1. Acknowledgment is made to the Donors of the American Chemical Society Petroleum Research Fund for support of this research. 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 PI: B. Mark Britt, Ph.D. Undergraduate Student Investigators: Stacey McNevin and Duong Nguyen Department of Chemistry and Physics, Texas Woman’s University, Denton, TX 76204 At 1.0 atm pressure and in 150 mM sodium phosphate (pH = 7.0), bovine carbonic anhydrase undergoes a nondenaturational conformational change at 30.3ºC and an unfolding transition from the physiological conformer to the molten globule state at 67.4ºC. The pressure dependences of the temperatures of these transitions have been studied under reversible conditions for the purpose of understanding ΔHº, ΔSº, 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 30ºC should not be regarded as the physiological structure (the normal bovine body temperature is 38.3ºC). These results should therefore have implications in any area which seeks to correlate the crystal structure of bovine carbonic anhydrase to physiological function. Partial phase diagram of BCA in 150 mM sodium phosphate (pH = 7.0). Shown are the T L→P values (blue circles), T P→MG values (green circles), and the approximate values for the temperature of conversion of the pre-molten globule state to the true unfolded state T PMG→U (red circles) as a function of pressure. Trend lines are second-order polynomial fits to the data. The solid trend lines emphasize that the T L→P and T P→MG values are obtained under reversible conditions; the dashed line emphasizes that the apparent transitions corresponding to the red circles are obtained from irreversible conditions and are thus only approximations. The regions that are associated with the low-temperature conformer L, the physiological-temperature conformer P, the molten globule MG and pre-molten globule PMG states, and the unfolded state U are indicated. The orange square is the normal body temperature of the cow at one atmosphere pressure.