Polyanionic characteristics of purified sulphated homofucans from brown algae

Polyanionic characteristics of purified sulphated homofucans from brown algae Bernard Kloareg*+, Maurice Demartyt and Serge Mabeau* *Centre d" Etudes Ockanographiques et de Biologie marine, C N R S (LP 4601) 29211 Roscoff France +Laboratoire des kchanges cellulaires, CNRS, (LA 203), Facultk des Sciences, Universitk de HauteNormandie, 76130, Mont St. Aignan, France

(Received 6 January 1986) Sulphated .[ucans were fractionated fi'om d!fferent brown algae using quaternary ammonium detergents. Purified /?actions .#om dif.lbrent species had the same ~teneral chemk'al composition, that of a typk'al sulphated homofiwan, or .fiwoidan. The interaction of unit~alent and dit~alent counter-ions with fi~coidan was investigated hy t,iscometl 3' and polarimetry, and by measuring the counter-inn activities and conductance coefficients in salt.fi'ee solutions. The activity and conductance coefficients were Jbund to be independent ~?]"the ~olyanion concentration and approximately the same within each family of counter-ions. This indicates that cation binding in salt-fi'ee solutions ~?[".[iwoidan is purely electrostatic. The conductit~ity results were in good agreement with the Manning's condensation polyelectrolyte model. The density charge parameter was 2.08 and the mean length ~?I the monomeric unit was 4.5 ~. Both the reduced riscosity and the optical rotation were dependent on the t'alency ~?["the counter-ions used. Coupled with the data obtained jkom light-scattering measurements, (~'1~ = 1.7 100 and Ra= 1760 ~), our results su.qgest that fi~coMan in aqueous solution is an extended, flexible coil. Keywonts: Sulphated homofucans: fucoidans: charge density parameter: equivalent conductance: viscosity: lightscattering

Introduction The cell walls of marine brown algae differ from those of land plants by the prevalence of the mucilaginous matrix compared with the skeletal components and by the abundance of charged polysaccharides compared with neutral polysaccharides ~. In brown algae, cell walls are made of cellulose, alginates, sulphated fucans and protein 2'3. Alginates are linear copolymers of ,8 (1,4)-Dmannuronic acid and its C5 epimer, ~ (1,4)-k-guluronic acid. They consist of about 20 unit blocks of poly-,8-r)mannuronic acid, poly-~-k-guluronic acid and mixtures of both 4"5. Fucans are a continuous spectrum of fucose containing sulphated polysaccharides, arbitrarily divided into three main families: homofucans, xylofucoglycuronans and glycuronofucoglycans. Homofucans, or fucoidans, are primarily composed of ~ (1,2)-linked units of 4-sulphuryl-L-fucose, with branching or sulphate at position 3. They also contain small proportions of Dxylose, J)-galactose and i)-mannose &. Xylofucoglycuronans, or ascophyllans, consist of a polyuronide backbone, mainly poly ,8 [1,4)-i)-mannuronic acid, branched with 3-O-~)-xylosyl-L-fucose-4-sulphate 7. Glycuronofucoglycans consist largely of linear chains of I 1-4)-linked ~)-galactose branched at C5 with t,-fucosyl-3sulphate or, occasionally, a uronic acid. probably r)glucuronic acid s. The chemical structure and the physicochemical properties ofalginates have been extensively studied. The general architecture of the cell wall as well as the self:i: Present address: Department of Botany and Plant Pathology. Oregon State University, Corwdlis. Oregon 97331, USA. 0141 8130/'86/060380~-07503.00 © 1986 Butterworth & Co. (Publishers) Ltd

