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The stopped-flow method is a specialised fluorescence spectroscopy technique used to measure the kinetics of fast reactions. By understanding the reaction kinetics, rate, and order; the underlying molecular mechanisms can be determined. A method for determining the kinetics of a reaction is by monitoring the concentration of the reactants over time using absorption or fluorescence spectroscopy.
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 specialised techniques must be used. The stopped-flow method is one of these techniques.
Using this method the reactants are forced into a cuvette. These are then mixed rapidly with the change in absorbance or fluorescence intensity monitored over time. Edinburgh Instruments offers a stopped-flow accessory for both the FLS1000 and the FS5 Fluorescence Spectrometers.
A model system for studying reaction kinetics is the fluorescence quenching of N-acetyltryptophanamide (NATA) by N-bromosuccinimide (NBS). NATA is oxidized by NBS to form a non-fluorescent bromohydrin and the kinetics of the reaction can be investigated by monitoring the change in fluorescence intensity of NATA as a function of time.
When an excess of NBS is used, the reaction exhibits pseudo first order kinetics with a rate constant that is proportional to the concentration of NATA. At the concentrations of NATA used in the following experiment, the reactions are too fast to be monitored using manual mixing and therefore serve as a convinient verification test of the stopped-flow accessory.
Figure 1. Oxidation of N-acetyl tryptophanamide (NATA) by N-bromosuccinimide (NBS).
Methods and Material
For this experiment, NATA and NBS were dissolved in phosphate buffered saline (PBS) at pH 7.3. Three NATA solutions were prepared with concentrations of 5 µM, 10 µM and 20 µM. The NBS quencher was kept in excess with concentrations of 50 µM, 100 µM and 200 µM.
Using the transmission detector of the FS5 Spectrofluorometer with an excitation bandwidth of 3 nm, the absorption spectrum of NATA was recorded. Fluorescence measurements were undertaken using the FLS1000 Photoluminescence Spectrometer equipped with double excitation and emission monochromators, photomultiplier tube detector (PMT-980), a 450 W Xe lamp and the N-M04MM stopped-flow accessory. The emission spectrum of NATA was recorded at an excitation wavelength of 280 nm using excitation and emission bandwidths of 3 nm and 1.5 nm.
The NATA and NBS solutions were loaded into the A & B syringes of the N-M04MM stopped-flow accessory to acquire the fluorescence kinetic measurements. Syringe C was filled with PBS buffer to serve as a reference. The TTL output trigger of the FLS1000 was used to initiate the N-M04MM injection and the total stop volume was set to 400 µl. The kinetic spectra were recorded using an excitation wavelength of 280 nm with a 5 nm bandpass, an emission wavelength of 360 nm with a 5 nm bandpass, and a temporal resolution of 0.2 ms.
Results and Discussion
It can be seen that NATA has an absorption maximum at 280 nm and an emission maximum close to 360 nm. These were chosen as the excitation and emission wavelengths for the subsequent fluorescence kinetic measurements.
Figure 2. Absorption (blue) and emission (red) spectra of NATA in PBS buffer.
The fluorescence quenching kinetic was recorded with a high temporal resolution of 0.2 ms. This greatly improved the quality of the recorded kinetic and enabled accurate fitting of the decay and allowed reliable rate constants to be extracted. The kinetic was fit with a single mono-exponential decay with a time constant of 28 ms. The FLS1000 offers flexible time ranges and resolutions for monitoring kinetics; equally capable of measuring fast decays with temporal resolutions of <0.1 ms and monitoring slower kinetics over the course of hours.
The data also shows that in the absence of NBS the fluorescence remains constant which confirms that the decay observed when NATA and NBS are injected must be fluorescence quenching and not caused by a change in absorbance of the solutions or an artifact in the system.
The difference in fluorescence intensity between the start of the decay and the baseline is due to the dead time of the stopped-flow system. The dead time is defined as the time between solution mixing and the first measurement. The dead time can be calculated from the intercept of the exponential fit and the baseline. This is approximately 12 ms.
Figure 3. Fluorescence kinetics after the injection of 10 µM NATA and 100 µM NBS (red) and after the injection of 10 µM NATA and PBS buffer (blue). Each kinetic trace (dots) is the superposition of 5 consecutive injections and acquisitions. The traces were fitted with a monoexponential fit (red) and a linear fit (blue) using Fluoracle. Each kinetic trace (dots) is the superposition of 5 consecutive injections and acquisitions.
The effect of the concentration of NATA on the rate of fluorescence quenching was then investigated. The fluorescence quenching kinetics of NATA with concentrations of 5 µM, 10 µM and 20 µM were measured and fit using Fluoracle with mono-exponential decays. As the concentration of NATA is increased the time constant of the decay decreases. This indicates that the reaction proceeds more rapidly. This is as expected for first order reaction kinetics. From the decay time constants the first order reaction rates can be calculated and are given in Table 1.
Table 1. Variation of first order rate constant with NATA concentration.
|Concentration of NATA (µM)
||Rate Constant (s-1)
Figure 4. Normalised fluorescence kinetics of NATA quenching by NBS at different concentrations. Solid lines are monoexponential fits using Fluoracle.
Using the FLS1000 with stopped-flow accessory, the fluorescence quenching kinetics of NATA with NBS were measured. The fluorescence quenching kinetics were fit using the FLS1000’s versatile Fluoracle operating software and the first order rate constants of the reaction determined.
This application demonstrates that the FLS1000 combined with the stopped flow accessory is an ideal tool for measuring rapid reaction kinetics. The flexible time ranges and temporal resolutions offered by the FLS1000 for measuring kinetics makes it ideally suited for measuring both fast decays with resolutions of <0.1 ms, as well as the monitoring of slower kinetics over many hours.
This information has been sourced, reviewed and adapted from materials provided by for Edinburgh Instruments.
References and Further Reading
- B. F. Peterman, Measurement of the Dead Time of a Fluorescence Stopped-Flow Instrument, Anal. Biochem. 93, 442-444 (1979)
- John J. Correia and H. William Detrich, III Biophysical Tools for Biologists Volume One: In Vitro Techniques, Academic Press, USA, 457 (2008)
- B. F. Peterman and K. J. Laidler, The Reactivity of Tryptophan Residues in Proteins Stopped-Flow Kinetics of Fluorescence Quenching, Biochem. Biophys. Acta 577, 314-323 (1979)
This information has been sourced, reviewed and adapted from materials provided by Edinburgh Instruments.
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