WEIGHT COMPLEXES BETWEEN BROWN ... - Wiley Online Library

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

A VANADIUM BROMOPEROXIDASE CATALYZES THE FORMATION OF HIGH-MOLECULAR-WEIGHT COMPLEXES BETWEEN BROWN ALGAL PHENOLIC SUBSTANCES AND ALGINATES 1 Leonardo Tavares Salgado Instituto de Pesquisas Jardim Botaˆnico do Rio de Janeiro, Diretoria de Pesquisas, MMA, 22460–030, Rio de Janeiro, Brasil

Leonardo Paes Cinelli Laborato´rio de Tecido Conjuntivo, Hospital Universita´rio Clementino Fraga Filho (HUCFF), Instituto de Bioquı´mica Me´dica (IBqM), 21941–590, UFRJ, Rio de Janeiro, Brasil

Nathan Bessa Viana ´ pticas-COPEA, ICB ⁄ Instituto de Fı´sica, 21941–972, UFRJ, Rio de Janeiro, Brasil Laborato´rio de Pinças O

Rodrigo Tomazetto de Carvalho Instituto de Pesquisas Jardim Botaˆnico do Rio de Janeiro, Diretoria de Pesquisas, MMA, 22460–030, Rio de Janeiro, Brasil

Paulo Antoˆnio de Souza Mouraõ Laborato´rio de Tecido Conjuntivo, HUCFF, IBqM, 21941–590, UFRJ, Rio de Janeiro, Brasil

Vale´ria Laneuville Teixeira Departamento de Biologia Marinha, Instituto de Biologia, 24001–970, Universidade Federal Fluminense, Niteroí, Brasil

Marcos Farina Laborato´rio de Biomineralizaçaõ, ICB, 21941–590, UFRJ, Rio de Janeiro, Brasil

and Gilberto Menezes Amado Filho2 Instituto de Pesquisas Jardim Botaˆnico 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 role in the structure of the cell walls of brown seaweeds. However, no clear evidence for this interaction was reported. Vanadium bromoperoxidase (VBPO) has been proposed as a possible catalyst for the binding of PS to ALG. In this work, we studied the interaction between PS and ALG from brown algae using size exclusion chromatography (SEC) and optical tweezers microscopy. The analysis by SEC revealed that ALG forms a high-molecular-weight complex with PS. To study the formation of this molecular complex, we investigated the in vitro interaction of purified ALG from Fucus vesiculosus L. with purified PS from Padina gymnospora (Ku¨tz.) Sond., in the presence or absence of VBPO. The interaction between PS and ALG only occurred when VBPO was added, indicating that the enzyme is essential for the binding process. The interaction of these molecules led to a reduction in ALG viscosity. We propose that VBPO promotes the binding of PS molecules to the ALG

uronic acids residues, and we also suggest that PS are components of the brown algal cell walls. Key index words: binding process; cell wall formation; haloperoxidases; optical tweezers; phenol; phloroglucinol; phlorotannins; polysaccharides; uronic acid; viscosity Abbreviations: A, absorbance; ALG, alginates; ALGFv, alginate from Fucus vesiculosus; ALG-Pg, alginate from Padina gymnospora; Apo, apochromatic; C16, 16 carbon atoms; CCD, charge-coupled device; CD 3 OD, deuterated methanol; DEAE, diethylaminoethyl; FPLC, flow pressure liquid chromatography; NA, number of aperture; Nd-YAG, neodymium-doped yttrium aluminum garnet; NIHUSA, National Institutes of Health of United States of America; Plan, low curvature of field; PS, phenolic substances; SEC, size exclusion chromatography; V 0 , elution point of higher molecular weight fractions; VBPO, vanadium bromoperoxidase; VIS, visible radiation; V t , elution point of lower molecular weight fractions

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Received 25 February 2008. Accepted 21 August 2008. Author for correspondence: e-mail [email protected].

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LEONARDO TAVARES SALGADO ET AL.

