CHARACTERIZATION OF ENZYMATICALLY ACTIVE ONCHOCERCA-VOLVULUS CU ZN SUPEROXIDE-DISMUTASE EXPRESSED IN ESCHERICHIA-COLI/

The Onchocerca volvulus superoxide dismutase was expressed in Escheric hia coli, using a protocol designed to produce the native enzyme rathe r than a fusion protein. The recombinant O. volvulus superoxide dismut ase (rOVSOD) was found in the cytosol of the disrupted bacteria and re presented > 10% of the total bacterial protein. The enzyme was purifie d to homogeneity using DEAE-Sepharose chromatography, followed by phen yl-Sepharose chromatography. The rOVSOD was enzymatically active which was demonstrated by its reactivity with O-2(.-) produced either by th e xanthine-xanthine oxidase system or by stimulated eosinophils. The s pecific activity was determined to be 4668 U mg(-1). This activity cou ld be blocked by rabbit antiserum raised against the rOVSOD. The maxim al activity was obtained upon supplementation of the bacterial growth media and enzyme buffer with copper and zinc ions. Activity characteri stics in the presence of inhibitors was also characteristic of a Cu/Zn superoxide dismutase. The rOVSOD has an apparent subunit molecular ma ss of 16000 in SDS-PAGE. The active enzyme behaves as a dimer of 32 kD a as determined by gel filtration.