Compositional heterogeneity of sulfated polysaccharides synthesized by the brown alga Costaria costata

Abstract

Brown algae contain sulfated polysaccharides, fucoidans, that exhibit various biological activities [1]. The variety of activities exhibited by fucoidans is related to their structural variations. It was shown that species of a single family (genus) of brown algae can contain sulfated polysaccharides that differ in structure and biological activity [2]. Correspondingly, several different fractions of fucoidans can be isolated from a single species [3, 4]. Our goal was to determine the composition of sulfated polysaccharides synthesized by the brown alga Costaria costata [Turn.] Saund (Laminariaceae), which is broadly distributed in seas of the Russian Far East. Polysaccharides were isolated by acid extraction at room temperature [5] from specimens of C. costata collected in July in Troits Bay (Sea of Japan). The polysaccharide fraction was separated into fucoidan (F) and laminaran by chromatography over the hydrophobic sorbent Polikhrom-1 [5]. The monosaccharide composition of the acid-hydrolysis products of the polysaccharides was determined by HPLC in an IC-5000 Biotronik carbohydrate analyzer (Shim-pack ISA-07/S2504 column, 0.4 25 cm, potassium borate buffer, flow rate 0.6 mL/min). Monosaccharides were detected using the bicinchoninate method. Monosaccharides (Rha, Man, Fuc, Gal, Xyl, Glc) were used as standards. The molecular weights (MWs) of the polysaccharides were determined by HPLC in a Shimadzu LC-20A instrument with an RID-10A refractometric detector. Fucoidan samples were dissolved in doubly distilled water and filtered through a membrane filter (0.45 m pore size). Fucoidans were separated over successively connected columns of Shodex Asahipak GS-520 HQ and GS-620 HQ (7.5 300 mm) at 50°C with elution by H2O (0.8 mL/min). Columns were calibrated using standard pullulans of MWs from 180 to 667,000 Da (Polymer Laboratories, USA) and blue dextran (Amersham, Sweden). Analysis of the monosaccharide composition of fraction F showed that fucose and galactose were the major monomers. Mannose, rhamnose, xylose, and glucose were detected in smaller quantities. The MW distribution of fraction F was heterogeneous. Three maxima with the strongest at 300 kDa and weaker ones at 80 and 560 were observed in the MW distribution curve. Fucoidan from C. costata collected in July had the following characteristics: yield 2.5% (% of dry alga); uronic acids, 2.2% (of fraction weight); protein, 3.1% (of fraction weight); SO3Na –, 17.3% (of fraction weight); monosaccharides (mol%): Fuc, 55.1; Gal, 18.1; Man, 9.2; Rha, 11.5; Xyl, 4.3; Glc, 1.8. Protein was determined by the Lowry method [6]; sulfates, by turbidimetry [7]; uronic acids, spectrophotometrically [8]. Fucoidan (F) was also fractionated by ion-exchange chromatography over DEAE-cellulose (3.5 14 cm). Retained polysaccharides were eluted by a linear gradient of H2O–NaCl (2 M) to afford fractions F-0.5 and F-1.5 (Table 1). Fraction F-0.5 contained a low-sulfated fucoidan with a heterogeneous monosaccharide composition and a high content of mannose and uronic acids (37 and 15.5%, respectively, of total monosaccharides) (Table 1). Uronic acid in F-0.5 and F-1.5 was identified after total hydrolysis as glucuronic acid using GC of the polyol acetates [9]. This fraction was characterized as uronofucomannans according to the monosaccharide composition determined using GC of the polyol acetates [9].