Detection of free radicals formation in haemolymph of insects by EPR spectroscopy

The generation of reactive oxygen species during oxidation of DOPA (3-(3,4-dihydroxyphenyl-DL-alanine) in haemolymph of insects has been studied by electron paramagnetic resonance with spin traps 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide and 1-hydroxy-3-carboxy-pyrrolidine. The data support the formation of DOPA-derived highly reactive intermediates during melanization in haemolymph of insects. The formation of superoxide has not been detected apparently due to reaction of DOPA with superoxide. The rate constant of the latter reaction was estimated ask≈5·105 M−1.c−1. The formation of DOPA-semiquinone in haemolymph has been measured using spin stabilization ofo-semiquinones by Mg2+. The comparative studies in haemolymph of intact insects and insects infected by fungal infection suggest an important role of DOPA-semiquinone production in the immune status of insects.