacuum ultraviolet circular dichroism o[alginate solutions, gels and solid films is repdrted. Two previously observed bands at ~ 215 and ~ 203 nm are assigned to n-,~z* transitions o] carboxy groups under d([J~,rent conditions tfflocal environment. Three bands not previously observed are at ~ 185 nm, assigned to carbox y ~z~ ~z* transitions, and at ~ 169 and ~ 149 nm, assigned to transitions o[the polymer backbone. In the course o[the sol (Na +) gel (Ca 2+), the sol (Na*) film (Na ~ ) and the gel (CaZ +) film (Ca 2+) transitions, intensity chan qes are observed in both the low energy and high energy hands. The c.d. changes during the three transitions d(ff~,r in magnitude, but are qualitatively the same,.J?om which we conclude that the chain co¢![brmations in the gel and films are similar, and that the principal spectral changes have their origin in perturbation q/ehromophores by sitebound cations.
Introduction In the study of polysaccharide conformation the process of gelation is of particular interest, and in recent years a great deal of attention has been paid to those with high gel formation tendency. Examples include carrageenan and agarose, which undergo heat-induced gel sol transitions 1'2, and pectin and alginates, which form gels by binding with divalent cations 3'4. The sol gel transition in many biopolymers arises from cooperative association and melting of extended regions of ordered tertiary structure 5. Such cooperative interactions in polysaccharide systems have been characterized by optical rotation 1'2. 1H and a3C n.m.r, relaxation 2'5, ultraviolet circular dichroism 2'5, and, more recently, by vacuum ultraviolet circular dichroism (v.u.c.d.)6. In the case of agarose, v.u.c.d, measurements were used to characterize the heat-induced gel sol transition 6. We now report v.u.c.d, measurements on alginate solutions, gels and solid films. Alginate is a (1 ~4)-linked copolymer of Ct-L-guluronate (I) and fl-D-mannuronate (II), with residues arranged v in homopolymeric blocks of each type, and in mixed se0
0
' C02H
0
o (I)
(IT)
quences in which both residues occur in an approximately alternating structure 8. Each block-type shows quite different c.d. behaviour in solution, thus providing a simple index of alginate composition 3. On gelation with Ca 2 ÷ or larger Group II cations pronounced c.d. changes are observed in the carboxy n~rt* spectral region above * Postdoctoral research associate 0!.41 --8130/80/040204 05502.00 © 1980 IPC Business Press
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Int. J. Biol. Macromol., 1980, Vol 2, August
~ 200 nm, which appear to arise principally from cation chelation to polyguluronate chain sequences 4. The spectroscopic origin of these changes, however, is still uncertain. Three possibilities can be suggested. (a) The presence of a cation tightly bound to the chromophore alters the rotational strength of the n--,zr* band, without greatly altering its position. (b) The energy of the transition is raised, moving part of the original band to below the short wavelength cut-off of commercial c.d. equipment. (c) The changes are due to exciton splitting. One objective in extending c.d. measurements into the vacuum ultraviolet is to establish which, if any, of these proposals is correct.
Experimental The alginate used was the same material as sample IV in the paper by Penman and Sanderson 9, who determined its block composition as 58.6~,, polyguluronate, 18.7'J'~(,polymannuronate, and 22.7°~, mixed sequences. Solutions were prepared by dispersing the alginate in water, adjusting the pH to 7, and filtering to optical clarity (1.2 /~m Millipore). Final solution concentrations were determined by total carbon analysis (Galbraith Laboratories, Knoxville, Tennessee). Solution c.d. measurements were made in 0.1 mm path length quartz cells. Gels were prepared by dialysis against 5 mM CaC1 z. Films were cast on calcium fluoride discs by evaporating to dryness, either in the atmosphere or in a dessicator. The absence of molecular orientation was confirmed by observing no change in signal when the film was rotated in the light beam. The v.u.c.d, spectrometer was operated as described elsewhere 6' l o.11, using a 3.2 nm spectral width, 10 or 30 s time constant, and 1.0 nm min- 1 scan speed. Molar ellipticities were calculated on the basis of a residue molecular weight of 198.
Alginate Circular dichroism." J. h¢. Liang et al.
