Cu/Zn superoxide dismutase in excretory-secretory products of the human hookworm Necator americanus. An electron paramagnetic spectrometry study.
EPR spectrometry was used to investigate the effect of excretory/secretory product from Necator americanus on superoxide radical anions generated by xanthine/xanthine oxidase as a measure of excretory/secretory product superoxide dismutase activity. Using 1,1',5,5'-dimethylpyrollidine-N-oxide (DMPO) as a superoxide spin-trapping agent a 12-line EPR spectrum characteristic of the DMPO-OOH adduct was observed to decrease in the presence of excretory/secretory product. Superoxide dismutase activity was proportional to excretory/secretory protein concentration, was inhibited with cyanide treatment and was progressively destroyed with increasing time of heat denaturation of excretory/secretory product. Using a purpose-built chamber the superoxide dismutase activity of excretory/secretory product from live worms in culture was shown to accumulate with time to a maximum at 4 h. The electron paramagnetic resonance spectrum obtained for the frozen excretory/secretory product of N. americanus recorded at 77 K is typical of Cu(II) in a protein matrix. The results are consistent with the presence of an active Cu/Zn superoxide dismutase in excretory/secretory product from N. americanus and demonstrate a method for the unequivocal determination of the fate of superoxide anions in the presence of live worms.
Taiwo
F A
FA
School of Pharmacy, De Montfort University, Leicester, UK. ttaiwo@dmu.ac.uk
Brophy
P M
PM
Pritchard
D I
DI
Brown
A
A
Wardlaw
A
A
Patterson
L H
LH
D000818:Animals; D002374:Catalase / Q000032:analysis; D003300:Copper / Q000032:analysis; D003486:Cyanides; D003497:Cyclic N-Oxides / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D005122:Exudates and Transudates / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D017221:Necator americanus / Q000201:enzymology*; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism; D013481:Superoxides / Q000032:analysis
D003486:Cyanides; D003497:Cyclic N-Oxides; D013481:Superoxides; C069977:2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxy; D003300:Copper; C055956:5,5-dimethyl-5-hydroperoxy-1-pyrrolidinyloxy; D002374:Catalase; D013482:Superoxide Dismutase