High-pressure spectrometry at subzero temperatures

A reliable, high-pressure bomb is described. The bomb has been designed such that it easily permits absorption and fluorescence measurements; operates between 1 bar and 8 kbar; can be used at temperatures between 30 and −40°C; can be rapidly opened and closed, thus permitting kinetic measurements; and can be easily interfaced with many commercially available instruments. The performance of the bomb was tested using the ice-water phase diagram as the experimental system and light scattering as the quantity measured. At 1.6 kbar, the freezing point of water was −27°C, a value lower than anticipated. Using the horseradish peroxidase-carbon monoxide (HRP-CO) association as a test system, the unexpectedly low freezing point was shown to be due to factors other than glass formation. At 1.6 kbar, association rate coefficients fit a straight line Arrhenius plot (Δ H ‡ = 8.6 ± 0.5kcal mol −1 ) down to −27°C which indicated that a glass was not formed. Applications of the bomb were demonstrated using both the HRP-CO system and the cytochrome P -450 high-spin-low-spin equilibrium. At a fixed temperature, an increase in pressure increased the rate of HRP-CO association, indicating a negative volume of activation (Δ V ‡ = −16.5 ± 0.5cm 3 /mol). In the P -450 system, volume changes (Δ V o ) associated with the high-spin-low-spin equilibrium were determined. At 4°C in Tris-water, pH 7, Δ V water o = 39.3 ± 0.5cm 3 /mol; at −20°C in Tris-water-glycerol (54.3% glycerol by weight), protonic activity, pa H = 9.1, Δ V water-glycerol o = 61.9 ± 0.5cm 3 /mol. The data suggest that major stereochemical changes accompany the transition of P -450 from high to low spin.