Journal of

Structural

Journal of Structural Biology 145 (2004) 216–225

Biology

www.elsevier.com/locate/yjsbi

Ultrastructure of acidic polysaccharides from the cell wallsof brown algae

Leonardo R. Andrade,a Leonardo T. Salgado,a Marcos Farina,a Mariana S. Pereira,b

Paulo A.S. Mour~ao,b and Gilberto M. Amado Filhoc,*

a Laborat�orio de Biomineralizac�~ao, Departamento de Histologia e Embriologia, Instituto de Ciencias Biom�edicas, CCS,Universidade Federal do Rio de Janeiro, Cidade Universit�aria, Rio de Janeiro, RJ 21941-590, Brazil

b Laborat�orio de Tecido Conjuntivo, Hospital Universit�ario Clementino Fraga Filho e Departamento de Bioqu�ımica M�edica,Instituto de Ciencias Biom�edicas, CCS, Cidade Universit�aria, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil

c Programa Zona Costeira, Instituto de Pesquisas Jardim Botanico/MMA, Rua Pacheco Le~ao 915, Jardim Botanico, Rio de Janeiro,

RJ 22460-030, Brazil

Received 10 June 2003, and in revised form 2 October 2003

Abstract

We have studied the ultrastructure of acidic polysaccharides from the cell walls of brown algae using a variety of electron mi-

croscopy techniques. Polysaccharides from Padina gymnospora present self assembled structures, forming trabecular patterns.

Purified fractions constituted by alginic acid and sulfated fucan also form well-organized ultrastructures, but the pattern of orga-

nization varies depending on the polysaccharide species. Alginic acid presents sponge-like structures. Sulfated fucan exhibits par-

ticles with polygonal forms with a polycrystalline structure. These particles are in fact constituted by sulfated fucan molecules since

they are recognized by a lectin specific for a-LL-fucosyl residues. X-ray microanalysis reveal that S is a constituent element, as ex-

pected for sulfated groups. Finally, an exhaustive purified sulfated fucan shows the same ultrastructure formed by polygonal forms.

Furthermore, elemental analyses of acidic polysaccharides indicate that they retain Zn, when algae were collected from a con-

taminated area. This observation is supported by direct quantification of heavy metal in the biomass and also in the solubilized

polysaccharides compared with the algae from a non-contaminated site. We conclude that these molecules have specific ultra-

structure and elemental composition; and act as metal binder for the nucleation and precipitation of heavy metals when the algae are

exposed to a metal contaminated environment.

� 2003 Elsevier Inc. All rights reserved.

Keywords: Alginic acid; Biomineralization; Brown algae; EDXA; Field emission scanning electron microscopy; Fucoidan; Heavy metals; Sulfated

fucan; Polysaccharides; TEM; Ultrastructure; Zinc

1. Introduction

Cell walls of brown algae have a fibrillar compart-

ment formed mainly of cellulose microfibrils, which is

embedded in an amorphous matrix of acid polysaccha-

rides linked each other by proteins (Kloareg et al.,1986). The acid polysaccharides are mainly composed of

alginic acids and sulfated fucans (also known as fucoi-

dan). The alginic acids are linear carboxylated copoly-

mers constituted by different proportions of 1,4-linked

* Corresponding author. Fax: +55-21-22942295.

E-mail address: gfilho@jbrj.gov.br (G.M. Amado Filho).

1047-8477/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.jsb.2003.11.011

b-DD-mannuronic acid (M-block) and a-LL-guluronic acid(G-block) (Haug et al., 1966). Algal sulfated fucans are

polymers mainly formed by sulfated a-LL-fucose but alsocontaining small proportions of other sugars such as

mannose and galactose (Dietrich et al., 1995; Kloareg

et al., 1986; Pereira et al., 1999, 2002).The chemical structure, physico-chemical properties,

and biological activities of alginic acids and sulfated

fucans have been widely studied, but the ultrastructure

of these molecules still unknown, especially in the case

of the sulfated fucans. The characterization of polysac-

charide morphology can contribute for the under-

standing of the cell wall architecture in the marine algae.

