Understanding fast reaction mechanisms
Stopped-flow spectroscopy plays a key role in deepening our understanding of reaction mechanisms and molecular structure. From the RX2000 accessory used as a teaching aid and to extend the capability of a UV or fluorescence spectrometer through to the high performance SX20 systems, stopped-flow solutions from Applied Photophysics are used in research labs throughout the world. Applications include investigations of protein-protein interactions, ligand binding, electron transfer, fluorescence resonance energy transfer (FRET), protein folding, as well as enzyme, chemical or coordination reactions.
Reactions are followed in solution over timescales in the range of one millisecond to hundreds of seconds. The resulting data enable researchers to determine reaction rates, reveal the complexity of a mechanism, gain information about short-lived reaction intermediates and more.
Enzyme mechanisms have multiple intermediate steps, typically involving substrate binding, product release and intermediate catalytic steps. These steps are all as fast or faster than the steady state turnover rate of the enzyme, so they can only be probed with techniques that can measure reactions on millisecond time-scales. Using stopped-flow spectroscopy to look at the details of the individual steps of an enzymatic reaction in an initial catalytic turnover, it becomes possible to separate and understand the individual reaction steps, such as substrate and product binding or reaction intermediates, identify any rate limiting steps and understand the reaction mechanism.
Pre-steady state kinetic analysis also offers the possibility of identifying individual chemical intermediates and the state of the cofactor in the complex multistep reaction mechanisms associated with redox active proteins and their cofactors, such as flavoproteins or metal complexes such as heme proteins.
Misfolded proteins often fail to function correctly, with excess misfolded proteins accumulating and interfering with cellular funcions. Consequently, they feature in many diseases, including Alzheimer's, Creutzfeldt-Jakob disease (CJD), cystic fibrosis, and many cancers. Understanding the differences in folding and the folding mechanism can bring insight into the causes of these diseases at the molecular level.