On the Stability of Bovine Superoxide Dismutase

Abstract

Abstract 1. Apo-superoxide dismutase was more labile toward a variety of inactivating stresses than was the holoenzyme. 2. Cu++ restored catalytic activity to the apoenzyme and markedly enhanced its thermal stability but Cu++ plus Zn++ were needed to attain the stability of the native enzyme. 3. Co++ or Hg++ were able to replace Zn++ in increasing the thermal stability of the Cu++-repleted apoenzyme. Indeed, the enzyme containing Hg++ + Cu++ was more stable than the native superoxide dismutase. In contrast no metal was found which could replace Cu++ in restoring activity to the apoenzyme. 4. Arrhenius plots showed that rates of irreversible inactivation were a biphasic function of temperature in the case of apo-superoxide dismutase but were monophasic in the presence of those metals which affected thermal stability. Metal binding apparently changed the pathway of thermal inactivation. 5. Since excess Cu++ could not substitute for Cu++ + Zn++ and since excess Zn++ could not prevent the effect of Cu++ we conclude that the Cu++ site cannot bind Zn++ and the Zn++ site cannot bind Cu++, although it can bind Co++ or Hg++. 6. Native superoxide dismutase retained much of its activity in the presence of 10.0 m urea or 4% sodium dodecyl sulfate and, although high concentrations of guanidinium chloride did inhibit, its effects were reversible by dialysis or dilution. 7. Guanidinium-chloride did however perturb the structure of the holoenzyme in a way which rendered its metals accessible to EDTA such that exposure to EDTA plus guanidinium chloride resulted in progressive inactivation. 8. It appears likely, on the basis of data available at present, that Zn++ plays a structural role in the bovine erythrocyte superoxide dismutase and lends it enhanced stability whereas Cu++ is directly involved in the catalytic cycle.