High-pressure stopped-flow spectrometry at low temperatures

Abstract

A stopped-flow instrument operating over temperature and pressure ranges of +30 to −20°C and 10 −3 to 2 kbar, respectively, is described. The system has been designed so that it can be easily interfaced with many commercially available spectrophotometers of fast response time, with the aid of quartz fiber optics. The materials used for the construction are inert, metal free and the apparatus has proven to be leak free at temperatures as low as −20°C under a pressure of 2 kbar. The performance of the instrument was tested by measuring the rate of reduction of cytochrome c with sodium dithionite and the 2,6-dichloroindophenol/ascorbate reaction. The dead time of the system has been evaluated to be 20, 50, and ⋍100 ms in water at 20°C, in 40% ethylene glycol/water, and at 20°C and −15°C, respectively. These values are rather pressure independent up to 2 kbar. Application of the bomb was demonstrated using the cytochrome c peroxidase/ethyl peroxide reaction. This process occurred in two phases and an increase in pressure decreased the rates of reactions indicating two positive volumes of activation (Δ V app ≠(fast) = 9.2 ± 1.5 ml·mol −1; Δ V app ≠(slow) = 14 ± 1.5 ml·mol −1, temperature 2°C). The data suggest that the fast reaction could involve a hydrophobic bond, whereas the slow process could be associated with a stereochemical change of the protein. The problem of temperature equilibrium for high-pressure experiments is also discussed.