Equipment available: Applied Photophysics SX20
Equipment location: New Biochemistry 00-064
Equipment coordinator: Dr David Staunton
Equipment charge: £100/day
Rapid reaction kinetics (Stopped-Flow)
Many biological reactions are rapid and UV/Visible spectroscopy has been widely used to measure rapid reactions and various methodologies developed to make the observations. One method for in widespread use is the ‘stopped flow’ method. This device can measure reactions after rapid mixing with a ‘dead’ time less than 1ms.
To study fast reactions like this it is necessary to mix the reactants quickly and effectively, then observe a spectroscopic change in the mixture as soon as possible after the mixing. This is done by holding the reactants in two syringe reservoirs which can be driven by a pneumatic ram. At the start of a run the solutions pass through a mixer then a closely situated observation cell into another syringe called the stop syringe. The reacting mixture passes from the mixer to the observation cell in less than a millisecond. When the stop syringe has filled, the flow has to stop, and so the material in the observation cell is no longer freshly mixed and changes with time. An electrical contact is made by the stop syringe plunger to trigger the start of measurements. The observation cell is made of quartz, and is arranged so that changes in absorbance or fluorescence at can be measured at visible or ultraviolet wavelengths.
Protein-protein binding: many proteins contain tryptophan residues whose fluorescence changes between bound and unbound states either through quenching, wavelength shift or anisotoropy.
Absorption can also be followed in reactions.
Stopped flow experiments can provide data on association rates for binding and hence on binding constants.
The Applied Photophysics SX20 is a pneumatically operated syringe system capable of 1:1 to 25:1 and with two additional syringes for sequential mixing experiments. The system uses a standard xenon arc lamp for UV and visible wavelengths and is capable of absorption and fluorescence emission measurements over 2 mm or 10 mm path length. The working parts are thermostatted by a recirculating water bath.
Before starting stopped-flow experiments it is essential to establish by conventional spectrophotometry or fluorimetry that a significant change in fluorescence or absorbance occurs as a consequence of the reaction. This will help define optimum wavelengths and conditions. Reaction rates are generally concentration dependent, but so is signal amplitude, hence conditions must be chosen with care. Solutions should be filtered to remove particles.
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