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blue shift of several nanometers is observed. Previous computer curve-fitting studies ~4, however, suggest that this may be due to overlap with the growing positive band at ~ 203 nm, leading to displacement of the apparent peak maxima from their true spectral positions. Alginate solutions also form gels at low pH on dialysis against acid solution, but the c.d. spectrum of the resulting gel (Figure 3) is quite different from that of the calcium gel, presumably largely due to partial replacement of the C O 0 - chromophore by COOH. The negative n~rt* band is enhanced rather than diminished, and there is no evidence of the appearance of a positive band at lower wavelength. On drying down such acid gels to solid films, band shape and position remain unchanged (Figure 3), and although exact quantitative comparison of film and gel spectra is complicated by uncertainties in film thickness, there is no evidence of any substantial change in intensity. Similarly films cast from Ca 2 + gels (Figure 4) show essentially the same c.d. behaviour as the parent gel. By contrast, on drying down sodium alginate solutions to solid films, appreciable c.d. changes are observed,
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Figure 1 Circular dichroism of alginate solution (Na +), and of
gels formed in the presence of different Group II cations: A, Ba2~-; B, Sr2+; C, CaZ+: D, Mg2+; E, Na + Im O
Results
As shown in Figure 1, sodium alginate solution displays two negative c.d. bands, one in the region 210-215 nm, and the other near "185 nm. The first has been assigned 3'4 to the carboxy n--*lt* transition. The second has not previously been observed, although its presence has been inferred 3 from the asymmetry of the n ~ * band above 200 nm. It is close in energy to the 182 nm band of glucuronic acid, which has been assigned 1° to the carboxy 7z~Tz* transition, and we therefore propose the same spectroscopic origin. Cation-induced gelation is accompanied by major c.d. changes (Figure 1). Ca z +, Sr 2 + and Ba 2 + produce spectral changes of comparable form and magnitude, while Mg z + shows a substantially smaller effect, consistent with previous studies of the relative binding affinities of Group II cations to polyguluronate chain sequences 12,13. During the course of gelation (Figure 2) a progressive decrease is observed in the intensity of the negative n-,rt* band at 215 nm, which is accompanied by the development of a positive band centred at ~ 203 nm. Little change in c.d. intensity is observed in the rt~Tt* spectral region around 185 nm, although at C a 2 + concentrations approaching the stoichiometric equivalent of the alginate, an apparent
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Figure 2 Spectral changes during the Ca" ~ gelation of alginate. Calcium ion concentrations as a percentage of the total stoichiometric cation requirement of the alginate were: AO , oo: -tL 2 8 ,o ,, , C, 41%; D, _%°'o, E, 83 °o," F, 103'~o
Int. Ji Biol. Macromol., 1980, Vol 2, August
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AI.qinate circular dichroism: J. N. Liang et al. Computer matching of the film spectra in Figure 4 gives a measure of the variation in intensity of these bands with Ca 2 ~ concentration, as shown in Figure 5. 4
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Competitive cation binding studies 12'14 indicate the following hierarchy of Ca z + chelation to alginate chains. ( 1) The primary mechanism of interchain association is by dimerization of polyguluronate sequences, with specific site binding of Ca/+ or larger G r o u p II cations between chains of regular two-fold symmetry 4'14. This process reaches completion at a Ca 2 + level equivalent to half the stoichiometric requirement of the polyguluronate sequences, since only the interior faces of the chains are involved in chelation. In the particular case of the alginate used in this study (~60~o polyguluronate) this will correspond to ~ 3 0 % of the total cation requirement of the alginate, and offers a direct explanation of the observed discontinuity in the Ca 2 + dependence of the ~ * transition (184 nm) at this calcium level (Figure 5).
