A study on the reconstitution of iron-superoxide dismutase from Pseudomonas ovalis.

Abstract

Iron-free aposuperoxide dismutase was prepared by two methods from a native enzyme from Pseudomonas ovalis. The first method was the incubation of the native enzyme with sodium carbonate buffer (pH 11.0) containing dithiothreitol and EDTA under anaerobic conditions with subsequent dialysis to return the pH to 7.8. The second method was the incubation of the native enzyme in 3-(N-morpholino)-propanesulfonic acid-NaOH buffer (pH 7.0) containing urea, dithiothreitol, and EDTA under anaerobic conditions with subsequent dialysis to remove the urea. The reconstituted enzyme was obtained by incubation of the apoenzyme with the corresponding buffer described above, with addition of ferrous ammonium sulfate under anaerobic conditions. The apoenzyme had neither any significant iron content not enzymatic activity. The reconstituted enzyme had the same amount of iron and the same enzymatic activity as the native one. The apoenzyme had no visible absorption, but the reconstituted enzyme had the same visible absorption as the native enzyme. The four sulfhydryl groups of the apoenzyme showed high reactivity with 5, 5‘-dithiobis-(2-nitrobenzoic acid), while those of the native and reconstituted enzymes had little reactivity. The fluorescence intensity of the apoenzyme was about three times that of the native and reconstituted enzymes. Circular dichroism spectra of the apo- and reconstituted enzymes in the 200–250 nm region were similar in shape to that of the native enzyme. In the 250–340 nm region, the circular dichroism spectrum of the apoenzyme was different from that of the native enzyme; this difference was greatly reduced in the reconstituted enzyme. The electron paramagnetic resonance spectrum of the reconstituted enzyme showed the same signal prameters as the native enzyme. Thermal inactivation kinetics of the reconstituted enzyme were compared with those of the native enzyme; the reconstituted enzyme was slightly less stable than the native enzyme.