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lnt, J. Biol. Macromol., 1986, Vol 8, December

assembly of cellulose and alginate chains is now well understood 9't°. In contrast, only a few data about the biochemical organization of fucans and their relationship with the other wall components are available t t-~3. Yet, they suggest that sulphated fucans might play a key role in wall architecture, cross-linking cellulose and alginates (Figure 1). An approach to assess this model would need to investigate the physicochemical properties of purified sulphated fucans and their conformational interactions with alginates. Physicochemical studies of sulphated fucans also are necessary to understand which specific functions, if any, these strong polyanions play in the ionic regulation of marine algae t . An approach to test this possible function would need to determine the cation-binding behaviour of the polyanionic wall components and such polyelectrolytic characteristics as their linear charge density parameter ~'~4. The physicochemical properties of alginates and their ion-exchange behaviour are well known. Polymannuronic blocks bind counter-ions only on the basis of electroselectivity, while polyguluronic blocks strongly complex with calcium and certain other divalent cations, readily leading to aggregation and gelation15 18. From the mean charge separation of polymannuronate and polyguluronate blocks ~9, cation condensation could be expected in both sequences. Indeed, the charge density parameter of whole alginates, calculated from potentiometric measurements is ~ = 1.43 in the sodium form 2° and ~ = 1.80 in the calcium form ~7. This discrepancy is probably due to the fact that, in calcium polyguluronate blocks, the apparent charge density parameter is shifted towards a higher value than the intrinsic one as a result of the autocooperati~)e

Polyanionic characteristics of fucoidans: B. Kloareq et al. Experimental

Preparation and analysis of fucoidan samples

(~:-

Cellulose microfibrils Alginate network

f ~ I

Xylofucoglucans Xylofucoglycuronans

(~"

Homofucans

©

Glycoproteic linkages

Figure 1 Semi-speculative model of the structure of the cell walls of brown algae. The cellulose chains are organized in crystalline, parallel microfibrils which lay tangentially to the cell surface and cross each other at a few definite angles9 (set arbitrarily at 90° here). Among the microfibrils or bundles of microfibrils, extends a three-dimensional continuous alginate network, constituted by calcium bridged polyguluronate blocks and more or less entangled polymannuronate chains 1°. The fucose-containing polymers probably play a key role in crosslinking cellulose and alginate. Hypothetically, to the cellulose microfibrils are firmly bound xyiofucoglucans (or galactans) in the same way as hemicellulose to cellulose in higher plants t ~; to alginates are linked xylofucoglycuronans (ascophyllans) by conformational association between their polyglycuronic backbone and the highly ordered polyguluronate sequences~2; eventually, the linkage between alginate and cellulose would involve glycoprotein linkages between the side chains of ascophyllans and those of xylofucoglucans t3. Homofucans (fucoidans) are either or both free and part of an acid-labile supramolecular complex with ascophyllan ~3

chelation of calcium ions by these sequences 17'Is. By contrast with the abundance of data about alginates, the polyelectrolytic and conformational behaviour of sulphated fucans has not yet been investigated. We report here on the ion-binding properties of fucoidans from various brown algae through their potentiometric and conductimetric behaviour in salt-free solutions. Preliminary investigations were also conducted on some of their viscometry, polarimetry and light-scattering characteristics. This study was restricted to the suiphated homofucans, namely fucoidans, mainly from Pelretia canaliculata, in which species these fucans account for about 40% of the dry weight of the isolated cell walls 2~