-
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~. (nm)
Figure 3 Comparison of the c.d. behaviour of alginic acid gels and films with that of sodium alginate at neutral pH. A, Sol(Na ÷); B, gel(H +); C, film(Na +); D, film(H +)
which are qualitatively similar to the changes observed on Ca 2+ gelation, but smaller in magnitude. Indeed, comparison of the sodium alginate film c.d. in Figure 4 with the spectrum obtained under hydrated conditions in the presence of 41'Y;, of the total stoichiometric Ca 2 + requirement [Figure 2) shows close similarity in position, widths and relative intensities of the bands at ~215, ~203 and 185 nm. In addition to these low energy transitions, two high energy bands, which are inaccessible in solutions and gels, are observed at ~ 170 and 150 nm in the c.d. spectra of both sodium and calcium alginate solid films (Figure 4). The relative magnitudes of these bands vary substantially with Ca 2 + level, as shown in Figure 4. Similar bands have been observed for iota carrageenan 11, agarose 6 and galactomannans 15, and are attributedto transitions of the polymer backbone. They are always observed to be of opposite sign, and have not yet been definitely assigned. It has been suggested, however, that they have t h e s a m e spectroscopic origin as the band seen in the vapour phase c.d. of tetrahydropyran ~''1i. i Quantitative analysis of alginate c.d. behaviour has previously been confined to the spectral region above 200 nm ~4. Similar computer curve-fitting in the vacuum ultraviolet region below 200 nm confirms that good quantitative agreement with the observed c.d. behaviour of both sodium and calcium alginate films in this spectral region can be obtained in terms of three Gaussian c.d. bands. These are all of width 10.2_+0.5 nm, and are centred at 149_+1, 169-t-1 and 184_+1 nm, respectively.
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!
A
x
% D
1
E 0
F
"~i" ~"
I
I 150
Ca z+ film
I
1
I
I
I 200
I
I
I
I
I 250
X(nm)
Figure 4 Cation-dependent spectral changes in the vacuum ultraviolet circular dichroism of algi-nate in the solid state. The agreement between observed ( ) and fitted (e) c.d. spectra is shown for Na + and Ca 2 + films. A similar quality of fit was obtained at intermediate Ca 2 + levels, which are shown as a percentage of the total cation requirement of the alginate: A, 0%; B, 14",,: C, 28",,: D, 56",,; E, 83%; F, 103%
Alginate circular dichroism: J. N. Liang et al.
x
0
%
-2
I 20
40
I
I
I
60
80
I00
Ca 2÷ (%)
Figure 5 Cation dependence of alginate v.u.c.d. The film spectra in Figure 4 may be fitted adequately by three bands below 200 nm, each of width 10.2 + 0.5 nm, and centred at 149 +1 m), 169+1 (A) and 184+1 (e) nm, respectively. The variation in intensity of these bands with Ca 2 ÷ level is as shown (Ca 2÷ concentrations expressed as a percentage of the total cation requirement ofthe alginate). Similar quantitative analysis of lower energy c.d. behaviour has already been reported elsewhere 14
data indicate stiff, extended chain geometry for polyguluronate in solution, suggesting only limited conformational mobility about the preferred solid state conformation, in contrast to polymannuronate and mixed chain sequences which show considerably greater flexibility 19. This is perhaps consistent with the observed cation dependence of the high energy transition at ~ 169 nm, which has previously been shown to provide a sensitive index of conformational changes during the sol-gel transition of agarose 6. Little variation in the magnitude of this band with Ca 2÷ level is observed up to the stoichiometric equivalent of the polyguluronate chain sequences (Figure 5), but at higher calcium concentrations there is a marked change in intensity, which may reflect conformational changes associated with Ca 2 ÷ chelation to mannuronate-containing chain sequences. If, as suggested by the above evidence, intermolecular association of polyguluronate involves little change in backbone geometry, then the observed spectral changes which accompany this process must have their origin in some other effect. We suggest that the most likely explanation lies in perturbation of chromophores by sitebound cations. Computer model-building studies, and visual inspection of physical models of polyguluronate chains in the preferred two-fold conformation, show 4 the existence of oxygen-lined pockets or 'nests' formed by O(1), (the glycosidic 'linking' oxygen), 0(2) and 0(3) of one sugar residue, and O(5), (the ring oxygen) and O(6), (one of the carboxy oxygens) of the adjacent residue in the reducing direction (see Figure 6). These 'nests' are periodically spaced along both sides of the chain, and their dimensions satisfy the criteria for Ca 2 ÷ chelation. From this it has been proposed that intermolecular association in alginate gels and in the solid state involves interchain chelation of extended arrays of site-bound cations (the 'eggrbox' model, shown schematically in Figure 6). The involvement of the ring oxygen and glycosidic oxygen of the polymer backbone in cation chelation may explain the sensitivity of the 149 nm band to ionic
