A VANADIUM BROMOPEROXIDASE CATALYZES THE FORMATION OFHIGH-MOLECULAR-WEIGHT COMPLEXES BETWEEN BROWN ALGAL

PHENOLIC SUBSTANCES AND ALGINATES1

Leonardo Tavares Salgado

Instituto de Pesquisas Jardim Botanico do Rio de Janeiro, Diretoria de Pesquisas, MMA, 22460–030, Rio de Janeiro, Brasil

Leonardo Paes Cinelli

Laboratorio de Tecido Conjuntivo, Hospital Universitario Clementino Fraga Filho (HUCFF), Instituto de Bioquımica Medica

(IBqM), 21941–590, UFRJ, Rio de Janeiro, Brasil

Nathan Bessa Viana

Laboratorio de Pincas Opticas-COPEA, ICB ⁄ Instituto de Fısica, 21941–972, UFRJ, Rio de Janeiro, Brasil

Rodrigo Tomazetto de Carvalho

Instituto de Pesquisas Jardim Botanico do Rio de Janeiro, Diretoria de Pesquisas, MMA, 22460–030, Rio de Janeiro, Brasil

Paulo Antonio de Souza Mourao

Laboratorio de Tecido Conjuntivo, HUCFF, IBqM, 21941–590, UFRJ, Rio de Janeiro, Brasil

Valeria Laneuville Teixeira

Departamento de Biologia Marinha, Instituto de Biologia, 24001–970, Universidade Federal Fluminense, Niteroi, Brasil

Marcos Farina

Laboratorio de Biomineralizacao, ICB, 21941–590, UFRJ, Rio de Janeiro, Brasil

and Gilberto Menezes Amado Filho2

Instituto de Pesquisas Jardim Botanico do Rio de Janeiro, Diretoria de Pesquisas, MMA, 22460–030, Rio de Janeiro, Brasil

The interaction between phenolic substances (PS)and alginates (ALG) has been suggested to play a rolein the structure of the cell walls of brown seaweeds.However, no clear evidence for this interaction wasreported. Vanadium bromoperoxidase (VBPO) hasbeen proposed as a possible catalyst for the bindingof PS to ALG. In this work, we studied the interactionbetween PS and ALG from brown algae using sizeexclusion chromatography (SEC) and optical twee-zers microscopy. The analysis by SEC revealed thatALG forms a high-molecular-weight complex with PS.To study the formation of this molecular complex,we investigated the in vitro interaction of purifiedALG from Fucus vesiculosus L. with purified PS fromPadina gymnospora (Kutz.) Sond., in the presence orabsence of VBPO. The interaction between PS andALG only occurred when VBPO was added, indicat-ing that the enzyme is essential for the binding pro-cess. The interaction of these molecules led to areduction in ALG viscosity. We propose that VBPOpromotes the binding of PS molecules to the ALG

uronic acids residues, and we also suggest that PS arecomponents of the brown algal cell walls.

Key index words: binding process; cell wall forma-tion; haloperoxidases; optical tweezers; phenol;phloroglucinol; phlorotannins; polysaccharides;uronic acid; viscosity

Abbreviations: A, absorbance; ALG, alginates; ALG-Fv, alginate from Fucus vesiculosus; ALG-Pg, algi-nate from Padina gymnospora; Apo, apochromatic;C16, 16 carbon atoms; CCD, charge-coupleddevice; CD3OD, deuterated methanol; DEAE, di-ethylaminoethyl; FPLC, flow pressure liquid chro-matography; NA, number of aperture; Nd-YAG,neodymium-doped yttrium aluminum garnet; NIH-USA, National Institutes of Health of United Statesof America; Plan, low curvature of field; PS,phenolic substances; SEC, size exclusion chroma-tography; V0, elution point of higher molecularweight fractions; VBPO, vanadium bromoperoxi-dase; VIS, visible radiation; Vt, elution point oflower molecular weight fractions

1Received 25 February 2008. Accepted 21 August 2008.2Author for correspondence: e-mail gfilho@jbrj.gov.br.

J. Phycol. 45, 193–202 (2009)� 2009 Phycological Society of AmericaDOI: 10.1111/j.1529-8817.2008.00642.x

193

The cell walls of brown seaweeds contain mainlycellulose microfibrils, several proteins, and two acidicpolysaccharides, named alginates (ALG) and sul-fated fucans (Kloareg and Quatrano 1988). Alginateis a linear polysaccharide composed of a-1,4-l-gulu-ronic acid and b-1,4-d-mannuronic acid units. Thepolymer contains blocks of polyguluronic and ⁄ orpolymannuronic sequences (Kloareg and Quatrano1988). This polysaccharide plays an essential role inthe cell wall of seaweeds, acting as an ionic barrierand structural framework (Kloareg and Quatrano1988, Andrade et al. 2004, Salgado et al. 2005).Recently, phenolic substances (PS), which are sec-ondary metabolites produced by brown algae, weredescribed as components of the ALG complexes inthe algal cell wall. Several works have proposed, onthe basis of indirect evidence, that PS link with ALGplaying an essential role in the algal cell wall struc-ture (Vreeland et al. 1998a, Schoenwaelder 2002,Arnold and Targett 2003, Koivikko et al. 2005,Salgado et al. 2005). The PS, also called phlorotan-nins, are mainly composed of phloroglucinol(Ragan and Glombitza 1986), and their main cellu-lar localization as soluble compounds is in theorganelle termed physode (Arnold and Targett2003). The PS play important roles in many physio-logical processes, such as protection of the algalcells against ultraviolet radiation (UV), inhibition ofherbivory, blocking of polyspermy, heavy-metal bind-ing and spore adhesion (Karez and Pereira 1995,Pavia et al. 1997, Schoenwaelder and Clayton 1998,Berglin et al. 2004, Ceh et al. 2005, Bitton et al.2006).

