Fingerprinting of polysaccharides attacked by hydroxyl radicals in vitro and in the cell walls of ripening pear fruit

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Abstract
Hydroxyl radicals (•OH) may cause non-enzymic scission of polysaccharides in vivo, e.g. in plant cell walls and mammalian connective tissues. To provide a method for detecting the action of endogenous •OH in vivo, we investigated the products formed when polysaccharides were treated with •OH (generated in situ by ascorbate-H2O2-Cu2+ mixtures) followed by NaB3H4. Treatment with •OH increased the number of NaB3H4-reacting groups present in citrus pectin, homogalacturonan and tamarind xyloglucan. This increase is attributed partly to the formation of glycosulose and glycosulosuronic acid residues, which are then reduced back to the original (but radioactive) sugar residues and their epimers by NaB3H4. The glycosulose and glycosulosuronic acid residues were stable for > 16h at 20°C in ethanol or buffer (pH4.7), but were destroyed in alkali. Driselase-digestion of the radiolabelled polysaccharides yielded characteristic patterns of 3H-products, which included galactose and galacturonate from pectin, and isoprimeverose, galactose, glucose and arabinose from xyloglucan. Pectin yielded at least eight 3H-labelled anionic products, separable by electrophoresis at pH3.5. The patterns of radioactive products form useful ‘fingerprints’ by which •OH-attacked polysaccharides may be recognized. Applied to the cell walls of ripening pear (Pyrus communis) fruit, the method gave evidence for progressive •OH radical attack on polysaccharides during the softening process.