Identification of the Free Radicals formed when Hylauronan Systems (including Hylan) interact with Hydroxyl Radicals (using Electron Paramagnetic Spectroscopy)

Saphwan Al-Assaf1, Clare L. Hawkins2, Barry J. Parsons1, Michael J. Davies2 & Glyn O. Phillips1

1North East Wales Institute, Mold Road, Wrexham, Clwyd LL11 2AW, UK.

2Heart Research Institute, 145 Missenden Road, Camperdown, Sydney NSW 2050, Australia.


Using a direct rapid flow experiment, OH radicals were generated using the Ti(III)-H2O2 couple. Such radicals can abstract H-atoms from carbohydrates. Experiments were carried out at pH 3.6 which resulted in the observation of a weak spectra, with both relatively broad and sharp, isotropic features detected. It is possible to assign the most intense features in the spectrum to two radicals; a doublet (aH 1.36mT, g 2.0049) and a doublet of doublets (aaH 1.86 mT, abH 0.81mT, g 2.0035. The doublet signal has parameters expected of those observed for the radical generated as a result of hydrogen abstraction from the C5 position of glucuronic acid. The doublet of doublet signal has parameters characteristic of a radical with an a-H and a b-H splitting, generated as a result of hydrogen abstraction from C6 on the N-acetyl-D-glucosamine monomer. The EPR data fully support the chain scission process which are observed after radiolysis of aqueous hyaluronan and hylan systems confirming that each OH radical results in a single chain scission. Finally, the ability of cross-linked hylan structure to withstand degradation better than the parent molecule (HA) after OH radical attack is confirmed.

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