Crude fucans were extracted from adult gametophytes of Pelvetia canaliculata, Fucus spiralis, F. vesiculosus, F. serratus, Ascophyllum nodosum, Bifurcaria bifurcata and Laminaria di#itata, as previously described 22. They were fractionated with quaternary ammonium detergents, Ncetyl-N-N-N-trimethyl ammonium bromide (CTAB)or N-cetylpyridinium chloride (CPC), then exhaustively dialysed and dried under reduced pressure at 40°C 22. In order to obtain the polyacid forms, aliquots were eluted through a Dowex AGW XS, H ÷, column. When necessary, any given salt was obtained by exact neutralization of the acid form using the corresponding hydroxide, or by dialysing against the appropriate chloride, then against distilled water. Because of the autodegradation of fucoidanic acid, the acid samples were utilized immediately or neutralized within a few hours after their elution from the Dowex H + column. L-Fucose was assayed by the cysteine-sulphuric acid method of Disches and Schettles 23. Half-ester sulphuryl groups, expressed as sulphate, were assayed with Ncetylpyridinium chloride at pH 1.5 according to Scott's procedure 24. The total polyanionic capacity was assayed by this same procedure but at pH 7.024 or by direct potentiometric or conductimetric titration of the polyanion H + form. Because of the high hygroscopicity of the purified fucoidans 22, the equivalent polyanion concentration, ne, calculated from titration, was usually preferred as reference concentration to the polymer concentration, C, which involved weight measurements. These two values were, however, found in good agreement. In view of a further assessment of the chemical purity, an aliquot (200 mg) of one of the samples (P. canaliculataIII) was eluted through a DEAE Sepharose CL-6B column (Pharmacia 1 6 m m x 3 0 0 m m ) with a linear gradient of sodium chloride (0-3 M, 1 litre, 2 ml min- ~) adjusted to pH 5.0 with N-chlorhydric acid. Each fraction (13 ml) was assayed for polyanionic sulphate according to the above described procedure. The polyanionic sulphate was eluted from the column within a single, broad pic corre~onding to a 1.2 M sodium chloride concentration. These fractions were pooled, exhaustively dialysed against distilled water and lyophilized. Aliquots (10mg) of the light cream lyophilizate were hydrolysed by 2 N trifluoracetic acid for 2 h at 100°C, then converted into alditol-acetates according to Sawardeker's procedure ~ and analysed by gas-liquid chromatography. Another aliquot (25rag) was methylated twice through Hakomori's 26"27 procedure for 4h. The methylated sample was hydrolysed at 10OC by 90"0 (v/v) formic acid then 2 N trifluoracetic acid for 1 h and 3 h respectively. The methylated sugars were then analysed by the alditolacetates procedure, using a gas-liquid chromatograph connected with a mass spectrograph. An aliquot of the lyophilized sample was also desulphated according to Rees' alkaline procedure 28.

Physicochemical measurements Specific rotations of aqueous solutions of fucoidans were measured at 589 nm at 2 2 C using a Perkin Elmer type 241 polarimeter with a l 0-cm optical pathlength cell. The increment index dn/dc of the CTAB purified

Int. J. Biol. Macromoi., 1986, Vol 8, December

381

Polyanionic characteristics of fi~coidans: B. KIoare9 et al. fucoidan from Pelvetia canaliculata in 0.1M NaC1 (3 g 1-- t < C < 20 g 1-1) was measured at 25°C using a Brice Phoenix differential refractometer. Its weight average molecular weight, A4w, was estimated from lightscattering measurements in 0.1 M NaC1 using a 546nm monochromatic light and incidence angles ranging from 37.5 to 135°. Before the measurements, the solutions were centrifuged for 3 h at 20 000 9. The viscosities of salt-free solutions of fucoidans were measured at 25°C using an automatic viscosimeter Fica MS with a capillary the flow time of which for water, was l12s. During potentiometric and conductimetric measurements, gaseous nitrogen was bubbled through the solutions. The activity coefficient of protons was directly determined by pH measurements at 25°C with a Tacussel TBC 112 HS pH combined electrode. The activity coefficients of sodium and calcium in salt-free fucoidan solutions were determined by a pseudo-zero method, with specific electrodes Orion 94-11 (Na +) or 93-20 (Ca 2+) and a double junction reference electrode Orion 90-02-00. Immediately after the measurement of the potential difference in each fucoidan solution, the specific electrode was calibrated with sodium or calcium chloride by progressive dilution until reaching that same potential difference as with the polyanionic salt. Conductimetry measurements were performed at 25°C with a Tacussel CN 78 conductimeter using a CM-01-G cell at 1 kHz. Assuming that at this frequency the conduction along the polyanion was nil, the equivalent conductance of salt-free fucoidan solutions was given by equation (1): ~L= A,= f()~p + )~,)

(1)

II e

where Z~is the measured conductance of fucoidan in the iionic form, f the conductance coefficient of the counterion species and )~p and )~ the equivalent conductance of the polyanion and the counter-ions, respectively 29.

