Stability of Myoglobin at Both Subambient and High Temperatures as Measured by Fluorescence

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We examined the cold and heat denaturation of horse heart metmyoglobin (HHM) using an improved version of our recently published fluorescence ratio intrinsic basis states analysis (FRIBSTA). This protein is of considerable interest because its thermodynamic properties have been extensively studied, and it is one of several globular proteins used as models to investigate native protein stability. According to the published analyses, at pH values close to neutral in dilute electrolyte solution native sperm whale metmyoglobin has a stability maximum relative to its unfolded form in non-denaturing conditions of about +50kJ mole^<-1> at 〜35℃. At 0℃ the native protein is estimated to have a somewhat decreased stability of +35kJ mole^<-1>. In contrast, FRIBSTA indicates a detectable level of denaturation at 0℃ and pH 7, and considerably more at 0℃ and pH 4. Comparison with far UV circular dichroism (CD) spectra recorded at -2℃, 35℃, and in the range of heat denaturation reinforce the view that HHM has an intact secondary structure at -2℃ and pH 7. This would indicate that a significant proportion of myoglobin molecules are in the molten globule configuration at modest subambient temperatures. FRIBSTA derived values of the heat denaturation enthalpy of myoglobin at pH 4 and pH 7 are consistent with the literature data. When utilizing the published thermodynamic parameters of myoglobin's heat denaturation, the two state model gives estimates of the extent of cold denaturation fundamentally different from the low temperature FRIBSTA estimates of the thermodynamic stability of the protein. We briefly discuss the implications of these findings.

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