The few available data about the morphology of these

L.R. Andrade et al. / Journal of Structural Biology 145 (2004) 216–225 217

polysaccharides are generally related to alginic acidbeads as scaffold to cell culture in tissue engineering;

carriers for immobilized proteins and polymers used for

the controlled release of drugs (Chung et al., 2002;

Magnani et al., 2000; Zhou and Zhang, 2001).

Acidic polysaccharides found in the cell wall of

brown algae have also been associated with the capacity

of these organisms to accumulate heavy metals when

exposed to an environment containing high concentra-tions of these elements (Amado Filho et al., 1999;

Andrade et al., 2002; Lignell et al., 1982; Pellegrini et al.,

1993). The negatively charged polysaccharides from the

mucilaginous matrix can adsorb and partially exclude

mobile anions, and consequently act as cation-ex-

changer and ionic barrier, selecting the elements that

will be absorbed by the algal cells (Kloareg et al., 1986;

Mariani et al., 1990; Percival, 1979).Different seaweeds from a heavy metal contaminated

area were analyzed by atomic absorption spectropho-

tometry (AAS) and the results indicated that the brown

alga Padina gymnospora presented the highest concen-

trations of Zn and Cd among the species tested (Amado

Filho et al., 1999). Analytical electron microscopy

showed that cell walls located in median regions of

thallus presented electron-dense granules composedmainly by Zn and S. Cd was not detected because of its

low concentration in the samples. According to this re-

sult, it was proposed that the main mechanism presented

by P. gymnospora to accumulate high heavy metal

concentrations from contaminated areas is related to the

precipitation of these elements into the cell walls, di-

minishing the availability of metals to cell absorption

(Amado Filho et al., 1996, 1999).In addition, adult individuals of P. gymnospora ex-

posed to sub-lethal concentrations of Cd and/or Zn in

vitro presented numerous crystalline granules along the

cell walls, formed mainly by Cd and/or Zn, which co-

localized with S and O (Andrade et al., 2002). This ob-

servation suggested that the acidic polysaccharides

(mostly sulfated fucans) play an important role in nu-

cleation of Cd and Zn. Perhaps the acid micro-envi-ronment assured by these polysaccharides in the cell

walls propitiates an increase of crystalline granules like a

biomineralization process, and consequently turning the

algae more tolerant to metals (Andrade et al., 2002).

In this work we have studied the ultrastructure of

alginic acid and sulfated fucan from the cell wall of

brown algae using different electron microscopy tech-

niques. Both polysaccharides presented a self-assem-bled, well-organized ultrastructure but the pattern of

organization varies depending on the polysaccharide

species. Alginic acid showed a sponge-like structure

whereas sulfated fucans presented particles with polyg-

onal forms. Furthermore, elemental analysis of these

ultrastructures indicates they retain heavy metals when

the algae were exposed to a contaminated area.

2. Materials and methods

2.1. Polysaccharides from P. gymnospora

Adult individuals of P. gymnospora were collected in

Sepetiba Bay, a heavy metal (mostly Zn and Cd) con-

taminated area located in Rio de Janeiro State. Algae

were also collected in an adjacent non-contaminated

area, Ribeira Bay, for comparative analysis. Fresh algaewere cleaned from epiphytes with a thin brush under a

stereoscopic microscope, briefly washed in distilled wa-

ter and dried at 50 �C. The glasses used in polysaccha-

ride extraction were previously washed in a detergent

solution (1% Extran) and an acid solution (2% HNO3).

