Hyaluronan degradation by copper(II) chloride and ascorbate: rotational viscometric, EPR spin-trapping, and MALDI–TOF mass spectrometric investigations

The degradation of high-molar-mass hyaluronan (HA) by copper(II) chloride and ascorbate was studied by means of rotational viscometry. It was found that even small amounts of CuCl2 present in the oxidative system led to the pronounced degradation of HA, reflected in a rapid decrease of the dynamic viscosity of the biopolymer solution. Such degradation was induced by free radicals generated in elevated amounts in the presence of copper ions. Electron paramagnetic resonance investigations performed on a model oxidative system containing Cu(II) and ascorbic acid proved the formation of relatively stable ascorbate anion radicals resulting from the reaction of ascorbic acid with hydroxyl radicals. In this way, by scavenging the hydroxyl radicals, ascorbic acid protected HA from their degradative action. Matrix-assisted laser desorption ionization–time-of-flight (MALDI–TOF) mass spectrometry was applied to analyze the degraded HA. The results showed that only regular fragmentation of hyaluronan occurred using the mentioned oxidative system that led to the formation of HA oligomers with unaffected primary chemical structure.