Assuming that fucoidan has no selectivity other than electroselectivity towards protons and sodium ions and from Manning's polyelectrolyte theory 29, equation (1/ was derived to give equation (2): AH+ _ )w + )~H+ ANa+ }~p-~ )~Na +

(2 }

)~ was taken to equal the zero concentration extrapolated equivalent conductance, )0 (Ref. 30). This allowed the calculation of )~p, then the estimation of the conductance coefficients of each of the counter-ions under study.

Results The purified fucans were very hygroscopic 2~. Their general chemical composition was, however, fairly homogeneous (Table 1). Whatever the specific source or the repetition, sulphate groups accounted for about 3540~o of the dry weight. The proportion of fucosyl residues ranged from 35 to 44 %. Consequently, the sulphate-tofucose molar ratio was quite constant, lying between 1.33 and 1.51 (Table 1). The proportion of total polyanionic groups, assayed with CPC at pH 7.0, was higher but very similar to the proportion of sulphate groups (Table 1). After chromatography and lyophilization, the fucose and sulphate contents of P. canaliculata-lI1 were 44 and 48 ~o respectively. After the alkaline desulphation, the sulphate content was still 25°/~,. On the alditol-acetates analysis, the carbohydrate composition of P. canaliculata-IlI was Lfucose, D-galactose, D-xylose, D-mannose and D-glucose in the molar ratio of 84:7:6:2:1. Because of the overpreponderance of fucose in the polysaccharide, only the derivatives of fucose could be taken in account among the methylated monosaecharides. The molar proportions of the methyl-fucosides were in percentages: unmethylated fucose, 3-O-methyl-fucose, 4-O-methyl-fucose, 2,4-di-O-methyl-fucose, 2,3-di-O-methyl-fucose, 3,4-di-O-methyl-fucose and 2,3,4-tri-O-methyl-fucose: 13:20:21:20:12:4:10.

Table 1 General chemical composition of purified fucoidans from brown algae (in % of dry weightl % Dry weight OSO3 Fucose

OSO~ Fucose

Sample

a

b

c

d

Pelvetia canaliculata-I P. canaliculata-ll P. canaliculata-Ill P. canaliculata-CTAB Fucus spiralis F. vesiculosus Ascophyllum nodosum F: serratus Bifurcaria rotunda Laminaria digitata

36.8 37.1 38.9 40.1 35.1 36.9 33.7 31.9 35.5 38.9

ND ND 38.9 ND 35.5 37.6 34.8 35.3 36.2 38.8

35.6 34.9 40.7 39.3 37.0 41.8 38.7 34.7 37.7 44.2

1.57 1.62 1.45 1.55 1.45 1.34 1.33 1.40 1.43 1.33

All the samples were purified using C PC, except P. canaliculata-C TAB. P. canaliculata-I, P. canaliculata-II and purified samples Sulphate groups assayed with CPC at pH 1.5 b Total polyanionic groups, expressed as sulphate, assayed with CPC at pH 7.0 c Fucosyl residues, determined by the cysteine-sulphuric acid method d Sulphate-to-fucose molar ratio. ND = Not determined.

382

Int. J. Biol. Macromol., 1986, Vol 8, December

P. canaliculata-lll wereindependent

Polyanionic characteristics of fucoidans: B. Kloare 0 et al. The specific rotations of aqueous solutions of fucoidans with different counter-ions are given in Table 2. They did not significantly change with the sample or the concentration but significant differences occurred according to the counter-ion valence. The optical rotation for the calcium form was - 143 ° while the N a + and H + forms yielded 118 ° and - 1 2 1 °, respectively

(Table 2).

