Differential scanning calorimetry of copper-zinc-superoxide dismutase, the apoprotein, and its zinc-substituted derivatives

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Abstract
We have employed differential scanning calorimetry (DSC) to investigate thermally induced unfolding of native Cu,Zn-superoxide dismutase (SOD), the apoprotein derived from native SOD, and the zinc-substituted derivatives of the apoprotein. We observe two overlapping melting transitions for native bovine SOD with heat capacity maxima at temperature (Tm) of 89 and 96.degree.C when a scanning rate of 0.82 deg/min is employed. By contrast, the dithionite-reduced native SOD (which contains Cu+ rather than Cu2+) exhibits only a single transition at 96.degree.C. Significantly, we find that the concentration of O2 present in native SOD samples influences the relative magnitudes of the 89 and 96.degree.C peaks. Specifically, the lower temperature transition becomes less pronounced as the concentration of O2 in the sample decreases. On the basis of these observations, we propose that the lower temperature peak corresponds to the melting of the oxidized native proteon, while the higher temperature peak reflects the melting of the reduced native protein, which forms spontaneously during the heating process. Our interpretation profoundly differs from that of Lepock et al. [Lepock, J. R., Arnold, L. D., Torrie, B.H., Andrews, B., and Kruuv, J. (1985) Arch. Biochem. Biophys. 241, 243-251], who have proposed that the low-temperature transition corresponds to the reduced form of the protein. We present evidence that suggests that their experiments were complicated by the presence of potassium ferrocyanide, which, in addition to reducing the cupric center, also perturbs the proteins. In contrast to native SOD, we observe that apo-SOD melts monophasically with a Tm of only 57.degree.C at the identical scan rate used for melting the native protein. This result demonstrates that metal ions play a significant role in enhancing the thermal stability of native SOD. A series of DSC melts on apo-SOD as a function of added Zn2+ reveals that binding of the first 2 equiv of Zn2+ ions induces most of the overall thermal stabilization observed for the binding of a total of four Zn2+ ions to the SOD protein dimer. While not altering the peak area, addition of the third and fourth equivalents of Zn2+ does cause the melting transition to sharpen and to exhibit a small increase in Tm. We also find that the DSC melting profiles for SOD exhibit a strong dependence on scan rate. Such a scan rate dependence can occur when the overall process is kinetically limited and/or irreversible. Consequently, we have considered these possibilities in our interpretation of the calorimetric data.