The polysaccharides were extracted from 4 g (dry

weight) of biomass in 250ml of ultra-pure water, under

agitation for 24 h at room temperature. The sampleswere centrifuged and the supernatants with soluble

components were precipitated with 70% ethanol. The

precipitates were removed by centrifugation, dissolved,

and dialyzed against distilled water for 24 h and freeze-

dried. The powder obtained is denominated as total

polysaccharides and used for ultrastructural observation

and elemental composition studies.

Alternatively, the polysaccharides were also extractedfrom the algae with 5% KOH solution (w/v) under agi-

tation for 24 h at room temperature. The solution was

centrifuged, and the polysaccharides precipitated from

the clear supernatant with 70% ethanol (v/v) and freeze-

dried. The powder was then dissolved in distilled water

and 4M CaCl2 was added to the solution in order to

precipitate the alginates. The sample was then centri-

fuged and both, the precipitated and supernatant con-taining alginic acid and sulfated fucan, respectively,

were dialyzed against distilled water for 24 h and freeze-

dried. The powders of fractioned polysaccharides (al-

ginic acids and sulfated fucans) were used for electron

microscopy studies.

2.2. Sulfated fucan from Laminaria brasiliensis

Polysaccharides were extracted from the cell wall of

the brown algae L. brasiliensis by papain digestion and

the sulfated fucan was purified by anion exchange

chromatography, as described (Pereira et al., 1999). The

purity of the sulfated fucan preparation was checked by

agarose gel electrophoresis and chemical analysis.

2.3. Transmission electron microscopy

For transmission electron microscopy (TEM) pur-

poses, the total and fractionated polysaccharides were

dissolved (5mg/ml) in ultra-pure water and 10 ll weretransferred to a Formvar film coated copper grids. The

polysaccharidic solutions were dried and the samples

were observed in a Jeol 1200 EX, operated at 80 kV.

218 L.R. Andrade et al. / Journal of Structural Biology 145 (2004) 216–225

2.4. Analytical TEM

The elemental composition of the extracted polysac-

charides was determined by energy dispersive X-ray

analysis (EDXA). The samples were prepared for TEM

as described above, and analyzed in a Jeol 1200 EX

equipped with a Noran–Voyager analytical system.

A focused spot (d � 100 nm) was used to analyze the

areas of interest in the polysaccharide samples. The ar-eas probed were representative of the whole sample (we

obtained several spectra from the individual particles).

Micrographs were obtained from untilted samples (be-

fore analysis). During EDXA the samples showed in this

work were stable under the electron beam.

The original specimen-retainer was changed for a

graphite one. Typical acquisition data were: accelerating

voltage¼ 80 kV, live-time¼ 300 s, dead-timeffi 20%, andsample tilt angle¼ 30�.

2.5. Field emission scanning electron microscopy

The extracted powder of the total polysaccharides

and sulfated fucan and alginic acid were directly de-

posited on a carbon tape coated aluminum stubs, and

sputtered with gold (Balzers/Union FL-9496) for 1min.High resolution field emission scanning electron mi-

croscopy (FESEM) was performed in a Jeol 6340F,

operated at 5 kV. A working distance of 8mm and slow

scan mode were used for imaging.

2.6. Labeling with lectins

The gold-labeled lectins (10 nm) from Ulex europaeus

(UEA), which recognizes specifically a-LL-fucosyl units,and Concanavalin A (Con A), specific to terminal resi-

dues of a-DD-mannosyl and a-DD-glucosyl were used for

identification of carbohydrate structures in the ultra-

structures of the molecules isolated from the brown al-

gae cell wall. The powders of the isolated molecules were

directly embedded in LRGold resin at )20 �C for 2 days

and polymerized under UV radiation at )20 �C for 3days. Ultra-thin sections were obtained in an ultrami-

crotome (Reichert Super-Nova) using a diamond knife

(Polyscience) and then collected on nickel grids (300

mesh).