Effect of the counter-ions on optical rotation of purified fucoidans from Pelvetia canaliculata

Table 2

Sample

[~t]

C (mg ml- 1 )

- 122° - 121 ° - 139°

1.0 3.9 0.96

- 121 ° - 116° - 146°

3.0 4.6 6.0

Pelvetia canaliculata4 H+ Na + Ca 2 +

Pelvetia canaliculata-ll H+ Na + Ca 2 +

Pelvetia canaliculata-CTAB Na + Ca 2 +

- 118° - 143°

I

_

I

I

131ppm

/

./

I

393 ppm

. -

5.2 3.8

,

./

628 pprn

./

./

,-

/,

-/--j I

I

l

I

1

2

3

4

The increment index dn/dc of the P. canaliculata.CTAB sample was 0.123g1-1. Results from light-scattering measurements were analysed using Zimm's plot 3t (Figure 2). The weight average molecular weight and the gyration radius were estimated to be M w = l . 7 x l 0 6 and / ~ = 1760 A, respectively. The slope of the zero incidence extrapolated line (C/1)O = 0 = f ( s i n 20/2 + kc) was slightly negative (Figure 2). The activity coefficient of protons in fucoidanic acid solutions, yH÷, was independent of the polyanion equivalent concentration, at least in the range studied, i.e. between 1 and 125 m E q 1-1. It varied with the samples between 0.4 and 0.6 (Table 3). The activity coefficients of sodium (YNa""" 0.47) and calcium (yc,,+ - 0.25) in the salt forms of fucoidans were also independent of the polyanion equivalent concentration (1 mEq 1- t < no< 10mEq 1-1) (Table 3). Whatever the counter-ion used, the conductance of fucoidan solutions varied linearly with respect to the polyanion concentration (1 m E q 1- ~< n~< 500 m E q 1-1 ). Equation (2) was tested with the measured conductances of the P. canaliculata-I sample in its acid and sodium forms, resulting in a value of 48.1 f~- 1 cm 2 E q - ~ for 2p. F r o m this value and equation (2), the conductance coefficients of various counter-ions in salt-free solutions of the P. canaliculata-I sample were calculated (Table 4). The conductance coefficients of the univalent counterions were very similar, about 0.46. Those of divalent counter-ions were about the half of this value (Table 4). Extended to the other samples, this procedure gave similar results, i.e. the 2p was about 50 f~- t c m 2 E q - ~ for all fucoidans (Table 5). However, the conductance coefficient of calcium was below half that of the univalent counter-ions. Reduced viscosity in salt-free solutions of fucoidans was dependent upon the polymer concentration and on the counter-ions (Figure 3). The reduced viscosity increased as the polymer concentration decreased, and at a given concentration, the reduced viscosity was significantly lower for the calcium salt (Figure 3). Discussion

sin 2 0/2 + kC

Figure 2 Zimm's plot of the light-scattering measurements from Pelvetia canaliculata-CTAB. The sample was dissolved in 0.1 M NaCI

The comparison between the proportion of total polyanionic groups and the proportion of sulphate groups (Table 1) shows that the purified fucans were virtually free from carboxylic groups, which would have been ionized at pH 7.0 but not at pH 1.524 . The

Table 3 Activity coefficients of counter-ions in salt-free solutions of purified fucoidans from brown algae Sample

Pelvetia canaliculata4 P. canaliculata-ll P. canaliculata4ll P. canaliculata-CTAB Fucus spiralis F. vesiculosus Ascophyllura nodosum F. serratus _Bifurcaria rotunda Laminaria digitata

7H+

"]Na+

;'Ca-'÷

0.52 0.61 0.47 0.50 0.46 0.53 0.44 0.50 0.49 0.48

0.47 0.52 ND 0.41 ND ND ND ND ND ND

0.25 0.28 ND 0.21 ND ND ND ND ND ND

(no= 10 mEq 1-1, T--- 25~'C) N D = N o t determined

Int. J. Biol. Macromol., 1986, Voi 8, December

383

Polyanionic characteristics

o/" jucoidans: B. Kloareg et al.

