Time-resolved analysis of macromolecular structures during reactions by stopped-flow electrooptics

Area of Science:

• Biophysical Chemistry
• Molecular Biology
• Analytical Instrumentation

Background:

• Understanding macromolecular structure changes during reactions is crucial.
• Existing methods may have limitations in time resolution or sample requirements.

Purpose of the Study:

• To describe a novel stopped-flow field-jump instrument for analyzing macromolecular structure dynamics.
• To demonstrate its application in studying DNA-ethidium intercalation using electrooptical measurements.

Main Methods:

• Development of a stopped-flow instrument with integrated electrodes in a quartz cuvette.
• Application of electric field pulses (up to 20 kV/cm, nanosecond rise times) after flow-stop.
• Time-resolved optical detection (nanosecond range) and electrooptical measurements.

Main Results:

• The instrument offers high time resolution (approx. 0.5 ms dead time) and low sample demand.
• Ethidium intercalation into DNA showed significant negative dichroism at 0.5 ms, increasing over milliseconds.
• Analysis revealed up to three relaxation processes, indicating ethidium insertion between DNA base pairs.

Conclusions:

• The developed instrument is effective for studying rapid macromolecular reactions.
• Electrooptical measurements provide valuable structural information, as demonstrated by ethidium-DNA intercalation.
• The study elucidates the mechanism and intermediate states of ethidium intercalation into DNA.