Recently, several papers have reported that theinteractions of PS with cell wall polysaccharides mayinterfere with some cellular processes or with somechemical properties of these polysaccharides (Karezand Pereira 1995, Moen et al. 1997a,b, Tam et al.2006, Salgado et al. 2007). For example, the bindingof heavy metals to physodes can be related to thepresence of cell wall polysaccharides in this orga-nelle (Karez and Pereira 1995). It has also been sug-gested that the viscosity of ALG can be modified bythe presence of PS in polysaccharide fractions(Moen et al. 1997a,b, Tam et al. 2006). Moreover, itwas also suggested that the capacity of PS to absorbUV in vitro is preserved due to the interactions withthe ALG (Salgado et al. 2007). However, the bind-ing process between PS and cell wall polysaccharidesand, consequently, the role of PS in the formationof cell wall structure is not yet supported by directevidence.

By some authors (Orive et al. 2002, Koivikkoet al. 2005, Tam et al. 2006), the association of PSwith ALG is considered a contamination caused bynonspecific interactions between these two com-pounds during polysaccharide extraction proce-dures. For others, the formation of these complexesis a specific event with a specific biological role. Inthe first case, random modifications of the chemical

structure of soluble PS, such as the oxidation of themolecule caused by degradation, is considered acondition that promotes the binding of PS to ALG(Koivikko et al. 2005). Other studies have proposedthat the binding mechanism between cell wall ALGand PS is mediated by the vanadium-dependentbromoperoxidase enzyme (VBPO) (Vreeland et al.1998a, Schoenwaelder 2002, Arnold and Targett2003). This enzyme was determined as being pres-ent in many brown and red algae species (Rushet al. 1995, Almeida et al. 1998, Shimonishi et al.1998, Vreeland et al. 1998b, Weyand et al. 1999,Berglin et al. 2004, Colin et al. 2004). In brownalgae, VBPO may act on the oligomerization of PS(Berglin et al. 2004) and also in the formation ofnatural algal adhesives (Bitton et al. 2006). In rela-tion to these natural adhesives, it was shown thatthe PS oxidized by VBPO undergo self-assembly andform with the ALG a type of macromolecular clus-ter, where the PS are encapsulated by the ALG gelnetwork (Bitton et al. 2006). However, the nature ofthe interactions between PS and ALG in this encap-sulation process is unknown, and, consequently,binding between PS and ALG remains undemon-strated.

In this regard, our goal was to contribute to abetter understanding of these interactions, address-ing two main questions. Does the PS specificallybind to the ALG? Does VBPO influence the inter-actions between these two molecules? In summary,the interactions between PS and ALG were analyzedin samples extracted from P. gymnospora by usingoptical tweezers microscopy and size exclusion chro-matography. We also developed an in vitro assay toevaluate the influence of VBPO on interactionsbetween PS from P. gymnospora and ALG fromF. vesiculosus, by measuring their molecular weightsand the viscosity of the solutions before and afterVBPO incubation, using optical tweezers.

MATERIALS AND METHODS

Algae samples. Adult individuals of P. gymnospora measuring5 cm length were collected at the upper region of the subtidalzone in Sepetiba Bay, located in Rio de Janeiro State, Brazil(22�57¢05¢ S, 43�54¢28¢ W). Fresh algae were cleaned ofepiphytes, briefly washed in Milli-Q H2O, and dried at 60�C.

Isolation and characterization of soluble PS from P. gymnospora.The extraction of soluble PS was carried out according to theprotocol described by Koivikko et al. (2005). The driedsample (50 g) was maintained in 70% aqueous acetonesolution (2 L) for 48 h. Thereafter, the acetone was evapo-rated, the residue was dissolved in Milli-Q H2O (Millipore,Billerica, MA, USA) and centrifuged (5,000g for 10 min, roomtemperature; Eppendorf, Hamburg, Germany), and the solu-ble fraction was lyophilized. The extracted PS was diluted inMilli-Q H2O and partially purified using a C-18 column (Sep-Pak C-18 Cartridge, Water Associates, Millipore

TM

, Billerica,MA, USA), which was preactivated with ethanol and washedwith Milli-Q H2O. The PS adsorbed to the column were elutedwith a stepwise gradient of ethanol, evaporated, dissolved inMilli-Q H2O, and lyophilized. This isolated PS sample waspurified further with size exclusion chromatography (SEC)

