Effect of Oxygen Free Radicals on Corneal Collagen

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Abstract
Corneal collagen was labeled in vivo by injection of 14C-proline into the anterior chamber of rabbit eyes. The isolated corneal collagen was incubated in iron-free phosphate buffered saline (pH 7.4) containing I mM axorbate and 0.1 mM CuSO4 for either 1 hour or 3 hours at 37°. Addition of 2 volumes of 8 M urea-I mM dithiothreitol and heating for 1 min at 100° solubilized virtually all of the collagen in the control incubations but left a significant amount of insoluble collagen in specimens exposed to the hydroxyl radical generating system. This residue amounted to 19% and 38% of the initial radioactivity in samples incubated for 1 h and 3 h, respectively. The chromatographic profiles (gel filtration on CL-4B) of the soluble fraction showed an increase in both aggregation and degradation products of collagen in the 1 h incubation mixture, whereas after 3 h there was an increase only in degradation products. These observations suggest that additional crosslinking of the soluble collagen aggregates observed at 1 h may be responsible for their subsequent disappearance at 3 h, with concomitant increase of the insoluble fraction. Collagen degradation by OH may play a role in corneal ulceration, whereas hydroxyl radical-mediated crosslinking is consistent with age-dependent increases in insoluble collagen.