Stopped Flow: Fluorescence Quenching Kinetics Using Stopped-Flow

Study of Fluorescence Quenching Kinetics Using Stopped-Flow

One of the most powerful methods of studying reaction kinetics is fluorescence quenching using the stopped flow technique. For slow reactions, the reactants can simply be mixed by hand or using a magnetic stirrer. However, for fast reactions, where the lifetime of the reaction is comparable with the mixing time, more rapid injection and mixing is required. In stopped flow the reactants are quickly forced into a cuvette where they rapidly mix and the change in absorbance or fluorescence intensity monitored over time. The understanding of reaction kinetics is an important area of study across biology and chemistry. Through the study of reaction kinetics the reaction rate, reaction order and the underlying molecular mechanisms can be determined.

Edinburgh Instruments offers a stopped flow accessory for both the FLS1000 and the FS5 Fluorescence Spectrometers, extending the capability of these systems for the measurement of rapid reaction kinetics. A model system for studying reaction kinetics is the fluorescence quenching of N-acetyltryptophanamide (NATA) by N-bromosuccinimide (NBS).  In this application note the fluorescence quenching of NATA by NBS is measured using the FLS1000 with stopped-flow accessory and rate constants of the reaction determined. By performing the reaction with an excess of NBS, the reaction exhibits pseudo-first order kinetics with a rate constant that is proportional to the concentration of NATA. At the high concentrations used here this reaction is too fast to be monitored using manual mixing and therefore serves as a useful verification test of the stopped-flow accessory.

Fluorescence Quenching of NATA by NBS Using Stopped Flow

Figure 1: Normalised fluorescence kinetics of NATA quenching by NBS stopped flow at different concentrations. Solid lines are monoexponential fits using Fluoracle.

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