194 LEONARDO TAVARES SALGADO ET AL.

using a Superose 6 column (Amersham Pharmacia Biotech,Buckinghamshire, UK). The SEC was also used to obtain anestimated molecular weight for these PS. The column wasequilibrated with 100 mM sodium acetate buffer (pH 6.3),20 mM EDTA, and 250 mM NaCl, linked to a HPLC Systemfrom Shimadzu (Tokyo, Japan). The molecular standards tomark Vo and Vt were dextran blue and glycine, respectively.The flow rate of the column was 15 mL Æ h)1. The fractionswere monitored by absorbance at 210 nm and also bycarbozole reaction (Diche 1947). During the extraction andisolation procedures, we were careful to protect PS againstlight degradation. Analysis performed on a UV-mini 1240UV-VIS Shimadzu spectrophotometer revealed that PS absorbsmainly at 210 nm. The soluble PS were identified by compar-ison of 1 H-NMR (300 MHz, CD3OD) spectral data withliterature data.

Isolation of ALG from P. gymnospora. Polysaccharides wereextracted from the dry algae by papain digestion (underagitation for 24 h at 60�C) and partially purified by threeconsecutive ethanol precipitations (Alves et al. 1997). Thepowder of total extracted polysaccharides was diluted in Milli-QH2O, and, thereafter, 3 M CaCl2 was added to the solution toprecipitate the ALG. The precipitated sample was dialyzedagainst 10% EDTA and thereafter against Milli-Q H2O for 72 heach, and then lyophilized. This sample was fractioned byanion-exchange chromatography on a Mono-Q column (HR5 ⁄ 5 – Amersham Pharmacia Biotech), equilibrated in 20 mMTris-HCl (pH 8.0), 5 mM EDTA, linked to an FPLC(Amersham Pharmacia Biotech). The column was eluted witha linear gradient of NaCl (0 to 3 M at a flow rate of0.5 mL Æ min)1). The fractions were monitored by absorbanceat 210 nm to check for the presence of PS and also for uronicacid by the carbozole reaction. The obtained fractions weredialyzed against Milli-Q H2O and lyophilized. These fractions(@10 lg) were applied to a 0.5% agarose gel to evaluate thesample purity. The gel was run for 1 h at 110 V in 0.05 M 1,3-diaminopropane ⁄ acetate (pH 9.0). The polysaccharides in thegel were fixed with 0.1% N-cetyl-N,N,N-trimethylammoniumbromide solution. After 12 h, the gel was dried and stainedwith 0.1% toluidine blue O in acetic acid ⁄ ethanol ⁄ water(0.1:5:5, v ⁄ v). The isolated ALG from P. gymnospora was namedALG-Pg.

Purification of ALG from F. vesiculosus. A crude ALGpreparation from F. vesiculosus was obtained from Sigma-Aldrich (St. Louis, MO, USA). The ALG was further purifiedusing anion exchange chromatography on DEAE celluloseequilibrated in 100 mM sodium acetate (pH 6.0), containing20 mM EDTA. The column was eluted with a linear gradient of0–3.0 M NaCl at a flow rate of 10 mL Æ h)1. Then, a high-molecular-weight fraction was purified from ALG using an SECcolumn Superose 6. The fractions were monitored by absor-bance at 210 nm and also by the carbazole reaction. Thepurified sample was named ALG-Fv.

Monosaccharides composition and molecular weight of ALG-Pg andALG-Fv. The monosaccharide composition of ALG-Pg andALG-Fv was estimated by paper chromatography in 1-buta-nol ⁄ pyridine ⁄ water (3:2:1, v ⁄ v) for 48 h after acid hydrolysis.The molecular weights of the purified ALGs were determinedby SEC on Superose 6. The purified fractions were dialyzedagainst Milli-Q H2O for 72 h and lyophilized.

In vitro binding assays with VBPO. The VBPO enzyme used inthis work (purchased from Fluka, ref. no. 17965; Fluka, Buchs,Switzerland) was extracted and purified from the red algaCorallina officinalis. The appropriate enzyme and substrates(KBr, H2O2, VO4

3)) concentrations, temperature, and pH ofthe incubation solutions were used as reported (Rush et al.1995). The binding assays were performed in solutionscontaining 100 mM sodium acetate (pH 6.4), 20 mM EDTA,250 mM NaCl, 8 mM KBr, 100 lM H2O2, 1 mM VO4

3), using

10 U Æ mL)1 VBPO, 1 mg Æ mL)1 PS, 1.5 mg Æ mL)1 ALG-Fv,and 1 mg Æ mL)1 BSA. Nine different incubation solutionswere maintained at 30�C for 24 h: (1) PS + ALG-Fv + VBPO;(2) PS + VBPO; (3) ALG-Fv + VBPO; (4) VBPO; (5) PS + ALG-Fv + BSA; (6) PS + BSA; (7) ALG-Fv + BSA; (8) BSA; and (9)PS + ALG-Fv. The products formed were analyzed by SEC onSuperose 6.

