Nanosecond time-resolved fluorescence measurements during protein denaturation

Area of Science:

• Biophysical Chemistry
• Biochemistry
• Analytical Chemistry

Background:

• Studying slow reaction kinetics (seconds to hours) with traditional nanosecond time-resolved fluorescence is challenging.
• Existing methods may lack the sensitivity or temporal resolution for long timescale processes.

Purpose of the Study:

• To develop and validate a modified pulse fluorometer for analyzing slow kinetic processes.
• To apply this new methodology to investigate the acid-induced unfolding of horse liver alcohol dehydrogenase (LADH).

Main Methods:

• Modification of a pulse fluorometer to collect sequential, short data sets stored on computer disk.
• Analysis of fluorescence decay curves as a function of time using a biexponential model.
• Monitoring changes in intrinsic fluorescence decay parameters of LADH.

Main Results:

• The modified fluorometer successfully captured fluorescence decay data over extended periods.
• Analysis revealed that acid-induced LADH unfolding could be described by a biexponential function.
• A decrease in steady-state fluorescence was attributed to a reduction in the amplitude of the longer decay constant.

Conclusions:

• Nanosecond time-resolved fluorescence, when adapted, can effectively study reactions on second-to-hour timescales.
• The method provides insights into the conformational changes of proteins like LADH during unfolding.
• This technique offers a valuable tool for kinetic studies in biophysical and biochemical research.