The ultra-thin sections were sequentially immersed in

0.1M phosphate buffer saline (PBS) for 10min, 3 and

1% bovine serum albumin (BSA) in PBS for 10min

each, and finally incubated with UEA or Con A over-night at 4 �C. Different concentrations of the lectins were

tested in order to determine the appropriated dilution.

The grids were washed in BSA, PBS, and distilled water.

The pH of solutions was previously adjusted to 6.3 and 8

for labeling with UEA and Con A, respectively. The

sections were stained with 2% uranyl acetate for 20min.

As a control, sections were incubated with pure lectins

but without the incubation procedures described above.The grids were observed in a Zeiss 900 EM operated at

80 kV.

2.7. Heavy metals quantification

Concentrations of Zn and Cd in the algae biomass

collected from a heavy metal contaminated area and an

adjacent non-contaminated area and of their freeze-dried polysaccharides were determined using 250mg

samples (triplicates) digested with 5ml of 65% HNO3

(Merck PA) in closed teflon flasks maintained in a mi-

crowave oven (CEM-MDS-2000) for 30min. The di-

gested solutions were evaporated in a hot plate and

thereafter re-dissolved in 10ml of 0.1N HCl. Metal

concentrations were measured by flame atomic absorp-

tion spectrophotometry (Varian AA-1475). The resultswere expressed as lg g�1 of dry weight. Analytical pro-

cedures were tested by comparative analysis of Inter-

national Atomic Energy Agency (IAEA) certified

reference material IAEA-140 (seaweed homogenate,

Fucus). The results obtained were within the confidence

limit (significance level a < 0:05).

3. Results

3.1. Polysaccharides extracted from the brown algae cell

wall present self-assembled structures with trabecular

pattern

Approximately 12 and 23% of the dry biomass of the

alga P. gymnospora were solubilized by extraction withdistilled water and 5% KOH solution, respectively. The

powder of solubilized polysaccharides varied from light

brown (water extraction) to dark brown color (KOH

extraction). TEM observations of the total polysaccha-

rides showed numerous stick-like structures joined to

each other (Fig. 1A). Electron diffraction images ob-

tained from this sample presented diffraction patterns

compatible with a polycrystalline material (inset inFig. 1A). FESEM of these polysaccharides showed self-

assembled structures, forming different lamellar or tra-

becular patterns (Figs. 1B–D). Ca and S were associated

with these well-organized structures as indicated by the

EDXA spectra (Figs. 1E and F). This confirm that al-

ginic acid and sulfated fucan are found in these trabec-

ular structures since calcium ions strongly bind to the

carboxyl groups of alginic acid while S is a constituentof sulfate groups found in the algal fucans.

3.2. Alginic acid and sulfated fucan differ in the pattern of

their ultrastructures

Alginic acid was separated from sulfated fucan in the

acidic polysaccharide mixture based on its property to

Fig. 1. Electron microscopy and EDXA of the acidic polysaccharides from the brown alga P. gymnospora. (A) TEM observation of the total

polysaccharides extracted from algae collected in SB presenting numerous stick-like structures joined to each other. The inset image is electron

diffraction pattern obtained in an area similar to figure 1, formed by spots regularly distributed. (B) FESEM of total polysaccharides extracted from

algae collected in SB showing self-assembled structures, forming different lamellar or trabecular patterns arranged transversally. (C) Low magni-

fication image of an assembly of total polysaccharide fraction extracted from algae collected in SB, showing a coil-like appearance. (D) Detailed view

of polysaccharides showing a plate-like arrangement. (E) and (F) EDXA spectra of total polysaccharides from Sepetiba Bay, showing Zn (E) and

from an adjacent non-contaminated site, Ribeira Bay (image of the polysaccharides not shown), (F) C (Ka ¼ 0:28 keV), O (Ka ¼ 0:52 keV), S

(Ka ¼ 2:31 keV), Cl (Ka ¼ 2:62 keV), and Ca (Ka ¼ 3:69 keV; Kb ¼ 4:01 keV). Inset in (E): Part of spectra showing the Zn peak (Ka ¼ 8:63 keV).