Characterization of the interaction between ALG and PS by usingSEC. The PS and ALG are low- and high-molecular-weightcompounds, respectively. The possible formation of a high-molecular-weight aggregate containing these two compoundswould be a direct evidence of their interaction. Thus, SECon Superose 6 column of ALG-Pg and of the products ofbinding assays was used to investigate the interactionbetween PS and ALG. If the PS and ALG moleculescoeluted, the binding between these molecules could beconfirmed.

Characterization of the interaction between ALG and PS bymeasuring solutions viscosity with optical tweezers. After elutionfrom SEC column Superose 6, the products obtained from invitro binding assays were analyzed in optical tweezersmicroscopy to measure the viscosity values of these samplesolutions. The analysis was performed with the followingsamples diluted in Milli-Q water (5 mg Æ mL)1): (1) ALG-Pg;(2) PS + ALG-Fv, incubated with VBPO; (3) PS + ALG-Fv, notincubated with VBPO; (4) PS; and (5) ALG-Fv. The sample‘‘3¢’’ was analyzed in five different PS concentrations (0.25,2.5, 5, 10, and 50 mg Æ mL)1) to test for a dose-depen-dence phenomenon influencing the ALG viscosity. Theisolated ALG fraction from P. gymnospora, a sample notused in the in vitro binding assays, was also analyzed at5 mg Æ mL)1.

Theory—solution viscosity measurement through Brownian motionevaluation with optical tweezers. The Brownian motion ofpolystyrene spheres (1.52 ± 0.05 lm radius) is used to mea-sure the viscosity of the medium in which they are immersed(Viana et al. 2006, 2007). A solution containing the polysty-rene spheres (10% v ⁄ v; Sigma-Aldrich) was diluted in each ofthe above described samples to a concentration of 10)4%(v ⁄ v). To hold the sphere solution a large glass coverslip(24 · 50 mm) with an O-ring of 1 cm internal diameter and0.3 cm high was glued onto the glass surface with siliconewax. The solution was placed in the region limited by the O-ring, and a second coverslip (24 · 24 mm) was placed on topto avoid evaporation. The sample was then observed in aninverted Nikon (Tokyo, Japan) Eclipse TE300 microscopeadapted to receiving a Nd-YAG laser beam (1,064 nmwavelength) entering by its EPI-fluorescence port. The useof a high numerical aperture objective lens (Plan Apo 100·;NA 1.4) made possible the creation of an optical trap nearthe objective lens focus (Ashkin and Dziedzic 1987) allowingthe manipulation of small dielectric objects (in the order offew micrometers in diameter). The whole system (micro-scope, laser source, and mirrors for the laser beam orienta-tion) was mounted on a Newport table (Newport RS 2000TM;Newport Co., Irvine, CA, USA) that minimizes the effects ofenvironmental vibrations. Digitized images were obtainedwith a CCD camera connected to an Argus-20 system(Hammamatsu; Hammamatsu City, Japan) and a LG3-16PCI CCIR Scion frame grabber (Scion Co., Frederick, MD,USA). The images obtained were processed and analyzedusing the ImageJ freeware software (NIH, Bethesda, MD,USA).

The mean square displacement of the variation of the centerof mass position in a time interval t is given by:

ðdqÞ2D E

¼ 4Dt ð1Þ

where

PHENOL AND ALGINATE COMPLEXES 195

D ¼ kbT

bð2Þ

and kbT is the thermal energy (4.14 · 10)21 Joules at roomtemperature); b, the Stokes friction coefficient, which is afunction of the bead radius a; h is the height distance of thepolystyrene sphere center to the bottom glass coverslip; andg is the fluid viscosity. The friction coefficient b is given bythe Faxen law (Feitosa and Mesquita 1991):

b ¼ 6pga 1� 9

16

a

h

� �þ 1

8

a

h

� �3� 45

256

a

h

� �4� 1

16

a

h

� �5� ��1

ð3Þ

For a fixed value of h, ðdqÞ2D E

is calculated using the digi-tized (1,000 frames; �30 frames Æ s)1) images of the spheresobserved through the microscope. From the linear fit of themeasured ðdqÞ2

D Evalues as a function of time t, using equa-

tion 1, the diffusion coefficient (D) is obtained. This valueallows the determination of the Stokes friction coefficient bby using equation 2. The sphere heights were then changedexperimentally, and the same calculation steps were repeated.From the fit of the b values as a function of sphere height(h) using equation 3, we obtained the sample viscosity g withan uncertainty of 5% to 10%. The system calibration wasdone by measuring the viscosity of Milli-Q water.

