Characteristics of xyloglucan after attack by hydroxyl radicals Original Research Article

It has been proposed that plant cell-wall polysaccharides are subject in vivo to non-enzymic scission mediated by hydroxyl radicals (radical dotOH). In the present study, xyloglucan was subjected in vitro to partial, non-enzymic scission by treatment with ascorbate plus H2O2, which together generate radical dotOH. The partially degraded xyloglucan appeared to contain ester bonds within the backbone, as indicated by an irreversible decrease in viscosity upon alkaline hydrolysis. Aldehyde and/or ketone groups were also introduced into the polysaccharide by radical dotOH-attack, as indicated by staining with aniline hydrogen-phthalate and by reaction with NaB3H4. The introduction of ester and oxo groups supports the proposed sequence of reactions: (a) radical dotOH-mediated H-abstraction to produce a carbon-centred carbohydrate radical; (b) reaction of the latter with O2; and (c) elimination of a hydroperoxyl radical (HO2radical dot). When the partially degraded xyloglucan was reduced with NaB3H4 followed by acid hydrolysis, several 3H-aldoses were detected ([3H]galactose, [3H]xylose, [3H]glucose, [3H]ribose and probably [3H]mannose), in addition to unidentified 3H-products (probably including anhydroaldoses). 3H-Alditols were undetectable, showing that few or no conventional reducing termini were introduced. Digestion of the NaB3H4-reduced, partially degraded xyloglucan with Driselase released 25 times more [3H]Xyl-α-(1→6)-Glc than Xyl-α-(1→6)-[3H]Glc, suggesting that the xylose side-chains of the xyloglucan had been more heavily attacked by radical dotOH than the glucose residues of the backbone. The radioactive xyloglucan was readily digested by cellulase, yielding 3H-products in the hepta- to nonasaccharide range. A fingerprinting strategy for identifying radical dotOH-attacked xyloglucan in plant cell walls is proposed.