The cell walls of brown seaweeds contain mainly cellulose microfibrils, several proteins, and two acidic polysaccharides, named alginates (ALG) and sulfated fucans (Kloareg and Quatrano 1988). Alginate is a linear polysaccharide composed of a-1,4-l-guluronic acid and b-1,4-d-mannuronic acid units. The polymer contains blocks of polyguluronic and ⁄ or polymannuronic sequences (Kloareg and Quatrano 1988). This polysaccharide plays an essential role in the cell wall of seaweeds, acting as an ionic barrier and structural framework (Kloareg and Quatrano 1988, Andrade et al. 2004, Salgado et al. 2005). Recently, phenolic substances (PS), which are secondary metabolites produced by brown algae, were described as components of the ALG complexes in the algal cell wall. Several works have proposed, on the basis of indirect evidence, that PS link with ALG playing an essential role in the algal cell wall structure (Vreeland et al. 1998a, Schoenwaelder 2002, Arnold and Targett 2003, Koivikko et al. 2005, Salgado et al. 2005). The PS, also called phlorotannins, are mainly composed of phloroglucinol (Ragan and Glombitza 1986), and their main cellular localization as soluble compounds is in the organelle termed physode (Arnold and Targett 2003). The PS play important roles in many physiological processes, such as protection of the algal cells against ultraviolet radiation (UV), inhibition of herbivory, blocking of polyspermy, heavy-metal binding 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 the interactions of PS with cell wall polysaccharides may interfere with some cellular processes or with some chemical properties of these polysaccharides (Karez and Pereira 1995, Moen et al. 1997a,b, Tam et al. 2006, Salgado et al. 2007). For example, the binding of heavy metals to physodes can be related to the presence of cell wall polysaccharides in this organelle (Karez and Pereira 1995). It has also been suggested that the viscosity of ALG can be modified by the presence of PS in polysaccharide fractions (Moen et al. 1997a,b, Tam et al. 2006). Moreover, it was also suggested that the capacity of PS to absorb UV in vitro is preserved due to the interactions with the ALG (Salgado et al. 2007). However, the binding process between PS and cell wall polysaccharides and, consequently, the role of PS in the formation of cell wall structure is not yet supported by direct evidence. By some authors (Orive et al. 2002, Koivikko et al. 2005, Tam et al. 2006), the association of PS with ALG is considered a contamination caused by nonspecific interactions between these two compounds during polysaccharide extraction procedures. For others, the formation of these complexes is a specific event with a specific biological role. In the first case, random modifications of the chemical

structure of soluble PS, such as the oxidation of the molecule caused by degradation, is considered a condition that promotes the binding of PS to ALG (Koivikko et al. 2005). Other studies have proposed that the binding mechanism between cell wall ALG and PS is mediated by the vanadium-dependent bromoperoxidase enzyme (VBPO) (Vreeland et al. 1998a, Schoenwaelder 2002, Arnold and Targett 2003). This enzyme was determined as being present in many brown and red algae species (Rush et 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 brown algae, VBPO may act on the oligomerization of PS (Berglin et al. 2004) and also in the formation of natural algal adhesives (Bitton et al. 2006). In relation to these natural adhesives, it was shown that the PS oxidized by VBPO undergo self-assembly and form with the ALG a type of macromolecular cluster, where the PS are encapsulated by the ALG gel network (Bitton et al. 2006). However, the nature of the interactions between PS and ALG in this encapsulation process is unknown, and, consequently, binding between PS and ALG remains undemonstrated. In this regard, our goal was to contribute to a better understanding of these interactions, addressing two main questions. Does the PS specifically bind to the ALG? Does VBPO influence the interactions between these two molecules? In summary, the interactions between PS and ALG were analyzed in samples extracted from P. gymnospora by using optical tweezers microscopy and size exclusion chromatography. We also developed an in vitro assay to evaluate the influence of VBPO on interactions between PS from P. gymnospora and ALG from F. vesiculosus, by measuring their molecular weights and the viscosity of the solutions before and after VBPO incubation, using optical tweezers. MATERIALS AND METHODS

Algae samples. Adult individuals of P. gymnospora measuring 5 cm length were collected at the upper region of the subtidal zone in Sepetiba Bay, located in Rio de Janeiro State, Brazil (2257¢05¢ S, 4354¢28¢ W). Fresh algae were cleaned of epiphytes, briefly washed in Milli-Q H2O, and dried at 60C. Isolation and characterization of soluble PS from P. gymnospora. The extraction of soluble PS was carried out according to the protocol described by Koivikko et al. (2005). The dried sample (50 g) was maintained in 70% aqueous acetone solution (2 L) for 48 h. Thereafter, the acetone was evaporated, the residue was dissolved in Milli-Q H2O (Millipore, Billerica, MA, USA) and centrifuged (5,000g for 10 min, room temperature; Eppendorf, Hamburg, Germany), and the soluble fraction was lyophilized. The extracted PS was diluted in Milli-Q H2O and partially purified using a C-18 column (SepTM Pak C-18 Cartridge, Water Associates, Millipore , Billerica, MA, USA), which was preactivated with ethanol and washed with Milli-Q H2O. The PS adsorbed to the column were eluted with a stepwise gradient of ethanol, evaporated, dissolved in Milli-Q H2O, and lyophilized. This isolated PS sample was purified further with size exclusion chromatography (SEC)