RESULTS

Isolation and characterization of soluble PS and ALG.The PS extracted from P. gymnospora were elutedfrom a C-18 column with 25%–30% ethanol. TheUV spectrum of the isolated PS revealed a majorabsorbance band at 210 nm. On SEC, the PS wereeluted closed to Vt of the column (continuous linein Fig. 1), and the molecular weight was estimatedas lower than 10 kDa. No uronic acid was detectedin the fractions from SEC by the carbazole reaction(open squares in Fig. 1). All proton bands due tothe ortho-acyl phloroglucinol functionality were pres-ent, and the remainder of the signals in the1H-NMR spectrum were high field methylenes, mostof which were observed at the same chemical shiftsof d 1.28 (22H, s). From the 1H NMR integrationdata, one methylene was shown to possess the satu-rated C16 side chain. The methylene protons a tothe carbonyl were observed as a triplet at d 3.24(2H, t = 7.0 Hz). Two high field aromatic protonsappeared as a doublet at d 5.92 (1H, J = 1.5 Hz)and 6.02 (1H, J = 1.5 Hz), indicating two meta-dis-posed aromatic protons. The metabolite found inour PS extract was 2-[1¢-Oxo-hexadecyl]-1,3,5-trihydr-oxybenzene (Fig. 2), which has a molecular weightof 350.5 Da (Gerwick and Fenical 1982).

The acidic polysaccharides extracted fromP. gymnospora were purified by anion exchange chro-matography on a Mono-Q column, eluted withincreasing NaCl concentrations. Two major compo-nents were detected when the fractions were assayedby the phenol sulfuric acid reaction for hexose. Thefraction eluted at low NaCl concentration containsuronic acid (as indicated by the positive carbazolereaction) and has no metachromatic property

(evaluated by using the reaction with 1,2-diamino-4,5-methylenedioxybenzene-DMB), indicative that itcontains ALG and not sulfated groups. The fractioneluted from the anion exchange column at highNaCl concentrations presented intense metachrom-asy and contained mainly the sulfated polysaccha-rides (mostly sulfated fucan). Agarose gelelectrophoresis (Fig. 3) confirmed the purity of thetwo polysaccharides obtained by anion exchangechromatography. ALG and sulfated fucan have lowand high electrophoretic mobility, respectively. Themonosaccharides found in these two fractions wereanalyzed by paper chromatography after acid hydro-lysis of the polysaccharides. ALG contained uronicacid while the sulfated polysaccharide was composedmainly of fucose, as expected (data not shown).Analysis of the ALG on a SEC showed that this poly-saccharide presented a high molecular weight(�5,000 kDa, Fig. 4). Proteins were not detected inthis purified fraction (absorbance measurement at280 nm).

In a parallel experiment, we also furtherpurified the ALG preparation from F. vesiculosus(purchased from Sigma-Aldrich). Anion exchangechromatography on DEAE-cellulose revealed two

Fig. 1. Size exclusion chromatogram of isolated phenolicsubstances (PS). Sample extracted from Padina gymnosporaperformed in a Superose 6 column (arrows indicateV0 = 5,000 kDa and Vt = 5 kDa, respectively). Fractions werechecked for uronic acid and PS detection, by carbazole reactionand by measuring absorbance at 210 nm, respectively (PS = bold-line marked with an asterisk and uronic acids = h.) Note thepeak close to the column Vt, which confirms that eluted PS havea low molecular weight.

Fig. 2. The molecular structure of 2-[1¢-Oxo-hexadecyl]-1,3,5-trihydroxybenzene.

196 LEONARDO TAVARES SALGADO ET AL.

major polysaccharides, eluted at 0.5 and 1.2 MNaCl, when the fractions were assayed by the phenylsulfuric acid reaction. The polysaccharide eluted atlow NaCl concentration is the ALG fraction, as indi-cated by the positive carbazole reaction for uronicacid and the absence of metachromasy (evaluatedby the DMB reaction). On SEC, this polysaccharideis eluted close to V0, indicative of a high molecularweight (�5,000 kDa, Fig. 5). When the fractionswere assayed by A210, we observed that no PS werecoeluted with the ALG (continuous line in Fig. 5).Proteins were not found in this purified fraction(absorbance measurement at 280 nm).

Characterization of the interaction between ALG and PSby using SEC. The SEC of the ALG purified fromP. gymnospora showed that PS coeluted with the

polysaccharide close to the column V0 and coinci-dent with the uronic acid peak (Fig. 4). This findingindicates the association of PS with ALG, and thatthe complex was not dissociated during the extrac-tion and purification procedures. In contrast withthis result, the ALG purified from F. vesiculosus didnot contain PS associated with the polysaccharide(Fig. 5). Therefore, the preparation of ALG purifiedfrom F. vesiculosus is appropriate for an in vitro assayaiming to evaluate the interaction of the polysaccha-ride with PS. In an initial attempt to detect the pos-sible formation of ALG-PS complex, we incubatedthese two molecules at 30�C for 12 h and analyzedthe mixture on a SEC (Fig. 6a). No evidence for theformation of a molecular complex was obtainedsince PS and ALG were eluted separately from thecolumn. However, when VBPO was added to thePS + ALG-Fv incubation mixture, and the productsformed were analyzed by SEC, we clearly detectedthe formation of the complex between the polysac-charide and PS by coelution of the two moleculesclose to the V0 of the column (Fig. 6b). The addi-tion of VBPO did not cause significant modificationon ALG-Fv molecular weight, as shown in Figure 7.In this sample, a peak corresponding to the enzymeelution was observed close to the Vt of the column(detected by absorbance measurement at 280 nm).When BSA replaced VBPO, we did not detect theformation of a complex by SEC (data not shown).

