Differential scanning calorimetry of Cu,Zn-superoxide dismutase, the apoprotein, and its zinc-substituted derivatives.
We have employed differential scanning calorimetry (DSC) to investigate the 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 temperatures (Tm) of 89 and 96 degrees 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 degrees C. Significantly, we find that the concentration of O2 present in native SOD samples influences the relative magnitudes of the 89 and 96 degrees 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 protein, 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., & 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 protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Roe
J A
JA
Department of Chemistry and Biochemistry, University of California, Los Angles 90024.
Butler
A
A
Scholler
D M
DM
Valentine
J S
JS
Marky
L
L
Breslauer
K J
KJ
D000818:Animals; D001051:Apoenzymes / Q000378:metabolism*; D001059:Apoproteins / Q000378:metabolism*; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning / Q000379:methods; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D013482:Superoxide Dismutase / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology*
D001051:Apoenzymes; D001059:Apoproteins; D013482:Superoxide Dismutase; D015032:Zinc