Fluorescence spectroscopy stopped-flow

School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton, S016 7PX 

The principles behind both fluorescence and stopped-flow techniques have been described in precedii chapters (2 and 8, respectively) and therefore readers should familiarize themselves with these chapters for some of the background information. In this chapter, we discuss the use of stopped-flow fluorescence spectroscopy and its application to a number of biochemical problems. 

A typical stopped-flow system is assembled from modular components of a conventional spectrophotometer/fluorimeter, a device permitting rapid mixing of the components of a reaction and a data recording system with a fest response. Commercially available instruments offer facilities for the observation of changes in absorption and/or fluorescence emission after rapid mixing of the 

It is important to test the reliability of the stopped-flow instrument using control experiments that test a range of parameters such as the dead time, mixing efficiency and signal output. In general, these tests will be the same for the instrument in the configuration for fluorescence studies as that for absorbance studies, and have been discussed in Chapter 8. 

To check the mixing efficiency of the stopped-flow instrument 

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Stopped-flow fluorescence spectroscopy was used to obtain insight into the interaction of fluoride with tyrosinase (Ty) under pseudo-first-order conditions, as well as to validate the use of fluorescence quenching in studying ligand-binding kinetics. ... 

In addition, many examples of binding systems were investigated by stopped-flow fluorescence spectroscopy, such as the binding of calmodulin to calcineuiin, the binding of guanine to calf spleen purine nucleoside phosphorylase, the nucleotide cofactor binding to the Escherichia coli PriA Helicase, etc. 

Garrity JD, Pauff JM, Crowder MW. 2004. Probing the dynamics of a mobile loop above the active site of LI, a metallo-beta-lactamase from Stenotrophomonas malto-philia, via site-directed mutagenesis and stopped-flow fluorescence spectroscopy. J Biol Chem 279 39663-39670. 

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