Chapter 3
Rapid Reaction Kinetic Techniques Christopher P. Toseland and Michael A. Geeves
Abstract Most biochemical processes occur on sub-second time scales. Relaxation and rapid mixing methods allow reactions from microsecond time scales onwards to be monitored in real time. This chapter describes the instrumentation for these techniques and it discusses general topics of sample excitation and signal detection. Keywords Kinetics • Stopped-flow • Fluorescence • Motor protein • Relaxation kinetics
Abbreviations CCD Deac DWR FRET kcat Kd Km kobs LED Mant NATA
Charge-coupled device Diethylaminocoumarin Direct wave recording Fo¨rster (or fluorescence) resonance energy transfer Catalytic turnover number Equilibrium dissociation constant Michaelis constant Observed rate constant Light-emitting diode 20 (30 )-O-(N-Methylanthraniloyl)N-Acetyl tryptophanamide
C.P. Toseland (*) Chromosome Organisation and Dynamics, Max-Planck Institute of Biochemistry, Martinsried 82152, Germany e-mail:
[email protected] M.A. Geeves School of Biosciences, University of Kent, Canterbury CT2 7NJ, UK e-mail:
[email protected] C.P. Toseland and N. Fili (eds.), Fluorescent Methods for Molecular Motors, Experientia Supplementum 105, DOI 10.1007/978-3-0348-0856-9_3, © Springer Basel 2014
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Pi PMT λex
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C.P. Toseland and M.A. Geeves
Inorganic phosphate Photomultiplier tube Excitation wavelength
Introduction
Many biological processes occur on sub-second time scales. These can be specific processes such as DNA replication by DNA polymerase which can replicate hundreds of bases every second. Or they can be multiple processes which enable a bird to flap its wings 50 times a second. In order to characterise these processes, it is important to be able to determine the dynamic individual steps of these reactions. Steady-state measurements are useful for determination of global kinetic parameters such as the dissociation constant, Kd, the Michaelis constant, Km, and the rate of catalytic turnover, kcat, but these parameters are made up of many individual molecular events which are only revealed by transient kinetics. These events include ligand binding (bimolecular reactions) and dissociation, protein isomerisations (conformational changes) and catalytic events (bond breaking and bond making). Most biological reactions are reversible and therefore reach an equilibrium, or steady state, under experimental conditions. This equilibrium can be perturbed through addition or removal of reactants or through changes in external parameters such as pressure and temperature [1]. Both the forward and reverse rate constants contribute to the rate at which equilibrium is achieved. Therefore, a more dynamic system, with faster forward and reverse rate constants, will reach equilibrium more quickly. It is this approach to equilibrium which is monitored using the methods to rapidly mix reactants or perturb the system. It is then possible to measure the forward and reverse rate constants of individual process to determine how rapidly the system can adapt. Fluorescence is ideal to study these reactions because fluorescence itself is a rapid process, pico- to nanosecond for the absorption and re-emission of the photon, compared to the micro- to second time scale of protein-based biochemical reactions [2]. But, standard spectrophotometers are not sufficient to measure these processes, in which manual mixing and initiating the measurement takes several seconds. The stopped-flow technique allows measurements of reactions typically from
