This application requests funds to purchase a rapid kinetic system that will allow determination of reaction rates having time constants from microseconds to minutes. It allows independent rate measurements by stopped-flow, quench-flow, temperature-jump, or stopped-flow followed by temperature-jump. The system is manufactured by Hi-Tech Scientific Ltd., and is equipped for either absorbance or fluorescence detection and for simultaneous detection of two optical signals (e.g. scattering and fluorescence, or fluorescence and absorbance), or a signal at two different wavelengths. The two-channel instrument is particularly useful for reactions that are accompanied by an increase in turbidity or a large change of heat, involve vesicular samples, or require monitoring at two absorption or emission peaks. A rapid scanning monochromator is linked to the system for recording spectra as a function of time starting from less than 10 milliseconds after mixing and thus providing spectral information on short-lived species. The quench-flow feature allows sequential termination of a reaction at well-defined intervals starting from several milliseconds. This capability is essential for reactions that cannot be monitored by optical signals. The requested instrument will be shared by ten groups of users involving thirteen senior investigators in six different departments whose current projects are supported by ten different NIH grants and three non-federal grants. Their projects range from solution studies of enzyme mechanism, ligand binding, DNA structure, protein-protein and protein-lipid interactions, and folding/unfolding of proteins to studies on membrane-bound ATPase and mobilization of cytosolic calcium with cell suspensions. The requested instrumentation will replace an obsolete home-built stopped-flow, extend the kinetic capability at the University of Alabama at Birmingham, and enable several investigators to expand the scope of their work and initiate new research directions.
| Lin, S H; Harzelrig, J B; Cheung, H C (1993) Transient kinetics of the interaction of actin with myosin subfragment-1 in the absence of nucleotide. Biophys J 65:1433-44 |
| Lin, S H; Cheung, H C (1992) The kinetics of a two-state transition of myosin subfragment 1. A temperature-jump relaxation study. FEBS Lett 304:184-6 |