P H E N O L A N D AL G I N A T E C O MP L E X E S

using a Superose 6 column (Amersham Pharmacia Biotech, Buckinghamshire, UK). The SEC was also used to obtain an estimated molecular weight for these PS. The column was equilibrated with 100 mM sodium acetate buffer (pH 6.3), 20 mM EDTA, and 250 mM NaCl, linked to a HPLC System from Shimadzu (Tokyo, Japan). The molecular standards to mark Vo and Vt were dextran blue and glycine, respectively. The flow rate of the column was 15 mL Æ h)1. The fractions were monitored by absorbance at 210 nm and also by carbozole reaction (Diche 1947). During the extraction and isolation procedures, we were careful to protect PS against light degradation. Analysis performed on a UV-mini 1240 UV-VIS Shimadzu spectrophotometer revealed that PS absorbs mainly at 210 nm. The soluble PS were identified by comparison of 1 H-NMR (300 MHz, CD3OD) spectral data with literature data. Isolation of ALG from P. gymnospora. Polysaccharides were extracted from the dry algae by papain digestion (under agitation for 24 h at 60C) and partially purified by three consecutive ethanol precipitations (Alves et al. 1997). The powder of total extracted polysaccharides was diluted in Milli-Q H2O, and, thereafter, 3 M CaCl2 was added to the solution to precipitate the ALG. The precipitated sample was dialyzed against 10% EDTA and thereafter against Milli-Q H2O for 72 h each, and then lyophilized. This sample was fractioned by anion-exchange chromatography on a Mono-Q column (HR 5 ⁄ 5 – Amersham Pharmacia Biotech), equilibrated in 20 mM Tris-HCl (pH 8.0), 5 mM EDTA, linked to an FPLC (Amersham Pharmacia Biotech). The column was eluted with a linear gradient of NaCl (0 to 3 M at a flow rate of 0.5 mL Æ min)1). The fractions were monitored by absorbance at 210 nm to check for the presence of PS and also for uronic acid by the carbozole reaction. The obtained fractions were dialyzed against Milli-Q H2O and lyophilized. These fractions (@10 lg) were applied to a 0.5% agarose gel to evaluate the sample purity. The gel was run for 1 h at 110 V in 0.05 M 1,3diaminopropane ⁄ acetate (pH 9.0). The polysaccharides in the gel were fixed with 0.1% N-cetyl-N,N,N-trimethylammonium bromide solution. After 12 h, the gel was dried and stained with 0.1% toluidine blue O in acetic acid ⁄ ethanol ⁄ water (0.1:5:5, v ⁄ v). The isolated ALG from P. gymnospora was named ALG-Pg. Purification of ALG from F. vesiculosus. A crude ALG preparation from F. vesiculosus was obtained from SigmaAldrich (St. Louis, MO, USA). The ALG was further purified using anion exchange chromatography on DEAE cellulose equilibrated in 100 mM sodium acetate (pH 6.0), containing 20 mM EDTA. The column was eluted with a linear gradient of 0–3.0 M NaCl at a flow rate of 10 mL Æ h)1. Then, a highmolecular-weight fraction was purified from ALG using an SEC column Superose 6. The fractions were monitored by absorbance at 210 nm and also by the carbazole reaction. The purified sample was named ALG-Fv. Monosaccharides composition and molecular weight of ALG-Pg and ALG-Fv. The monosaccharide composition of ALG-Pg and ALG-Fv was estimated by paper chromatography in 1-butanol ⁄ pyridine ⁄ water (3:2:1, v ⁄ v) for 48 h after acid hydrolysis. The molecular weights of the purified ALGs were determined by SEC on Superose 6. The purified fractions were dialyzed against Milli-Q H2O for 72 h and lyophilized. In vitro binding assays with VBPO. The VBPO enzyme used in this work (purchased from Fluka, ref. no. 17965; Fluka, Buchs, Switzerland) was extracted and purified from the red alga Corallina officinalis. The appropriate enzyme and substrates (KBr, H2O2, VO43)) concentrations, temperature, and pH of the incubation solutions were used as reported (Rush et al. 1995). The binding assays were performed in solutions containing 100 mM sodium acetate (pH 6.4), 20 mM EDTA, 250 mM NaCl, 8 mM KBr, 100 lM H2O2, 1 mM VO43), using