Copper peaks (Ka ¼ 8:04 keV; Kb ¼ 8:91 keV) were originated from the grid. Arrows in (A) point to typical region that were analyzed by a focused

beam (�100 nm). (A) Bars in figures (A)–(D) correspond to 1lm.

L.R. Andrade et al. / Journal of Structural Biology 145 (2004) 216–225 219

precipitate with CaCl2 (see Section 2). Macroscopically,the appearance of total polysaccharide sample was a fine

powder, while sulfated fucan was granular and alginic

acid presented flocculated forms. The two purified

polysaccharides presented different ultrastructure and

elemental composition.

In the alginates fraction, TEM showed amorphous

structures containing numerous pores (Fig. 2A). FE-

SEM revealed a sponge-like form (Fig. 2B) with theirsurfaces having a sheet appearance (Fig. 2C). The pores

of alginate scaffold varied between 10 and 50 lm. Ca was

determined as the characteristic element (Fig. 2D). In

contrast, sulfated fucans showed numerous particles

with polygonal projections, individualized or arranged

in chains (Figs. 3A and B). Electron diffraction images

revealed a polycrystalline structure of this fraction (inset

Fig. 2. Electron microscopy and EDXA of alginate. (A) TEM of the alginates fraction presenting amorphous material with numerous pores. (B)

FESEM of alginates fraction revealing a sponge-like structure. (C) FESEM of alginate fraction revealing a surface with a sheet appearance. (D)

EDXA spectrum from alginate fraction. A focused beam of circa 100 nm was used (arrows point to typical analyzed regions). Ca was determined as

the characteristic element. C, O, Cl, K, and Cu were also detected. (A) Bar¼ 20lm; (B) bar¼ 10 lm; and (C) bar¼ 50 lm.

220 L.R. Andrade et al. / Journal of Structural Biology 145 (2004) 216–225

in Fig. 3B). FESEM showed numerous cubic particleswith sizes ranging of 4–20 lm (Figs. 3C and D). High

magnification images of these particles showed a discrete

undulated surface (Fig. 3E). Sulfur was detected as the

characteristic element (Fig. 3F).

One possible explanation for the unusual ultrastruc-

ture of the sulfated fucan extracted from brown algae is

either the presence of covalent-linked proteins or others

molecules which would bring together the polysaccha-ride chains. In order to clarify this aspect we extracted

acidic polysaccharide from the brown algae using ex-

haustive protease digestion and the sulfated fucan was

purified by anion exchange chromatography (Fig. 4A).

For these experiments we used the species L. brasiliensis

since the purification and analytical procedures for the

sulfated fucan from this brown alga were already es-

tablished (Pereira et al., 1999, 2002). The purity of thefinal product was assured by agarose gel electrophoresis

(Fig. 4B). Again, TEM observations of the exhaustive

purified sulfated fucan revealed the same ultrastructure,

composed of numerous particles with polygonal pro-

jections (Fig. 4C), as already described for the polysac-

charide obtained from P. gymnospora. X-raymicroanalysis detected the presence of S, besides C and

O (Fig. 4D). The Na and Cl peaks originated from saline

solution added to sulfated fucan during the purification

procedures while Cu peaks are from the copper grids, as

already mentioned.

Further evidence that the observed ultrastructure is

in fact constituted by the sulfated fucan from brown

algae came from studies with specific lectin. The ob-servations of ultra-thin sections of sulfated fucan ul-

trastructure revealed an intense labeled with UEA, a

lectin specific for a-LL-fucosyl (Fig. 5A). When ConA

instead of UEA was used we observed a less intense

label (Fig. 5B), as expected since mannose is only a

minor component of the algal fucans (Dietrich et al.,

1995; Pereira et al., 1999). The polygonal particles

were labeled less intensively with both tested lectins(Figs. 5C and D). Ultra-thin sections of alginic acid

incubated with the two lectins presented no significant

label (data not shown). The grids previously incubated

with pure lectins (control tests) did not exhibit any

label.