Characterization of the interaction between ALG andPS by measuring solution viscosity with optical tweezers.Isolated PS solution analyzed with the optical twee-zers presented an average viscosity value of 1.0 ± 0.1cPoise (see Materials and Methods for details),while purified ALG-Fv presented an average viscosityvalue of 8.3 ± 0.9 cPoise (Table 1). The addition ofPS to ALG-Fv solutions did not cause a significant

Fig. 4. Size exclusion chromatograph of ALG-Pg. Analysis per-formed in a Superose 6 column (arrows indicate V0 = 5,000 kDaand Vt = 5 kDa, respectively). Fractions were checked for uronicacids and PS, by carbazole reaction and by measuring absorbanceat 210 nm, respectively (PS = boldline marked with an asteriskand uronic acids = h.) ALG-Pg, alginate from Padina gymnospora.

Fig. 5. Size exclusion chromatogram of ALG-Fv. Analysis per-formed in a Superose 6 column (arrows indicate V0 = 5,000 kDaand Vt = 5 kDa, respectively). Fractions were checked for uronicacids and PS, by carbazole reaction and by measuring absorbanceat 210 nm, respectively (PS = boldline marked with an asteriskand uronic acids = h.) ALG-Fv, alginate from Fucus vesiculosus.

Fig. 3. Agarose gel electrophoresis of polysaccharides extra-cted from Padina gymnospora showing the total polysaccharides(T), ALG-Pg (1) and the sulfated fucans (2). Note that ALG (1)has a lower electrophoretic mobility than sulfated fucan (2).ALG, alginates; ALG-Pg, alginate from P. gymnospora.

PHENOL AND ALGINATE COMPLEXES 197

change (P > 0.05) in the viscosity values (Table 1).Isolated ALG-Pg sample presented viscosity valuesnear 1 cPoise (Table 1), which were very similar tothe PS values and significantly different from ALG-Fv values (P < 0.05).

When the solution of ALG-Fv was mixed with PSin the presence of the enzyme VBPO, the viscositydropped significantly (P < 0.05, see Fig. 8 andTable 1) compared to the value obtained from anal-ysis of ALG-Fv mixed with PS without VBPO addi-tion (Fig. 8 and Table 1).

DISCUSSION

The molecular weight of the PS extracted frombrown algae varies considerably. In the case of a

single species, the molecular weight of PS variedfrom 0.32 up to 400 kDa (Ragan and Glombitza1986). This variation may be a consequence of theprocedures used to extract PS, which can produceextracts with soluble and nonsoluble PS fractions.When the analysis is restricted to soluble PS, therange of their molecular weights is significantly morenarrow and close to a lower molecular weight (Berg-lin et al. 2004), as also shown in our study. By usingSEC, the molecular weight of the purified PS samplewas estimated as being close to 10 kDa. The analysis

Fig. 6. In vitro analysis of PS-ALG interactions. (A) Size exclu-sion chromatogram of mixed ALG-Fv and PS samples performedin a Superose 6 column (arrows indicate V0 = 5,000 kDa andVt = 5 kDa, respectively). (B) Size exclusion chromatogram ofmixed ALG-Fv and PS samples, with VBPO addition, performedin a Superose 6 column. Fractions were checked for uronic acidsand PS, by carbazole reaction and by measuring absorbance at210 nm, respectively. (PS = boldline marked with an asterisk anduronic acids = h.) In (B), note the presence of a PS peak closelyassociated with the ALG peak, thus revealing the binding medi-ated by VBPO activity. ALG, alginates; ALG-Fv, alginate fromFucus vesiculosus; PS, phenolic substances; VBPO, vanadium brom-operoxidase.

Fig. 7. In vitro analysis of ALG-VBPO interaction. Size exclu-sion chromatogram of ALG-Fv sample mixed with VBPO per-formed in a Superose 6 column (arrows indicate V0 = 5,000 kDaand Vt = 5 kDa, respectively). (VBPO = boldline marked with anasterisk and uronic acids = h.) The VBPO was detected by mea-suring absorbance at 280 nm. Note that the molecular weight ofALG-Fv was not modified after incubation with VBPO and alsothat the two compounds did not form a high-molecular-weightcomplex. ALG, alginates; ALG-Fv, alginate from Fucus vesiculosus;VBPO, vanadium bromoperoxidase.

Table 1. Viscosity values expressed in cPoise measured byusing optical tweezer.

SampleViscosity in

cPoise (±SD)

PSa 1.0 ± 0.1ALG-Pga (with PS linked in vivo) 1.1 ± 0.1ALG-Fvb 6.7 ± 0.5ALG-Fvb mixed with PS(values at mg Æ mL)1)0.25 7.6 ± 0.62.50 7.8 ± 0.65.00 7.6 ± 0.510.0 6.8 ± 0.550.0 6.9 ± 0.5

ALG-Fvb linked with PSc 1.6 ± 0.1

ALG, alginates; ALG-Fv, alginate from Fucus vesiculosus; PS,phenolic substances; VBPO, vanadium bromoperoxidase.

aSample concentration of 5 mg Æ mL)1.bIn these analyzed samples, the ALG were diluted at

5 mg Æ mL)1.cThe concentration of PS in the assay using VBPO (last row

in the table) was 5 mg Æ mL)1 (see Materials and Methodssection for details).

