Stopped-flow fluorescence spectroscopy

Abstract

Biochemical reactions, such as substrate or coenzyme binding to enzymes are usually completed in no more than 50-100 ms and thus require rapid reaction techniques such as stopped-flow instrumentation for their study. Fortunately, many such reactions can be followed by changes in the absorption properties of the substrate, product or coenzyme, and examples of these have been described in Chapters 1, 7 and 8. An alternative possibility is that during the reaction there is a change in the fluorescence properties of the substrate, coenzyme or the protein itself. Some reactions, particularly those involving the oxidation/ reduction of coenzymes, involve both changes in absorption and changes in fluorescence emission intensity. In many cases, the fluorescence properties of the ligand or protein itself may change when a complex is formed, even in the absence of a full catalytic reaction occurring, e.g. the protein fluorescence emission of most pyridine or flavin nucleotide-dependent dehydrogenases is quenched when NAD(P)H or FADH (respectively) binds to them, due to resonance energy transfer from the aromatic amino acids of the protein to the coenzyme. Conversely, the fluorescence emission from the reduced-coenzymes is usually enhanced on formation of the complex with these enzymes (1-3). The principles behind both fluorescence and stopped-flow techniques have been described in preceding 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 fast response. Commercially available instruments offer facilities for the observation of changes in absorption and/or fluorescence emission after rapid mixing of the reagents. These measurements can often be made simultaneously due to the different optical requirements of the two spectroscopic techniques. Figure 1 gives a generalized diagram of the geometry of a stopped-flow system able to simultaneously measure changes in absorption and fluorescence intensity of a reaction.