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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 solutions were maintained at 30C for 24 h: (1) PS + ALG-Fv + VBPO; (2) PS + VBPO; (3) ALG-Fv + VBPO; (4) VBPO; (5) PS + ALGFv + BSA; (6) PS + BSA; (7) ALG-Fv + BSA; (8) BSA; and (9) PS + ALG-Fv. The products formed were analyzed by SEC on Superose 6. Characterization of the interaction between ALG and PS by using SEC. The PS and ALG are low- and high-molecular-weight compounds, respectively. The possible formation of a highmolecular-weight aggregate containing these two compounds would be a direct evidence of their interaction. Thus, SEC on Superose 6 column of ALG-Pg and of the products of binding assays was used to investigate the interaction between PS and ALG. If the PS and ALG molecules coeluted, the binding between these molecules could be confirmed. Characterization of the interaction between ALG and PS by measuring solutions viscosity with optical tweezers. After elution from SEC column Superose 6, the products obtained from in vitro binding assays were analyzed in optical tweezers microscopy to measure the viscosity values of these sample solutions. The analysis was performed with the following samples diluted in Milli-Q water (5 mg Æ mL)1): (1) ALG-Pg; (2) PS + ALG-Fv, incubated with VBPO; (3) PS + ALG-Fv, not incubated 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-dependence phenomenon influencing the ALG viscosity. The isolated ALG fraction from P. gymnospora, a sample not used in the in vitro binding assays, was also analyzed at 5 mg Æ mL)1. Theory—solution viscosity measurement through Brownian motion evaluation with optical tweezers. The Brownian motion of polystyrene spheres (1.52 ± 0.05 lm radius) is used to measure the viscosity of the medium in which they are immersed (Viana et al. 2006, 2007). A solution containing the polystyrene spheres (10% v ⁄ v; Sigma-Aldrich) was diluted in each of the 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 and 0.3 cm high was glued onto the glass surface with silicone wax. The solution was placed in the region limited by the Oring, and a second coverslip (24 · 24 mm) was placed on top to avoid evaporation. The sample was then observed in an inverted Nikon (Tokyo, Japan) Eclipse TE300 microscope adapted to receiving a Nd-YAG laser beam (1,064 nm wavelength) entering by its EPI-fluorescence port. The use of a high numerical aperture objective lens (Plan Apo 100·; NA 1.4) made possible the creation of an optical trap near the objective lens focus (Ashkin and Dziedzic 1987) allowing the manipulation of small dielectric objects (in the order of few micrometers in diameter). The whole system (microscope, laser source, and mirrors for the laser beam orientation) was mounted on a Newport table (Newport RS 2000TM; Newport Co., Irvine, CA, USA) that minimizes the effects of environmental vibrations. Digitized images were obtained with a CCD camera connected to an Argus-20 system (Hammamatsu; Hammamatsu City, Japan) and a LG3-16 PCI CCIR Scion frame grabber (Scion Co., Frederick, MD, USA). The images obtained were processed and analyzed using the ImageJ freeware software (NIH, Bethesda, MD, USA). The mean square displacement of the variation of the center of mass position in a time interval t is given by: D E ðdqÞ2 ¼ 4Dt ð1Þ where

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D¼

kb T b

ð2Þ

and kbT is the thermal energy (4.14 · 10)21 Joules at room temperature); b, the Stokes friction coefficient, which is a function of the bead radius a; h is the height distance of the polystyrene sphere center to the bottom glass coverslip; and g is the fluid viscosity. The friction coefficient b is given by the Faxen law (Feitosa and Mesquita 1991): 9 a 1 a 3 45 a 4 1 a 5 1 b ¼ 6pga 1 þ 16 h 8 h 256 h 16 h ð3Þ D E 2 is calculated using the digiFor a fixed value of h, ðdqÞ tized (1,000 frames; 30 frames Æ s)1) images of the spheres observed through D E the microscope. From the linear fit of the measured ðdqÞ2 values as a function of time t, using equation 1, the diffusion coefficient (D) is obtained. This value allows the determination of the Stokes friction coefficient b by using equation 2. The sphere heights were then changed experimentally, 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 with an uncertainty of 5% to 10%. The system calibration was done by measuring the viscosity of Milli-Q water.

Fig. 1. Size exclusion chromatogram of isolated phenolic substances (PS). Sample extracted from Padina gymnospora performed in a Superose 6 column (arrows indicate V0 = 5,000 kDa and Vt = 5 kDa, respectively). Fractions were checked for uronic acid and PS detection, by carbazole reaction and by measuring absorbance at 210 nm, respectively (PS = boldline marked with an asterisk and uronic acids = h.) Note the peak close to the column Vt, which confirms that eluted PS have a low molecular weight.

RESULTS

Isolation and characterization of soluble PS and ALG. The PS extracted from P. gymnospora were eluted from a C-18 column with 25%–30% ethanol. The UV spectrum of the isolated PS revealed a major absorbance band at 210 nm. On SEC, the PS were eluted closed to Vt of the column (continuous line in Fig. 1), and the molecular weight was estimated as lower than 10 kDa. No uronic acid was detected in the fractions from SEC by the carbazole reaction (open squares in Fig. 1). All proton bands due to the ortho-acyl phloroglucinol functionality were present, and the remainder of the signals in the 1 H-NMR spectrum were high field methylenes, most of which were observed at the same chemical shifts of d 1.28 (22H, s). From the 1H NMR integration data, one methylene was shown to possess the saturated C16 side chain. The methylene protons a to the carbonyl were observed as a triplet at d 3.24 (2H, t = 7.0 Hz). Two high field aromatic protons appeared as a doublet at d 5.92 (1H, J = 1.5 Hz) and 6.02 (1H, J = 1.5 Hz), indicating two meta-disposed aromatic protons. The metabolite found in our PS extract was 2-[1¢-Oxo-hexadecyl]-1,3,5-trihydroxybenzene (Fig. 2), which has a molecular weight of 350.5 Da (Gerwick and Fenical 1982). The acidic polysaccharides extracted from P. gymnospora were purified by anion exchange chromatography on a Mono-Q column, eluted with increasing NaCl concentrations. Two major components were detected when the fractions were assayed by the phenol sulfuric acid reaction for hexose. The fraction eluted at low NaCl concentration contains uronic acid (as indicated by the positive carbazole reaction) and has no metachromatic property

