Cationic Dye for the Specific Determination of Sulfated Polysaccharides

ABSTRACT

A new, highly specific toluidine blue assay for the determination of sulfated polysaccharides is reported. Sulfated polysaccharides may be specifically determined at pH 1 in the presence of carboxylated polysaccharides and phosphorylated molecules such as nucleic acids. In addition, performing the assay at pH 1 extended the linear dynamic range for fucoidan up to 3 g/L but lowered the sensitivity in comparison with pH 7. For a color change at neutral pH, at least a sulfated disaccharide is required, whereas at pH 1, the detection was negative even for a sulfated maltose. A comparison of the fucoidan concentration in crude algae determined by the toluidine blue assay at pH 1 and gel permeation chromatography confirmed the accuracy and specificity of the colorimetric method. The limit of quantitation for the toluidine blue assay at pH 1 was 0.62 g/L for fucoidan. For a deeper understanding of the interaction between the sulfated polysaccharides and the toluidine blue, spectroscopic constants were evaluated by Ketelaar plots for sulfate esters. The maximum value for the molar absorptivity was 4.07 × 10⁴ L/(mol cm), indicating strong affinity. Similar behavior was obtained for fucoidan at pH 7 with a value of 4.46 × 10³ L/mol.

KEYWORDS:

• Colorimetric assay
• spectrophotometry
• spectroscopic constants
• sulfated polysaccharides
• toluidine blue