Fig. 3. Electron microscopy and EDXA of sulfated fucans. (A) TEM image of sulfated fucan fraction showing isolated particles with polygonal

projections. (B) TEM of fucan fraction with particles arranged in chains. Inset in (B) electron diffraction image of sulfated fucans revealing the

polycrystalline nature of the sample. (C) FESEM of sulfated fucans showing numerous particles with polyhedrical morphologies. (D) FESEM of a

fucan particle. (E) High magnification of a fucan particle showing an undulated surface. (F) EDXA spectrum from sulfated fucans fraction obtained

by TEM. The regions analyzed are the typical individual particles in (B) indicated by the arrows. S was detected as the characteristic element. The

elements C, O, K (Ka ¼ 3:31 eV) (from KOH extraction), and Cu (from the grids) were also observed. (A) Bar¼ 5 lm; (B) bar¼ 18 lm; (C)

bar¼ 10 lm; (D) bar¼ 1lm; and (E) bar¼ 0.1lm.

L.R. Andrade et al. / Journal of Structural Biology 145 (2004) 216–225 221

3.3. Heavy metals are accumulated into the acidic

polysaccharides ultrastructures

EDXA spectra showed the presence of Zn in the ul-trastructure of polysaccharides extracted from algae of

heavy metal contaminated area (Fig. 1E) while this el-

ement was absent from samples of control site (Fig. 1F).

The elements C, O, S, K, and Ca were detected in

samples obtained from the two sites and Cu peaks areprovided from the copper grids. This observation is

Fig. 4. Biochemical and microscopic analysis of sulfated fucans from L. brasiliensis. (A) Anion exchange chromatography on DEAE–cellulose

column of sulfated fucan. Fractions were checked by the phenol–H2SO4 (s) and carbazole (m) reactions, for metachromasia (d) and NaCl con-

centration (- - -) as described (Pereira et al., 1999). (B) Agarose gel electrophoresis of the crude polysaccharide (1) and the purified sulfated fucan (2)

(�15 lg of each) were applied to a 0.5% agarose gel and the electrophoresis was run and stained as described (Pereira et al., 1999). (C) TEM image

showing particles presenting similar structures as observed in sulfated fucans fractions of P. gymnospora. (D) EDXA spectrum showing the elements

C, O, and S. Na and Cl peaks were probably originated from saline solution added to fucans fraction during biochemical purification. Cu peaks are

from the grids. (C) Bar¼ 20lm.

222 L.R. Andrade et al. / Journal of Structural Biology 145 (2004) 216–225

support by direct quantification of heavy metal in the

biomass and also in solubilized polysaccharides. Zn and

Cd contents were marked higher in algae from con-

taminated area comparing to the control algae (Table 1).

Heavy metals were not detected in the samples of puri-

fied polysaccharides. Possibly, these elements were dis-

rupted from the polysaccharides during the purificationprocedures.

4. Discussion

4.1. Acidic polysaccharides from the algal cell wall

possess a well-organized ultrastructure

The architecture of marine algae cell walls is main-

tained by the self-assembly of cellulose and alginic acid

chains. Possibly, sulfated fucans play a key role cross-

linking these two macromolecules (Kloareg and Quatr-

ano, 1988). Studies about cell wall deposition during

morphogenesis in Palvatia compressa reveal the wall

strength in young zygotes depends on F-actin, whereas

cellulose and a sulfated polysaccharide (probably a

sulfated fucan) are more important in tip growing

zygotes (Bisgrove and Kropf, 2001).

