Hyaluronan‐mediated protective effect against cell damage caused by enzymatically produced hydroxyl (OH·) radicals is dependent on hyaluronan molecular mass

Abstract

Hyaluronan (HA) protected tendon fibroblasts against cell damage mediated by hydroxyl radicals (OH·) as demonstrated by release of ⁵¹Cr from labelled cells. Protection afforded by high molecular mass (M_r) HA (1218 kDa) was much more effective than that provided by lower (176 kDa and 668 kDa) M_r HA. OH· was generated by coupling H₂O₂ produced by glucose oxidase: glucose to [Fe²⁺-EDTA] chelate in a Fenton-type system. The flux of OH· was measured by a spectrofluorimetric assay of salicylate produced by the reaction of benzoate with OH·. Cell damage caused by the OH· generating system was prevented in the presence of catalase, which destroyed H₂O₂. Damage caused in a standard incubation time increased with increased amounts of glucose oxidase. Protection against OH·-mediated cell damage increased with increasing concentration of HA. The presence of HA did not interfere with the enzyme-Fenton system, as monitored by production of gluconate. On the other hand, HA scavenged OH· produced by the enzyme-Fenton system, as shown by competition with benzoate, which produced less salicylate in the spectrofluorimetric assay in the presence of HA. The reaction of OH· with HA was measured directly by a pulse radiolysis technique in which a hydrated electron (e) produced OH· by the reaction with nitrous oxide. Second order rate constants obtained in distilled H₂O or in phosphate buffer showed no dependence on HA M_r. Similarly, fluorimetric assay of the flux of in the enzyme-Fenton system confirmed that HA competed with benzoate, thus lowering salicylate production, and the flux was also independent of the molecular mass of HA. These results demonstrate that part of the HA-mediated protection against enzyme-Fenton produced OH· and other reactive oxygen-derived toxic species was not a consequence of either the primary or secondary structure of HA, but rather depends on higher order HA organization. Some aspects of the formation of the HA meshwork (tertiary structure) are M_r dependent. We therefore propose that cell-anchored HA meshworks excluded relatively large enzyme molecules from the immediate environment of the cell, thus reducing the flux of OH· etc. at the cell surface and diminishing cell damage.