carbohydrate composition of the P. canaliculata4ll sample was very similar to that previously shown for the P. canaliculata-I, P. canaliculata-II and P. canaliculataC T A B samples 22. About half the sulphate groups of that sample were stable upon alkaline desulphation, thus located at C 4 on the fucopyranose ring 32. This is consistent with the i.r. spectra of the P. canaliculata-IlI and P. canaliculata-CTAB samples between 800 and 900cm -~ (Ref. 22). Considering these results and the sulphate to fucose molar ratio of the P. canaliculata-lll sample (Table 1), the methylation analysis is consistent with a.highly branched fucan with 1-2, 1-3 and 1-4 fucosidic linkages and sulphate groups primarily at C4 but also at C2 and C3. This structure is a good agreement with that described in previous studies of fucoidans 32 34. The specific rotation of our samples (Table 2) was very similar to that of other investigators on purified fucoidans obtained from Fucus vesiculosus (Ca 2+, - 1 4 0 ° ) 35, Pelvetia wrightii (Mg 2 +, - 141 :,)30, Ascophyllum nodosum (Mg 2 +, - 144°) 37, Himanthalia lorea (Ca 2 +, - 14if} 3° and Lessonia flavk'ans (K ÷, - 1 1 3 ' ) 38. They indicate ~linkages, which is consistent with the ~3C n.m.r, spectra of the P. canaliculata-I sample 22. From both the chemical analysis and the polarimetry results, we thus concluded that our samples were typical fucoidans, with sufficient purity to allow further physicochemical analyses.

From Fiyure 2, the second virial coefficient was slightly negative, which is quite unlikely for a very hygroscopic polysaccharide such as fucoidan. This suggests that the solutions were not perfectly freed from microparticles. However, this probably did not critically affect the molecular weight and the gyration radius estimations. Such dimensions as M,~= 1.7 10~' and R g = 1760A correspond to an extended polyion and suggest that fucoidan, though a highly branched polymer, adopts an extended coil conformation in aqueous solutions. This allows us to interpret the potentiometry and conductimetry results using the models developed for largely stretched polyions.

c(g1-1 ) 2.5 J

5.0 I

Na + •

Table 4 Measured conductance and calculated conductance coefficients of salt-free solutions of Pelvetia canaliculata-I (ne = 3.37 mEq I- 7, T= 25°C). From fN~• and fn ÷ and equation (2), 20 was 48.1 f~-l cm 2 Eq-1 Counter-ion species

zi (mf~- l cm- 2 )

ii

H+

0.640

0.48

Alkaline Rb + Cs + K+ Na ~ Li *

0.204 0.200 0.190 0.158 0.148

0.48 0.47 0.46 0.48 0.48

(CH3) 3 N +

0.160

0.51

(CH 3 CH2) 3 N + [CH3 CH3 CH2) N +

0.145 0.116

0.53 0.48

0.098 0.089 0.084

0.27 0.24 0.23

H + [] 2.0

Ca2+ 0

A

7

1.0

Quaternary ammoniums k~'~D~O~'o~<~o___o_ o

Earth-alkaline Sr 2 + Ca 2 * Ba 2 +

Table 5

Figure 3 Dependence on the concentration and the counterions of the reduced viscosity of salt-free aqueous solutions of Pelvetia canaliculata-I

Polyanion equivalent conductances and conductance coefficients in salt-free solutions of purified fucoidans from brown algae

Sample

)op~ - t cm 2 Eq - I

.]~4+, . [ - ,

.i~<+

fc,2 -

Pelvetia canaliculata-lll Fucus spiralis F. vesiculosus Ascophyllum nodosum F. serratus B([hrcaria rotunda Laminaria di.qitata

