Characteristics of xyloglucan after attack by hydroxyl radicals

It has been proposed that plant cell-wall polysaccharides are subject in vivo to non-enzymic scission mediated by hydroxyl radicals ((OH)-O-.). In the present study, xyloglucan was subjected in vitro to partial, non-enzymic scission by treatment with ascorbate plus H2O2, which together generate (OH)-O-.. 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 (OH)-O-.-attack, as indicated by staining with aniline hydrogen-phthalate and by reaction with (NaBH4)-H-3. The introduction of ester and oxo groups supports the proposed sequence of reactions: (a) (OH)-O-.-mediated H-abstraction to produce a carbon-centred carbohydrate radical; (b) reaction of the latter with O-2; and (c) elimination of a hydroperoxyl radical (HO2.). When the partially degraded xyloglucan was reduced with (NaBH4)-H-3 followed by acid hydrolysis, several H-3-aldoses were detected ([H-3]galactose, [H-3]xylose, [H-3]glucose, [H-3]ribose and probably r3Hlmannose), in addition to unidentified H-3-products (probably including anhydroaldoses). H-3-Alditols were undetectable, showing that few or no conventional reducing termini were introduced. Digestion of the (NaBH4)-H-3-reduced, partially degraded xyloglucan with Driselase released 25 times more [H-3]Xyl-alpha-(1 -->6)-Glc than Xyl-alpha-(1 -->6)-[H-3]Glc, suggesting that the xylose side-chains of the xyloglucan had been more heavily attacked by (OH)-O-. than the glucose residues of the backbone. The radioactive xyloglucan was readily digested by cellulase, yielding H-3-products in the hepta- to nonasaccharide range. A fingerprinting strategy for identifying (OH)-O-.-attacked xyloglucan in plant cell walls is proposed. (C) 2001 Elsevier Science Ltd. All rights reserved.