Microsecond time-scale kinetics of transient biochemical reactions

Area of Science:

• Biophysical Chemistry
• Biochemistry
• Spectroscopy

Background:

• Enzyme kinetics and protein folding studies require high time resolution.
• Existing methods have limitations in capturing rapid molecular events.

Purpose of the Study:

• To develop a continuous-flow instrument for microsecond time-scale mixing.
• To achieve unprecedented dead-time for mechanistic studies.
• To enable comparison of experimental data with molecular dynamics simulations.

Main Methods:

• Developed a continuous-flow microsecond time-scale mixing instrument.
• Integrated a micro-mixer with a 2.7 μs mixing time and a 30 mm flow-cell.
• Utilized ultraviolet-visible spectroscopy for reaction monitoring.
• Achieved a dead-time of 3.8 ± 0.3 μs and spectral resolution of 0.4 nm.

Main Results:

• Studied spontaneous protein refolding of pH-denatured cytochrome c.
• Identified three folding intermediates with time constants of 4.7 μs, 83 μs, and 345 μs.
• Demonstrated the instrument's capability for microsecond time-resolved measurements.

Conclusions:

• The developed instrument provides a powerful tool for mechanistic studies in enzyme kinetics and protein folding.
• It enables the investigation of rapid events in biomacromolecules.
• Opens possibilities for comparing experimental results with molecular dynamics calculations on overlapping timescales.