Partial Phase Diagram of Aqueous Bovine Carbonic Anhydrase: Analyses of the Pressure-Dependent Temperatures of the Low- to Physiological-Temperature Nondenaturational Conformational Change and of Unfolding to the Molten Globule State

Abstract

At 1.0 atm pressure and in 150 mM sodium phosphate (pH = 7.0), bovine carbonic anhydrase undergoes a nondenaturational conformational change at 30.3 °C and an unfolding transition from the physiological conformer to the molten globule state at 67.4 °C. The pressure dependences of the temperatures of these transitions have been studied under reversible conditions for the purpose of understanding ΔH°, ΔS°, and ΔV for each conformational change. Temperatures for the low-temperature to physiological-temperature conformational change T_L→P are obtained from physiologically relevant conditions using slow-scan-rate differential scanning calorimetry. Temperatures for the physiological-temperature conformation to molten globule state conversion T_P→MG are obtained from differential scanning calorimetry measurements of the apparent transition temperature in the presence of guanidine hydrochloride extrapolated to zero molar denaturant. The use of slow-scan-rate differential scanning calorimetry permits the calculation of the activation volume for the conversion of the low-temperature conformer to the physiological-temperature conformer ΔV^‡ _L→p. At 1.0 atm pressure, the transition from the low-temperature conformer to the physiological- temperature conformer involves a volume change ΔV_L→P = 15 ± 2 L/mole, which contrasts with the partial unfolding of the physiological-temperature conformer to the molten globule state (ΔV_p→MG = 26 ± 9 L/mole). The activation volume for this process ΔV^‡ _L→P = 51 ± 9 L/mole and is consistent with a prior thermodynamic analysis that suggests the conformational transition from the low-temperature conformation to the physiological-temperature conformation possesses a substantial unfolding quality. These results provide further evidence the structure of the enzyme obtained from crystals grown below 30 °C should not be regarded as the physiological structure (the normal bovine body temperature is 38.3 °C). These results should therefore have implications in any area that seeks to correlate the crystal structure of bovine carbonic anhydrase to physiological function.