Identification of associating carbohydrate sequences with labelled oligosaccharides: Localization of alginate-gelling subunits in cells walls of a brown alga

The gelling subunit of alginate, the major cell-wall polysaccharide of brown algae, was used as a molecular marker for identification of this cell-wall carbohydrate subunit at the cellular level. Short polyguluronate chains were conjugated to fluorescein and used as a probe to identify the gelling regions of alginate in tissue sections from a brown alga. The specificity of the probe for gelling subunits was demonstrated by lack of cell-wall labelling in the absence of calcium, correlation between divalent-cation binding affinities of polyguluronate with labelling intensity, and lack of labelling by fluorescein-conjugated nongelling subunits. The probe labelling-pattern also differed from sulfated fucan distribution. Extracellular matrix and cell walls were labelled on sections of vegetative blade, stipe and reproductive frond of Fucus gardneri Silva. Probe labelling was rapid, being virtually complete within 5 min. Probe labelling in seawater differed markedly from labelling at lower ionic strength and is interpreted as reflecting alginate-gelling properties in natural conditions. High- and low-affinity binding sites are discussed in terms of gelling-subunit length and steric availability. Fluorescein-conjugated polygalacturonate, which also forms calcium dimers, labelled extracellular alginate by formation of mixed polygalacturonate-polyguluronate dimers. Binding by the alginate hybridization probe differs from nucleic-acid hybridization in divalent-cation bridging and the lack of both a conformational transition and polymer polarity.