198 LEONARDO TAVARES SALGADO ET AL.

of the isolated PS fraction by NMR revealed that themain PS constituent is 2-(1¢-Oxo-hexadecyl)-1,3,5-tri-hydroxybenzene (�350.5 Da), a compound that waspreviously found in another brown seaweed, Lobopho-ra variegata (Gerwick and Fenical 1982). The pres-ence of a long fatty acid chain (C16) attached to thephloroglucinol molecule region indicates a putativeamphipathic character of this PS extracted from P.gymnospora.

Here, we observed that ALG from two species ofbrown algae, P. gymnospora and F. vesiculosus, differin the degree of bound PS molecules and also havesignificantly different viscosities. The analysis onSEC of the ALG extracted from P. gymnosporarevealed the coelution of uronic acids and PS indi-cating that a significant amount of PS can be boundto the ALG. Hence, we suggest that these two mole-cules form a high-molecular-weight complex in theseaweed cell wall. On the other hand, the samplepurified from F. vesiculosus did not present PS mole-cules bound to the ALG. The absence of PS in thisALG fraction could be related to different extrac-tion and purification procedures that can disruptPS-ALG linkages—for example, the utilization ofhigh alkaline solutions (Koivikko et al. 2005) com-monly used in prepurification process.

It has been suggested that the interaction betweenPS and ALG is accomplished through weak bonds,

such as hydrogen bonds or hydrophobic interaction(McManus et al. 1985, Le Bourvellec et al. 2004,Koivikko et al. 2005). However, it was also proposedthat these interactions may involve covalentbonds, possibly an ester bond or hemiacetal bond(Vreeland et al. 1998a, Schoenwaelder 2002, Arnoldand Targett 2003, Koivikko et al. 2005). The link ofpolysaccharides to phenolic compounds by esterand ⁄ or ether bonds has been demonstrated forsome groups of plants (Lewis and Yamamoto 1990,Lozovaya et al. 1999, de Ascensao and Dubery 2003,Kerr and Fry 2004, Xu et al. 2005).

Some authors suggest that linkages between PSand cell wall polysaccharides occur immediatelyafter the secretion of PS into the cell wall and arerelated to a degradation of PS, which causes modifi-cations of its structure, such as oxidation, necessaryfor PS to establish the linkages with ALG (Koivikkoet al. 2005). Other studies suggested that a specificagent is necessary to catalyze the linkages betweenPS and ALG (Vreeland et al. 1998a, Schoenwaelder2002, Arnold and Targett 2003, Koivikko et al.2005), with VBPO proposed as being the ‘‘catalyst’’for this interaction. However, no strong experimen-tal evidence for this proposition was reported(Vreeland et al. 1998a, Schoenwaelder 2002, Arnoldand Targett 2003, Koivikko et al. 2005, Bitton et al.2006). The three types of haloperoxidases (bromo-peroxidases, chloroperoxidases, and iodoperoxi-dases) were described in several red and brownalgae, such as Corallina, Laminaria, Fucus, and Dict-yota (Rush et al. 1995, Almeida et al. 1998, Shimoni-shi et al. 1998, Vreeland et al. 1998b, Weyand et al.1999, Colin et al. 2004). Despite the diversity amongalgae divisions, the evolution and similarity of thevanadium-dependent haloperoxidases were demon-strated by phylogenetic analysis (Colin et al. 2005).It was observed that the overall protein structuresare highly conserved and also that the active sitesand reaction mechanisms are quite identical (Colinet al. 2005).

In our work, when the ALG preparation fromF. vesiculosus devoid of PS-associated molecules wasused for in vitro binding assays, we observed thatVBPO is required for the formation of PS-ALG com-plexes. The SEC of PS mixed with ALG and VBPOshowed a coelution of PS and ALG (Fig. 6b). WhenVBPO was removed from the incubation mixture,no complex was detected. Thus, we could mimic, invitro, the conditions necessary for the in vivo bind-ing between ALG and PS.

The viscosity measurements revealed that theALG obtained from P. gymnospora had a compara-tively low viscosity, while the one obtained fromF. vesiculosus had a relatively high viscosity. The sam-ples obtained from the in vitro assays revealedthat the addition of PS to solutions of ALG fromF. vesiculosus in the absence of VBPO did notchange the viscosity of the solution. However, whenthe enzyme was added to the solution, a significant

Fig. 8. Graph showing friction coefficient (b) as a function ofsphere height (h) measured using laser tweezers. From thesemeasurements the viscosity (g) is obtained (see eq. 3 in Materialsand Methods for details). Continuous lines in (A) and (B) arecurve fits of the results of b using least square method. Dashedlines in (A) and (B) correspond to the theoretical curvesobtained if we consider that the viscosity is twice the standarddeviation (which means �100% of the data) in both sides of thecurve. All the experimental data (filled circles) fall between thoselines for each set of measurements demonstrating that the experi-mental error corresponds to the standard deviation. (A) b · hcurve obtained from an alginate-water solution (5 mg Æ mL)1),g = 6.6 ± 0.5 cPoise; (B) b · h curve obtained from an alginate-water solution (5 mg Æ mL)1) incubated with VBPO and phenoliccompounds g = 1.6 ± 0.1 cPoise. VBPO, vanadium bromoperoxi-dase.