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

(evaluated by using the reaction with 1,2-diamino4,5-methylenedioxybenzene-DMB), indicative that it contains ALG and not sulfated groups. The fraction eluted from the anion exchange column at high NaCl concentrations presented intense metachromasy and contained mainly the sulfated polysaccharides (mostly sulfated fucan). Agarose gel electrophoresis (Fig. 3) confirmed the purity of the two polysaccharides obtained by anion exchange chromatography. ALG and sulfated fucan have low and high electrophoretic mobility, respectively. The monosaccharides found in these two fractions were analyzed by paper chromatography after acid hydrolysis of the polysaccharides. ALG contained uronic acid while the sulfated polysaccharide was composed mainly of fucose, as expected (data not shown). Analysis of the ALG on a SEC showed that this polysaccharide presented a high molecular weight (5,000 kDa, Fig. 4). Proteins were not detected in this purified fraction (absorbance measurement at 280 nm). In a parallel experiment, we also further purified the ALG preparation from F. vesiculosus (purchased from Sigma-Aldrich). Anion exchange chromatography on DEAE-cellulose revealed two


Fig. 3. Agarose gel electrophoresis of polysaccharides extracted 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.

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

major polysaccharides, eluted at 0.5 and 1.2 M NaCl, when the fractions were assayed by the phenyl sulfuric acid reaction. The polysaccharide eluted at low NaCl concentration is the ALG fraction, as indicated by the positive carbazole reaction for uronic acid and the absence of metachromasy (evaluated by the DMB reaction). On SEC, this polysaccharide is eluted close to V0, indicative of a high molecular weight (5,000 kDa, Fig. 5). When the fractions were assayed by A210, we observed that no PS were coeluted 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 PS by using SEC. The SEC of the ALG purified from P. gymnospora showed that PS coeluted with the

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

polysaccharide close to the column V0 and coincident with the uronic acid peak (Fig. 4). This finding indicates the association of PS with ALG, and that the complex was not dissociated during the extraction and purification procedures. In contrast with this result, the ALG purified from F. vesiculosus did not contain PS associated with the polysaccharide (Fig. 5). Therefore, the preparation of ALG purified from F. vesiculosus is appropriate for an in vitro assay aiming to evaluate the interaction of the polysaccharide with PS. In an initial attempt to detect the possible formation of ALG-PS complex, we incubated these two molecules at 30C for 12 h and analyzed the mixture on a SEC (Fig. 6a). No evidence for the formation of a molecular complex was obtained since PS and ALG were eluted separately from the column. However, when VBPO was added to the PS + ALG-Fv incubation mixture, and the products formed were analyzed by SEC, we clearly detected the formation of the complex between the polysaccharide and PS by coelution of the two molecules close to the V0 of the column (Fig. 6b). The addition of VBPO did not cause significant modification on ALG-Fv molecular weight, as shown in Figure 7. In this sample, a peak corresponding to the enzyme elution was observed close to the Vt of the column (detected by absorbance measurement at 280 nm). When BSA replaced VBPO, we did not detect the formation of a complex by SEC (data not shown). Characterization of the interaction between ALG and PS by measuring solution viscosity with optical tweezers. Isolated PS solution analyzed with the optical tweezers presented an average viscosity value of 1.0 ± 0.1 cPoise (see Materials and Methods for details), while purified ALG-Fv presented an average viscosity value of 8.3 ± 0.9 cPoise (Table 1). The addition of PS to ALG-Fv solutions did not cause a significant

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Fig. 7. In vitro analysis of ALG-VBPO interaction. Size exclusion chromatogram of ALG-Fv sample mixed with VBPO performed in a Superose 6 column (arrows indicate V0 = 5,000 kDa and Vt = 5 kDa, respectively). (VBPO = boldline marked with an asterisk and uronic acids = h.) The VBPO was detected by measuring absorbance at 280 nm. Note that the molecular weight of ALG-Fv was not modified after incubation with VBPO and also that the two compounds did not form a high-molecular-weight complex. ALG, alginates; ALG-Fv, alginate from Fucus vesiculosus; VBPO, vanadium bromoperoxidase.

