Sam Lehrer, Ph.D., Senior Scientist
Often a biochemist or biophysicist wishes to measure how fast a substance interacts with another, for example, a substrate binds to an enzyme. This can be done in a simple spectrophotometer if either molecule contains a probe whose spectral properties change during the binding process and if the contents can be mixed faster than the substrate binds to the enzyme. Hand mixing usually takes several seconds; if the reaction is more rapid than that, a faster mixing system becomes necessary. Also, most proteins do not have a sensitive amino acid side chain located in the right place in the structure to have its spectral properties perturbed by the interaction (i.e., an intrinsic probe). To solve these problems, we use a Stopped-Flow instrument which can mix solutions in a couple of milliseconds and use extrinsic probes that we attach to the protein which are sensitive to the interaction under study. Fluorescent probes are good choices because of their high sensitivity. A simplified schematic of the basic components of a stopped-flow fluorescence instrument is shown below:
After filling the drive syringes with solutions A and B (one of which contains molecules with the fluorescent probe), the syringes are pushed within a millisecond. Solution A is rapidly mixed with B and enters a spectrophotometer cell. After the flow is stopped the change in fluorescence is monitored.
The stopped-flow instrument measures the dynamics of interacting proteins. With this instrument we can measure, for example, how fast myosin interacts with actin thin filaments using mutant proteins, e.g. those associated with muscle diseases. Since many biologically important processes function by producing changes in the millisecond time range, it is useful for studying a variety of physiological and pathological systems.