Formation of compound 305 requires the simultaneous generation of both alloxan and GSH radicals

This in vitro study investigates the conditions under which "compound 305" is formed. Using HPLC, ESR as well as UV spectroscopy, "compound 305" was largely separated and characterized. It has an absorption peak at 314 nm, which changes after reoxygenation to shorter wavelengths within hours. The retention time of "compound 305" amounts to 10.93 +/- 0.042 min. The formation of "compound 305" does not depend on alloxan (ALX) or reduced glutathione (GSH), but most likely on the steady-state concentration of the paramagnetic derivatives of both reactants (ALX* and GS*). The alloxan radical (ALX*) is formed by either a one-electron transfer from e. g. GSH to alloxan or oxidation of dialuric acid. The concentration of the ALX* was determined to be 12 +/- 3.6 micromol/l using the stable ultramarine radical as an ESR standard. ALX* is stable only under anaerobic conditions. It disappears within 2 min in air. Since formation of "compound 305" needs both ALX* as well as GS*, which are also necessary for the generation of reactive oxygen species (ROS), it is assumed that formation of "compound 305" diminishes the toxicity of alloxan.