A microfibrillar arrangement of the brown algae cell

walls is clearly observed by TEM at routine prepara-

tions. The cellulose microfibrils are embedded in an

‘‘amorphous’’ matrix, composed mostly by solublepolysaccharides. In some regions of the cell wall high

amounts of this matrix masks the microfibrils, especially

near the external region. The term ‘‘amorphous’’ is

widely applied to the matrix polysaccharides because it

is difficult to discriminate these compounds within the

microfibrillar arrangement.

The ‘‘amorphous’’ polysaccharides are easily ex-

tracted from the algal cell wall and purified as two majorcomponents, named alginic acid and sulfated fucan. The

chemical structure and several biological properties of

these polysaccharides have been widely studied. How-

ever no data is available concerning the ultrastructure

and/or conformation of these soluble polysaccharides

from the cell wall of brown algae, especially in the case

of the sulfated fucans. It is generally assumed that the

Table 1

Concentrations of Zn and Cd (determined by atomic absorption

spectrophotometry) in samples of the brown algae P. gymnospora

collected from a heavy metal contaminated area (Sepetiba Bay) and an

adjacent non-contaminated area (Ribeira Bay)

Sample Collection site Metals

Zn Cd

Total biomass Sepetiba Bay 242� 34 1.30� 0.20

Ribeira Bay 23� 8 0.36� 0.09

Solubilized

polysaccharides

Sepetiba Bay 95� 12 0.21� 0.05

Ribeira Bay 9.3� 0.4 ND

ND, not detected.

Results in lg g�1 of dry weight (mean�SD).

Fig. 5. TEM of ultra-thin sections (LRGold resin) of sulfated fucans fraction incubated with specific lectins labeled with colloidal gold. (A)

Amorphous-like material intensively labeled with UEA. (B) Amorphous-like material intensively labeled with Con A. (C) Particle with a polygonal

outline labeled in the border by UEA. (D) Particles with polygonal outlines also labeled in the border by Con A. Bar represents 200 lm in (A); 150lmin (B); 100lm in (C), and 100lm in (D).

L.R. Andrade et al. / Journal of Structural Biology 145 (2004) 216–225 223

physiological role of these molecules depends exclusively

of the overall negative charged groups. Only very recent

studies using synthetic oligosaccharides have suggested

that sulfated fucans may assume specific conformation

(Gerbst et al., 2001, 2002).

We now used a different approach to investigate

whether the algal polysaccharides present specific ul-

trastructure. The polysaccharides were visualized by a

variety of electron microscopy techniques. We observed

that sulfated fucan and alginic acid present well-orga-

nized ultrastructure but the pattern of organization

varies depending on the polysaccharide species. Alginic

acid showed a sponge-like structure whereas sulfatedfucan presented particles with polygonal forms.

We have conclusive evidence that the ultrastructure

composed of numerous particles with polygonal pro-

jection is in fact formed by sulfated fucan molecules.

First, EDXA spectra reveal the presence of S in these

structures, as expected for the sulfate groups found in

the algal fucans. Second, these ultrastructures were in-

tensively labeled with UEA, a specific lectin that rec-ognize a-LL-fucosyl residues. Finally, an exhaustive

purified sulfated fucan showed the same morphology,

composed of polyhedrical forms.

A well-known example of polysaccharides assuming

organized ultrastructure is the proteoglycans from

224 L.R. Andrade et al. / Journal of Structural Biology 145 (2004) 216–225

vertebrate extracellular matrix (Rothenburger et al.,2002). However, the ultrastructure is observed exclu-

sively on the intact molecules, which are glycosamino-

glycan chains covalently attached to a protein core.

Once the proteoglycan is digested with protease, the

released glycosaminoglycan chains (molecular mass

ranging from 10 to 40 kDa) no longer show ultrastruc-

ture organization under microscopic examination. The

sulfated fucans still show the characteristic ultrastruc-ture even after exhaustive protease digestion. These algal

polysaccharides possess high molecular weight

(>100 kDa, Pereira et al., 1999), well above the size of

vertebrate glycosaminoglycan chains and similar to

several intact proteoglycans.

