1. Academic Validation
  2. Fingerprinting of hydroxyl radical-attacked polysaccharides by N-isopropyl-2-aminoacridone labelling

Fingerprinting of hydroxyl radical-attacked polysaccharides by N-isopropyl-2-aminoacridone labelling

  • Biochem J. 2014 Oct 15;463(2):225-37. doi: 10.1042/BJ20140678.
Robert A M Vreeburg 1 Othman B Airianah 1 Stephen C Fry 1
Affiliations

Affiliation

  • 1 *The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences, Daniel Rutherford Building, The King's Buildings, Edinburgh EH9 3JH, U.K.
Abstract

Hydroxyl radicals (•OH) cause non-enzymic scission of Polysaccharides in diverse biological systems. Such reactions can be detrimental (e.g. causing rheumatic and arthritic diseases in mammals) or beneficial (e.g. promoting the softening of ripening fruit, and biomass saccharification). Here we present a method for documenting •OH action, based on fluorescent labelling of the oxo groups that are introduced as glycosulose residues when •OH attacks Polysaccharides. The method was tested on several Polysaccharides, especially pectin, after treatment with Fenton reagents. 2-Aminoacridone plus cyanoborohydride reductively aminated the oxo groups in treated polysaccharides; the product was then reacted with acetone plus cyanoborohydride, forming a stable tertiary amine with the carbohydrate linked to N-isopropyl-2-aminoacridone (pAMAC). Digestion of labelled pectin with 'Driselase' yielded several fluorescent products which on electrophoresis and HPLC provided a useful 'fingerprint' indicating •OH attack. The most diagnostic product was a disaccharide conjugate of the type pAMAC·UA-GalA (UA=unspecified uronic acid), whose UA-GalA bond was Driselase-resistant (product 2A). 2A was clearly distinguishable from GalA-GalA-pAMAC (disaccharide labelled at its reducing end), which was digestible to GalA-pAMAC. The methodology is applicable, with appropriate Enzymes in place of Driselase, for detecting natural and artificial •OH attack in diverse plant, animal and microbial Polysaccharides.

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