49.5 52.0 48.5 49.7 55.4 48.6 48.4

0.41 0.39 0.37 0.39 0.43 0.40 0.54

0.38 0.38 0.36 0.39 0.44 0.39 0.49

0.15 0.14 0.12 0.13 0.19 0.16 0.15

384

Int. J. Biol. Macromol., 1986, Vol 8, December

Polyanionic characteristics of fucoidans: B. Kloareg et al. Most ionic properties of polyelectrolytes are driven by their linear density charge parameter, defined as (3) =

e2 ~ ek T

=7.13

R ff

at 25°C

(3)

where e is the electronic charge, IV the mean number of ionized fixed groups by monomeric unit, e the dielectric constant, k the Boltzmann's constant, T the absolute temperature and b the mean length (A) of the monomeric unit, taken along the mean axis of the polyion 27. It has been demonstrated that, in salt-free polyelectrolyte solutions, the counter-ions do 'condense' onto the polyion line as long as the net value of the charge density parameter is higher than the opposite of the counter-ions valency. The activity coefficient of counter-ions is then expressed as (4): 1n7~=-0.5

-lnlz~[~,

i f ~ > z i -1

(4)

where z~ is the algebric valency of counter-ions 29. From this equation and the value found for 7H+ and 7N,+ (Table 3), fucoidan does not exhibit any selectivity between H + and Na +. Moreover, since the activity coefficient of calcium ions is roughly half of those of protons or sodium ions, the interactions between fucoidans and cations seem to be purely electrostatic. This result agrees with the previous conclusions of equilibrium dialysis experiments 39 41. The introduction of the 7H+ values into equation (4) allows the estimation of the charge density parameter. By using the sulphate to fucose molar ratios from Table I and assuming that fucose accounts for 9 0 ~ of total carbohydrates, it is possible from equation (3) to estimate the parameter b. The results are given in Table 6. The mean value for ~ is 1.22 and the corresponding value of b is 7.8/~,. To our knowledge, there are no data about the monomeric dimensions of fucans, but, by reference to other polysaccharides ~2, this result is not consistent with the expected length of a fucose unit even in an extended ~t (l,2)-L-fucan. Our calculation procedure to estimate the equivalent conductance of fucoidan assumed from the potentiometric evidence of pure electroselectivity between H + and Na + that fH+=fN~+. The internal

consistence of the conductance coefficients for the P. canaliculata-I sample within each family of cations (Table 4), and the good agreement of the 2p values among the different fucoidans (Table 5) also support this hypothesis. The fact that the conductance coefficients of univalent counter-ions are approximately equal among all samples also provides additional evidence of a purely electrostatic binding phenomenon, at least within the univalent counter-ions. F r o m Manning's model, the conductance coefficients of univalent counter-ions are related to the charge density parameter by equation (5) f = 0 . 8 7 ~ -1,

if ~>1 (Ref. 29)

(5)

In the same way as above, ~ and the monomeric length parameter b were calculated by introducing the experimental values offH+ in equation (5) (Table 6). The mean value of the density charge parameter was ~ = 2.08, corresponding to a monomeric length of b = 4.5 A. This length is more consistent with what is expected for an (1,2)-L-fucan in an extended conformation. The changes in optical rotation of fucoidans when a monovalent ion was replaced by a divalent one (Table 2) probably indicate some modifications in the macromolecular conformation, due to electrostatic interactions. It is well known that the optical activity of polysaccharides not only depends on the carbohydrate sequence and linkages, but also upon the overall conformation of the macromolecule 42. The amplitude or the direction of changes shown in Table 2 is, however, difficult to interpret in relation to the counter-ion used. Such modifications could also be shown by the dependence of the reduced viscosity of fucoidan on the counter-ion valency (Fioure 3), probably because of differences of coil expansion due to intramolecular Coulomb repulsion 43. Such an effect might also explain why in Table 5 the conductance coefficients of calcium are below half the conductance coefficients of the univalent ions. In the calcium form, fucoidan might be allowed to undergo some local folding. This would not destroy the overall chain geometry but decrease the contour length of the polyelectrolyte chain and significantly lower the b value from that of the more extended acid or alkaline fucoidans.