TEM images showed that the sulfated fucans of the

two species studied in this work had similar morpho-

logies (i.e., polyhedrical particles arranged in chains). Itmeans that probably, the morphology of micron sized

crystals of sulfated fucans from the two species was not

influenced by the distinct proportions of the polysac-

charides in these two algae, or the differences in poly-

saccharide composition is not significantly high to

induce differences in macromolecular arrangement at

the ultrastructural level.

Our approach in the study of the algal polysaccha-rides may help the comprehension of some their physi-

ological and biological role in terms of molecular

structure. This is especially noteworthy for the sulfated

fucans, which reveal an unusual polycrystalline struc-

ture. The chemical structure of these molecules may not

reveal the spatial distribution of sulfate groups but this

aspect may be clarified using structural analysis. It may

clarify the drastic differences in biological activities be-tween sulfated fucans with similar chemical structures

(Alvez et al., 1997; Pereira et al., 2002).

4.2. Acidic polysaccharides form the algal cell wall retain

heavy metals

Different species of brown algae have been used as

biomonitor of heavy metal pollution due to their highcapability of long-term uptake comparing with other

seaweeds (Amado Filho et al., 1999; Engdahl et al.,

1998; Karez et al., 1994; Murugadas et al., 1995). In a

previous work, we have demonstrated that a brown al-

gae collected from a heavy metal contaminated area

accumulated Zn as electron-dense granules in the cell

walls in median regions of thallus (Amado Filho et al.,

1999). The polyanionic polysaccharides have been pro-posed as the molecules responsible for the binding of

heavy metals when the algae are exposed to high con-

centrations of these elements (Amado Filho et al., 1996,

1999; Andrade et al., 2002; Ballan-Dufranc�ais et al.,

1991; Haug et al., 1974; Lignell et al., 1982; Pellegrini

et al., 1991, 1993; Tretyn et al., 1996; Stengel and

Dring, 2000).

In the present work, Zn was detected in the totalbiomass and in the solubilized polysaccharides from

algae exposed to a heavy metal contaminated environ-

ment. In this sample, EDXA spectra showed that Zn

was associated with Ca and S (besides C and O), indi-

cating that alginic acid and sulfated fucan are the mol-

ecules responsible for the binding of heavy metal.

Mariani et al. (1985) have studied the elemental

composition of polysaccharides isolated from Fucus

virsoides by X-ray microanalysis in SEM, and suggested

that Ca2þ and Mg2þ were strongly complexed with al-

ginic acid in algal cell walls, stabilizing the wall archi-

tecture. In contrast, the seawater cations were mainly

bound to the sulfated polysaccharides, which regulate

the passive ion disposed to the algal cells. Andrade et al.

(2002) showed that when P. gymnospora was exposed

to sub-lethal concentrations of Cd isolated and addedwith Zn in vitro, other cations such as Naþ, Kþ, Ca2þ

found naturally in cell walls were substituted by Cd2þ

and Zn2þ.In summary, the present work describes for the first

time the ultrastructure of sulfated fucans and alginic

acid extracted from the cell wall of brown algae. Alginic

acid presented a sponge-like form with numerous pores

while sulfated fucans showed polyhedrical forms with aunique crystalline nature. In addition, Zn was detected

in these polysaccharides when the algae were collected

from a heavy metal contaminated environment. This

demonstrates the role of polyanionic polysaccharides as

metal binders for the nucleation and precipitation of

heavy metals in brown algae cell walls.

Acknowledgments

We thank Ms. Mair Machado for technical support,

Ricardo Thomas for AAS measures and Dr. W. de

Souza for electron microscopy facilities. This work was

supported by PRONEX—CNPq, FAPERJ, JBRJ/

MMA. Paulo A.S. Mour~ao is a fellow of the JohnSimon Guggenheim Memorial Foundation.

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