In this paper, we demonstrate that one of the effects of the reaction of hydroxyl radicals with proteins is that the imidazole ring sidechain of histidine residues is progressively opened up and destroyed. Eventually, histidine turns into glycine, with supporting evidence from amino acid analysis data. In intermediate stages, the opened up sidechain of histidine forms isopeptide linkages with sidechains on other protein chains leading to the formation of protein-protein crosslinks. These crosslinks survive treatment with beta-mercaptoethanol during preparation of hydroxyl-radical treated protein samples for SDS-PAGE electrophoretic analyses. The involvement of histidine in the formation of crosslinks is additionally confirmed through the observation that treatment of proteins with diethypyrocarbonate (DEPC) reduces and obviates crosslink formation, by protecting the imidazole sidechain from oxidation. Besides this insight, a key highlight of the paper is the manner of production of hydroxyl radicals in situthrough the generation and release of the radicals into aqueous solution from a potent photosensitizer adsorbed onto a solid substrate (resin beads) kept in suspension through stirring and in the light path. The photosensitizer creates the radicals from water. The paper shows how hydroxyl radical-mediated damage to the lens crystallins causes these proteins to crosslink, lose stability and precipitate, offering insight into the development of cataracts in lens tissue.

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