Fig. 6. In vitro analysis of PS-ALG interactions. (A) Size exclusion chromatogram of mixed ALG-Fv and PS samples performed in a Superose 6 column (arrows indicate V0 = 5,000 kDa and Vt = 5 kDa, respectively). (B) Size exclusion chromatogram of mixed ALG-Fv and PS samples, with VBPO addition, performed in a Superose 6 column. Fractions were checked for uronic acids and PS, by carbazole reaction and by measuring absorbance at 210 nm, respectively. (PS = boldline marked with an asterisk and uronic acids = h.) In (B), note the presence of a PS peak closely associated with the ALG peak, thus revealing the binding mediated by VBPO activity. ALG, alginates; ALG-Fv, alginate from Fucus vesiculosus; PS, phenolic substances; VBPO, vanadium bromoperoxidase.

single species, the molecular weight of PS varied from 0.32 up to 400 kDa (Ragan and Glombitza 1986). This variation may be a consequence of the procedures used to extract PS, which can produce extracts with soluble and nonsoluble PS fractions. When the analysis is restricted to soluble PS, the range of their molecular weights is significantly more narrow and close to a lower molecular weight (Berglin et al. 2004), as also shown in our study. By using SEC, the molecular weight of the purified PS sample was estimated as being close to 10 kDa. The analysis Table 1. Viscosity values expressed in cPoise measured by using optical tweezer. Sample

change (P > 0.05) in the viscosity values (Table 1). Isolated ALG-Pg sample presented viscosity values near 1 cPoise (Table 1), which were very similar to the PS values and significantly different from ALGFv values (P < 0.05). When the solution of ALG-Fv was mixed with PS in the presence of the enzyme VBPO, the viscosity dropped significantly (P < 0.05, see Fig. 8 and Table 1) compared to the value obtained from analysis of ALG-Fv mixed with PS without VBPO addition (Fig. 8 and Table 1). DISCUSSION

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

PSa ALG-Pga (with PS linked in vivo) ALG-Fvb ALG-Fvb mixed with PS (values at mg Æ mL)1) 0.25 2.50 5.00 10.0 50.0 ALG-Fvb linked with PSc

Viscosity in cPoise (±SD)

1.0 ± 0.1 1.1 ± 0.1 6.7 ± 0.5 7.6 7.8 7.6 6.8 6.9 1.6

± ± ± ± ± ±

0.6 0.6 0.5 0.5 0.5 0.1

ALG, alginates; ALG-Fv, alginate from Fucus vesiculosus; PS, phenolic substances; VBPO, vanadium bromoperoxidase. a Sample concentration of 5 mg Æ mL)1. b In these analyzed samples, the ALG were diluted at 5 mg Æ mL)1. c The concentration of PS in the assay using VBPO (last row in the table) was 5 mg Æ mL)1 (see Materials and Methods section for details).


Fig. 8. Graph showing friction coefficient (b) as a function of sphere height (h) measured using laser tweezers. From these measurements the viscosity (g) is obtained (see eq. 3 in Materials and Methods for details). Continuous lines in (A) and (B) are curve fits of the results of b using least square method. Dashed lines in (A) and (B) correspond to the theoretical curves obtained if we consider that the viscosity is twice the standard deviation (which means 100% of the data) in both sides of the curve. All the experimental data (filled circles) fall between those lines for each set of measurements demonstrating that the experimental error corresponds to the standard deviation. (A) b · h curve obtained from an alginate-water solution (5 mg Æ mL)1), g = 6.6 ± 0.5 cPoise; (B) b · h curve obtained from an alginatewater solution (5 mg Æ mL)1) incubated with VBPO and phenolic compounds g = 1.6 ± 0.1 cPoise. VBPO, vanadium bromoperoxidase.

of the isolated PS fraction by NMR revealed that the main PS constituent is 2-(1¢-Oxo-hexadecyl)-1,3,5-trihydroxybenzene (350.5 Da), a compound that was previously found in another brown seaweed, Lobophora variegata (Gerwick and Fenical 1982). The presence of a long fatty acid chain (C16) attached to the phloroglucinol molecule region indicates a putative amphipathic character of this PS extracted from P. gymnospora. Here, we observed that ALG from two species of brown algae, P. gymnospora and F. vesiculosus, differ in the degree of bound PS molecules and also have significantly different viscosities. The analysis on SEC of the ALG extracted from P. gymnospora revealed the coelution of uronic acids and PS indicating that a significant amount of PS can be bound to the ALG. Hence, we suggest that these two molecules form a high-molecular-weight complex in the seaweed cell wall. On the other hand, the sample purified from F. vesiculosus did not present PS molecules bound to the ALG. The absence of PS in this ALG fraction could be related to different extraction and purification procedures that can disrupt PS-ALG linkages—for example, the utilization of high alkaline solutions (Koivikko et al. 2005) commonly used in prepurification process. It has been suggested that the interaction between PS and ALG is accomplished through weak bonds,