PHENOL AND ALGINATE COMPLEXES 199

decrease in the viscosity of the ALG was detected(Fig. 8, Table 1) and was similar to that observedfor ALG from P. gymnospora.

The oxidation of uronic acid molecules by VBPOwas proposed as one of the possible reasons for theviscosity decrease (Bouhadir et al. 2001, Lee et al.2002). The oxidation process could cause the dis-ruption of ALG polymers, which also means areduction of ALG molecular weight (Bouhadir et al.2001, Lee et al. 2002). However, in our experi-ments, a significant reduction in ALG molecularweight was not observed, even when ALG was incu-bated only with VBPO.

Due to the biotechnological applications of algi-nates, the influence of PS on rheological propertiesof ALG has been widely studied. Some authors haveshown that PS can inhibit microbial development,thus inhibiting the secretion of alginate lyase by bac-teria and, indirectly, ALG degradation (Moen et al.1997b). Consequently, it was suggested that PS pre-serves the viscous property of ALG (Moen et al.1997b). In contrast, other authors suggested thatthe PS found in ALG fractions is a contaminant thatinduces, in some cases, the reduction in ALG viscos-ity (Moen et al. 1997a,b, Davis et al. 2004, Tamet al. 2006). Nevertheless, we showed that the sim-ple addition of PS to an ALG solution did not causea significant reduction in ALG viscosity, even when

a PS concentration 10 times higher than the ALGconcentration was used. Another study presented asimilar result, revealing that the addition of oxi-dized phenolic polymers (oxidation mediated by aVBPO enzyme) to an ALG solution did not modifyALG viscosity (Bitton et al. 2006).

We propose that the reduction of ALG viscosity iscaused by the binding between PS and ALG medi-ated by VBPO. The PS isolated from P. gymnosporapossess a long fatty acid chain, a structural charac-teristic that increases significantly its hydrophobicproperty. This fatty acid chain may interfere at theinteraction between uronic acid molecules and, con-sequently, inhibit the ALG gelling process that nor-mally occurs in the presence of divalent cationsaccording to the ‘‘egg-box model’’ (Kloareg andQuatrano 1988).

As we propose in a schematic way in Figure 9, theVBPO activity is essential to promote the modifica-tions on PS structure necessary to the binding pro-cess with the anionic groups of the uronic acids(ALG units). These modifications are very similar tothe ones demonstrated in previous works for themechanism of phloroglucinol polymerization (Grossand Sizer 1959, Eickhoff et al. 2001, Oudgenoeget al. 2002, Berglin et al. 2004). It is possible thatoxidation and isomerization are the first modifica-tions of the phloroglucinol units (performed by

Fig. 9. Schematic model for binding process between PS and ALG from brown algae mediated by VBPO enzyme. The first step showsthe oxidation and isomerization of a phloroglucinol unit caused by VBPO. The second step presents the possible binding mechanismrelated to the combination of uronic acid (guluronic acid is shown) and phloroglucinol, reducing the chemical vacancies in both mole-cules. The third step represents the halogenation process performed by VBPO in the remaining vacancy of phloroglucinol. The final stepconsists of the rearrangement of the halogen atom present in the phloroglucinol structure and shows the final molecular structure config-uration. ALG, alginates; PS, phenolic substances; VBPO, vanadium bromoperoxidase.

200 LEONARDO TAVARES SALGADO ET AL.

VBPO). These modifications can produce two chem-ical vacancies in the carbon atom of the cyclic struc-ture of phloroglucinol (step 1). Thereafter, thecombination of oxidized phloroglucinol with uronicacid characterizes the beginning of the binding pro-cess. At this step, one of the vacancies of phloroglu-cinol is reduced, and also the vacancy of the uronicacid anionic group is eliminated (step 2). In step 3,the VBPO activity is related to the halogenation pro-cess in the remaining carbon vacancy of the phloro-glucinol unit. Then, after the rearrangement of thehalogen in the phloroglucinol structure, thebinding process between PS and ALG is completed(step 4).

In conclusion, on the basis of our results of thePS and ALG binding process, we suggest that PS is acomponent of the brown algae cell walls (Vreelandet al. 1998a, Schoenwaelder 2002, Arnold andTargett 2003, Koivikko et al. 2005). However, wecannot judge how important the PS are to cell walldevelopment and if its participation in cell wallformation is a programmed cellular event. Furtherstudies are needed to answer this question. Forexample, specific inhibitors of VBPO activity and PSsynthesis can be used to evaluate the importance ofthese substances in cell wall formation.

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