Table 6

Charge density and monomeric length parameters of purified fucoidans from brown algae, according to the Manning's condensation polyelectrolyte model b (A) Sample

.N

,

h

,,

h

Pelvetia canaliculata-I P. canaliculata-ll P. canaliculata-Ill P. canaliculata-CTAB Fucus spiralis F. vesiculosus Ascophyllum nodosum F. serratus Bi.furcaria rotunda Laminaria digitata

1.41

1. i 6

1.89

8.7

1.46 .3 i .40 .31 .23 .23 .38 .31

1.00 1.29 1.21 1.32 1.14 1.38 1.21 1.24

ND 2.12 ND 2.23 2.35 2.23 2.02 2.18

1.20

1.26

1.61

10.4 7.2 8.2 7.1 7.7 6.4 8.2 7.5 6.8

5.3 ND 4.4 ND 4.2 3.7 3.9 4.9 4.3 5.3

" Calculated from 7H+ values h Calculated from )'h * values N, the averagenumberof anionic group per monomerwas calculated using the data of Table I and taking the proportion of fucosylresiduesas 90",, of the total glycosylresidues

Int. J. Biol. Macromol., 1986, Vol 8, December

3115

Polyanionic characteristics of jucoidans." B. Kloareg et al. However, this was not shown in Table 3 or in Table 4. Thus, this interpretation clearly needs further investigation.

Conclusion Fucoidans, the highly sulphated homofucan fraction of brown algal wall mucilages, are high molecular weight polymers. Light-scattering, viscosity and polarimetry measurements suggest that they behave as extended but flexible coils in aqueous solutions. Further investigation, however, is needed to assess this preliminary result and to determine if fucoidan can adopt more ordered conformations in the presence of added salt or when associated with calcium alginate, the other major charged cell wall polysaccharide of brown algae. From the conductance coefficients of salt-free solutions and according to Manning's condensation polyelectrolyte model, the charge density parameter of fucoidans is about 2.08. Since the dissociation characteristics of fucoidans were not affected by polyanion concentration, this result probably remains true even at very high concentrations such as those found in the cell walls of some brown algae 3. Considering that, in the presence of calcium, the charge density parameter of alginates is 1.80, the charge density parameter of the whole wall is probably about 1.8 2.0. Strong cation condensation is thus expected in cell walls of brown algae equilibrated against seawater or other electrolyte solutions. The interactions between fucoidans and counter-ions are purely electrostatic, at least within each family of the univalent or the divalent cations. In other words, fucoidans do not exhibit any significant selectivity between the main cations of seawater except the electroselectivity, while alginates strongly bind protons, and, to a lesser degree, calcium 39. Considering that the cell walls of brown algae account for nearly half the thallus dry weight 3 and that their alginate/fucan ratio is highly correlated with the species zonation on the shore 4°, these major differences of cation exchange behaviour between fucans and alginates suggest that both polysaccharides have a specific function in relation with the intertidal environment 4°. In order to assess this hypothesis, we are investigating the cation exchange properties of isolated cell walls from various brown algae.

Acknowledgements This work was partly supported by funds from the PIRSEM (ATP 'Connaissance des Algues'). The structural analysis of the purified fucoidan was carried out during a residence of one of us (S.M.) at the CERMAV, Grenoble, France and we are grateful to Dr J. P. Joseleau for his collaboration in this study. We also wish to express our gratitude to Dr D. Kropfand Dr R. S. Quatrano (Oregon State University) and to Dr J. Schellman (University of Oregon) for critical reading of the manuscript.

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Symbols Charge density parameter Algebraic valency of counter-ions Equivalent polyanion concentration I1e Measured conductance of fucoidan solutions Zi Equivalent conductance of fucoidan solutions mi 2p, 2 i Equivalent conductance of fucoidan and counterions, respectively Conductance coefficient of counter-ions Activity coefficient of counter-ions 7, N Mean number of ionized groups per monomeric unit Mean length (A) of monomeric unit Zi