High-pressure stopped-flow spectrometry at low temperatures

Area of Science:

• Biophysical Chemistry
• Chemical Kinetics
• Spectroscopy

Background:

• Studying reaction kinetics under extreme conditions (high pressure, low temperature) is crucial for understanding molecular mechanisms.
• Existing instrumentation often has limitations in pressure range, temperature control, or interfacing capabilities.

Purpose of the Study:

• To describe a novel stopped-flow instrument designed for kinetic measurements over a wide temperature (-20 to +30°C) and pressure (10⁻³ to 2 kbar) range.
• To demonstrate the instrument's performance and applicability in studying complex biochemical reactions.

Main Methods:

• Construction of a leak-free, inert, metal-free stopped-flow apparatus.
• Interfacing the instrument with commercial spectrophotometers using quartz fiber optics.
• Kinetic measurements of model reactions (cytochrome c reduction, 2,6-dichloroindophenol/ascorbate) and a two-phase enzymatic reaction (cytochrome c peroxidase/ethyl peroxide).
• Evaluation of system dead time and pressure-dependent activation volumes.

Main Results:

• The instrument operates reliably between -20°C and +30°C up to 2 kbar, with pressure-independent dead times (20-100 ms).
• Kinetic analysis of cytochrome c peroxidase revealed two phases with positive activation volumes, suggesting hydrophobic interactions and stereochemical changes.
• The study discusses challenges in achieving temperature equilibrium for high-pressure experiments.

Conclusions:

• The developed stopped-flow instrument is a valuable tool for investigating reaction mechanisms under challenging physical conditions.
• High-pressure kinetic studies provide insights into the molecular events governing enzyme catalysis, such as hydrophobic bond formation and conformational changes.
• The findings contribute to a deeper understanding of protein dynamics and reaction pathways.