[Purification, characterization and induction by oxygen of superoxide dismutase from Bacteroides gingivalis 381].

Several strains of Bacteroides gingivalis had strong activities of superoxide dismutase (SOD) and were markedly tolerant in the presence of oxygen in 13 strains of black-pigmented Bacteroides species tested. Thus, the strains were maintained and incubated in the either anaerobic or aerobic system. It was found that the SOD activity was significantly induced by oxygen, especially in B. gingivalis 381. The SODs, anaero-SOD and aero-SOD from the extracts of B. gingivalis 381 cells, each was purified by hydrophobic chromatography followed by anion exchange chromatography, and then by gel filtration, respectively. Both the purified enzymes having molecular weight of about 46,000 consisted of two subunits of equal sizes. Spectral analysis revealed that anaero-SOD had the characteristic A350 of Fe-SOD, but aero-SOD exhibited A475 of Mn-SOD. Both samples contained three isozymes with identical isoelectric points of 5.25, 5.10 and 5.00. On the basis of inactivation of SOD by H2O2, it was shown that aero-SOD consisted of one Mn-SOD and a small quantity of two Fe-SODs, whereas anaero-SOD contained only Fe-SOD. To ascertain whether or not the apoprotein of aero-SOD is the same as that of anaero-SOD, each apoprotein was prepared by dialysis in guanidinium chloride plus 8-hydroxyquinoline. Only one protein band with the same isoelectric point of 5.30 on an isoelectric focusing gel was obtained in each purified SOD sample. Subsequent reconstitution of both apoenzymes with either Fe (NH4)2 (SO4)2 or MnCl2 significantly restored their activity. These reconstituted SODs showed only one protein band with SOD activity on Native-PAGE. The complete amino acid sequence of anaero-SOD was determined by automated Edman degradation of the protein, Achrombacter protease I, endoproteinase Asp-N and tryptic digestion. The sequence consisted of 191 residues corresponding to a molecular weight of 21,500 per subunit. Furthermore, the first 36 amino acid sequence of aero-SOD was determined following N-terminal analysis. The two enzymes had similar amino acid compositions, and their amino-terminal sequences were identical through the first 36 amino acids in which methionine residue was present at N-terminal. These results suggest the three isozymes of either anaero-SOD or aero-SOD in B. gingivalis 381 may be formed from the same apoprotein.