What is stopped-flow fluorescence?

Stopped-flow fluorescence is a technique used in biochemical research to monitor the rapid kinetics of enzyme-catalyzed reactions and protein interactions. It involves the rapid mixing of two solutions, one containing a fluorescent molecule and the other containing an enzyme or protein, in a stopped-flow device. The resulting fluorescence signal is then measured in real-time to determine the kinetics of the reaction.

Typically, the fluorescence signal is collected using a photomultiplier tube and recorded using computer software. The technique allows for the measurement of fluorescence changes on a millisecond timescale, making it useful for studying fast biological reactions.

One of the advantages of stopped-flow fluorescence is that it can be used to study both static and kinetic properties of biomolecules. For example, it can be used to measure the equilibrium dissociation constant (Kd) of protein-protein or protein-ligand interactions, as well as the rate constants (kcat and Km) of enzymatic reactions.

Stopped-flow fluorescence also has some limitations, including the need for specialized equipment and the requirement for a fluorescent probe that is sensitive to the reaction being studied. Additionally, the presence of other fluorescent molecules in the sample may interfere with the measurement.