dime,
(2) Subsequent association of these dimers into larger aggregates would involve further cation chelation, reaching a theoretical maximum of stoichiometric equivalence (calcium polyguluronate), in the present case at ~ 6 0 ~ of the total cation requirement for the entire chain. As shown in Figure 5, discontinuities in the Ca 2 ÷ dependence of both the high energy backbone transitions (at 149 and 169 nm) are, in fact, observed at this calcium level. (3) At higher Ca 2 ÷ levels some binding to polymannuronate and mixed chain sequences may occur. In solutions and gels this may be weak and non-specific, but in the solid state is likely to involve at least some degree of site binding, on loss of mobility of the participating chain sequences. X-ray fibre diffraction and i.r. dichroism studies ~7'18 have established an extended two-fold conformation for polyguluronic acid, which appears to persist in all salt forms so far investigated. Viscosity and light scattering
Random coil 2
o
co~ ~z
Aggregation of climers o
Figure 6 Schematic representation of cooperative intermolecular cation binding to alginate polyguluronate chain sequences
Int. J. Biol. Macromol., 1980, Vol 2, August
207
AI,qinate circular dichrosim: J. N. Lian,q et al. environment (see Fi,qure 5), although differences in the behaviour of this band and of the other backbone transition at 169 nm must remain the subject of further investigation. The positions and widths of these far vacuum u.v. bands are in excellent agreement with the values which we have recently obtained for the corresponding backbone transitions in the galactomannan family of plant polysaccharides 1s. The lower energy c.d. bands of alginate all involve transitions of the carboxy group, which is likely to experience the most severe perturbations from cation binding. Our results show no evidence of displacement of these bands into the vacuum u.v. region on cation binding, nor of exciton splitting, and thus the observed spectral changes must have their origin in changes in rotational strength. As indicated in the following scheme, four states of alginate may be considered, each having its own characteristic c.d. behaviour from the carboxy chromophore.
Solution (Na+)~Film (Na +) Gel (Ca2 +)+Film (Ca z+) The major change in c.d. is observed on going from solution to gel. Little further change occurs on drying down to the solid state (Ca 2+ film), consistent with previous evidence 2 that association of polyguluronate sequences in alginate gels approaches completion. The principal spectral change is inversion of sign of the n--, rt* band, with a change in position from ~ 215 nm to ~ 203 nm. Similar energy differences are observed in the n--,rt* transitions of uronate monomers, and have been interpreted in terms of rotational isomerism about the C(5)C(6) bond 3. It seems probable, therefore, that the spectral changes associated with Ca 2+ chelation by polyguluronate have their origin in locking of the carboxy group in a fixed orientation relative to the sugar ring, which is different from the preferred orientation in solution. The proximity of the divalent cation is likely to have a substantial effect on the asymmetric environment of the carboxy group, and hence on the rotational strength of the transition. It is perhaps significant, therefore, that the spectral changes on going from sodium alginate solution to the Na + film are closely similar in form to those observed with Ca 2+, but of approximately half the magnitude, particularly since Na + and Ca 2 + are almost isomorphous, and may therefore be capable of occupying the same binding sites, with little change in local geometry. Thus the major differences in their influence on c.d. behaviour (Figures 4 and 5) may arise from differences in ionic charge. In summary, therefore, we conclude that both the high energy transitions of the alginate backbone, and the low
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energy transitions of the carboxy groups show appreciable changes on intermolecular association, and that for polyguluronate sequences these changes arise principally from perturbations of chromophores by site-bound cations. Our results also indicate the possibility of conformation-sensitive c.d. changes for mannuronatecontaining chain sequences, and this is currently the subject of further research. The data described here support a model of alginate gel structure previously proposed, i.e. the work is put forth as confirming evidence for that model. It is possible that our results are compatible with some other model for the gel structure, but the data displayed in Figure 5 must be considered a very strong constraint on alternative models.
Acknowledgements Acknowledgement is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research. Partial support was also received from the US National Institute of Health through Grant G M 24862.
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