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such as hydrogen bonds or hydrophobic interaction (McManus et al. 1985, Le Bourvellec et al. 2004, Koivikko et al. 2005). However, it was also proposed that these interactions may involve covalent bonds, possibly an ester bond or hemiacetal bond (Vreeland et al. 1998a, Schoenwaelder 2002, Arnold and Targett 2003, Koivikko et al. 2005). The link of polysaccharides to phenolic compounds by ester and ⁄ or ether bonds has been demonstrated for some groups of plants (Lewis and Yamamoto 1990, Lozovaya et al. 1999, de Ascensaõ and Dubery 2003, Kerr and Fry 2004, Xu et al. 2005). Some authors suggest that linkages between PS and cell wall polysaccharides occur immediately after the secretion of PS into the cell wall and are related to a degradation of PS, which causes modifications of its structure, such as oxidation, necessary for PS to establish the linkages with ALG (Koivikko et al. 2005). Other studies suggested that a specific agent is necessary to catalyze the linkages between PS and ALG (Vreeland et al. 1998a, Schoenwaelder 2002, Arnold and Targett 2003, Koivikko et al. 2005), with VBPO proposed as being the ‘‘catalyst’’ for this interaction. However, no strong experimental evidence for this proposition was reported (Vreeland et al. 1998a, Schoenwaelder 2002, Arnold and Targett 2003, Koivikko et al. 2005, Bitton et al. 2006). The three types of haloperoxidases (bromoperoxidases, chloroperoxidases, and iodoperoxidases) were described in several red and brown algae, such as Corallina, Laminaria, Fucus, and Dictyota (Rush et al. 1995, Almeida et al. 1998, Shimonishi et al. 1998, Vreeland et al. 1998b, Weyand et al. 1999, Colin et al. 2004). Despite the diversity among algae divisions, the evolution and similarity of the vanadium-dependent haloperoxidases were demonstrated by phylogenetic analysis (Colin et al. 2005). It was observed that the overall protein structures are highly conserved and also that the active sites and reaction mechanisms are quite identical (Colin et al. 2005). In our work, when the ALG preparation from F. vesiculosus devoid of PS-associated molecules was used for in vitro binding assays, we observed that VBPO is required for the formation of PS-ALG complexes. The SEC of PS mixed with ALG and VBPO showed a coelution of PS and ALG (Fig. 6b). When VBPO was removed from the incubation mixture, no complex was detected. Thus, we could mimic, in vitro, the conditions necessary for the in vivo binding between ALG and PS. The viscosity measurements revealed that the ALG obtained from P. gymnospora had a comparatively low viscosity, while the one obtained from F. vesiculosus had a relatively high viscosity. The samples obtained from the in vitro assays revealed that the addition of PS to solutions of ALG from F. vesiculosus in the absence of VBPO did not change the viscosity of the solution. However, when the enzyme was added to the solution, a significant

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decrease in the viscosity of the ALG was detected (Fig. 8, Table 1) and was similar to that observed for ALG from P. gymnospora. The oxidation of uronic acid molecules by VBPO was proposed as one of the possible reasons for the viscosity decrease (Bouhadir et al. 2001, Lee et al. 2002). The oxidation process could cause the disruption of ALG polymers, which also means a reduction of ALG molecular weight (Bouhadir et al. 2001, Lee et al. 2002). However, in our experiments, a significant reduction in ALG molecular weight was not observed, even when ALG was incubated only with VBPO. Due to the biotechnological applications of alginates, the influence of PS on rheological properties of ALG has been widely studied. Some authors have shown that PS can inhibit microbial development, thus inhibiting the secretion of alginate lyase by bacteria and, indirectly, ALG degradation (Moen et al. 1997b). Consequently, it was suggested that PS preserves the viscous property of ALG (Moen et al. 1997b). In contrast, other authors suggested that the PS found in ALG fractions is a contaminant that induces, in some cases, the reduction in ALG viscosity (Moen et al. 1997a,b, Davis et al. 2004, Tam et al. 2006). Nevertheless, we showed that the simple addition of PS to an ALG solution did not cause a significant reduction in ALG viscosity, even when

a PS concentration 10 times higher than the ALG concentration was used. Another study presented a similar result, revealing that the addition of oxidized phenolic polymers (oxidation mediated by a VBPO enzyme) to an ALG solution did not modify ALG viscosity (Bitton et al. 2006). We propose that the reduction of ALG viscosity is caused by the binding between PS and ALG mediated by VBPO. The PS isolated from P. gymnospora possess a long fatty acid chain, a structural characteristic that increases significantly its hydrophobic property. This fatty acid chain may interfere at the interaction between uronic acid molecules and, consequently, inhibit the ALG gelling process that normally occurs in the presence of divalent cations according to the ‘‘egg-box model’’ (Kloareg and Quatrano 1988). As we propose in a schematic way in Figure 9, the VBPO activity is essential to promote the modifications on PS structure necessary to the binding process with the anionic groups of the uronic acids (ALG units). These modifications are very similar to the ones demonstrated in previous works for the mechanism of phloroglucinol polymerization (Gross and Sizer 1959, Eickhoff et al. 2001, Oudgenoeg et al. 2002, Berglin et al. 2004). It is possible that oxidation and isomerization are the first modifications 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 shows the oxidation and isomerization of a phloroglucinol unit caused by VBPO. The second step presents the possible binding mechanism related to the combination of uronic acid (guluronic acid is shown) and phloroglucinol, reducing the chemical vacancies in both molecules. The third step represents the halogenation process performed by VBPO in the remaining vacancy of phloroglucinol. The final step consists of the rearrangement of the halogen atom present in the phloroglucinol structure and shows the final molecular structure configuration. ALG, alginates; PS, phenolic substances; VBPO, vanadium bromoperoxidase.


VBPO). These modifications can produce two chemical vacancies in the carbon atom of the cyclic structure of phloroglucinol (step 1). Thereafter, the combination of oxidized phloroglucinol with uronic acid characterizes the beginning of the binding process. At this step, one of the vacancies of phloroglucinol is reduced, and also the vacancy of the uronic acid anionic group is eliminated (step 2). In step 3, the VBPO activity is related to the halogenation process in the remaining carbon vacancy of the phloroglucinol unit. Then, after the rearrangement of the halogen in the phloroglucinol structure, the binding process between PS and ALG is completed (step 4). In conclusion, on the basis of our results of the PS and ALG binding process, we suggest that PS is a component of the brown algae cell walls (Vreeland et al. 1998a, Schoenwaelder 2002, Arnold and Targett 2003, Koivikko et al. 2005). However, we cannot judge how important the PS are to cell wall development and if its participation in cell wall formation is a programmed cellular event. Further studies are needed to answer this question. For example, specific inhibitors of VBPO activity and PS synthesis can be used to evaluate the importance of these substances in cell wall formation. We thank Mauro Sola-Penna for helping us with enzymatic assays. We also thank Sandra M. F. O. Azevedo and Vale´ria F. Magalha˜es (Laborato´rio de Ecofisiologia e Toxicologia de Cianobacte´rias, IBCCF, UFRJ) for laboratory facilities. Financial support: CNPq and FAPERJ to G. M. Amado Filho and M. Farina, Instituto do Mileˆnio de Nanotecnologia, Instituto do Mileˆnio de Avanço Global e Integrado da Matema´tica Brasileira. Almeida, M., Humanes, M., Melo, R., Silva, J. A., da Silva, J. J. R. F. & Vilter, H. 1998. Saccorhiza polyschides (Phaeophyceae; Phyllariaceae): a new source for vanadium dependent haloperoxidases. Phytochemistry 48:229–39. Alves, A. P., Mulloy, B., Diniz, J. A. & Mourao, P. A. S. 1997. Sulfated polysaccharides from the egg jelly layer are species-specific inducers of acrosomal reaction in sperms of sea urchins. J. Biol. Chem. 272:6965–71. Andrade, L. R., Salgado, L. T., Farina, M., Pereira, M. S., Mouraõ, P. A. S. & Amado Filho, G. M. 2004. Ultrastructure of acidic polysaccharides from the cell walls of brown algae. J. Struct. Biol. 145:216–25. Arnold, T. M. & Targett, N. M. 2003. To grow and defend: lack of tradeoffs for brown algal phlorotannins. Oikos 100:406–8. de Ascensaõ, A. R. F. D. C. & Dubery, I. A. 2003. Soluble and wallbound phenolics and phenolic polymers in Musa acuminata roots exposed to elicitors from Fusarium oxysporum f. sp. cubense. Phytochemistry 63:679–86. Ashkin, A. & Dziedzic, J. M. 1987. Optical trapping and manipulation of viruses and bacteria. Science 235:1517–20. Berglin, M., Delage, L., Potin, P., Vilter, H. & Elwing, H. 2004. Enzymatic cross-linking of a phenolic polymer extracted from the marine alga Fucus serratus. Biomacromolecules 5:2376– 83. Bitton, R., Ben-Yehuda, M., Davidovich, M., Balazs, Y., Potin, P., Delage, L., Colin, C. & Bianco-Peled, H. 2006. Structure of algal-born phenolic polymeric adhesives. Macromol. Biosci. 6:737–46. Bouhadir, K. H., Lee, K. Y., Alsberg, E., Damm, K. L., Anderson, K. W. & Mooney, D. J. 2001. Degradation of partially oxidized

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