Steady-state measurements only give the rate limiting step in a pathway, but the individual rate constants which establish the steady-state are not determined.  Many biological processes occur in less than a second, and in order to understand these processes, they need to be measured.  Rapid-reaction (relaxation) techniques have been developed to measure these systems.  In these cases, the reaction under investigation is perturbed by an external factor such as pressure or temperature, and can be initiated by rapid change in either of these properties. 

 

Stopped-Flow

 

Stopped-flow uses optical signals to measure a process in which the optical properties change during the time course of the reaction.  This is often achieved by using fluorophores.  These can be intrinsic to the system under investigation, an example being tryptophan, or extrinsic, such as Cy3 covalently attached to a cysteine.  The key principle is that the fluorescent changes report the progress of the reaction. 

 

Principle:

 

For a single mixing experiment, two solutions are rapidly pushed through a mixer into an observation chamber.  The new mixture displaces the old solution into the stop syringe.  This forces the plunger against the stop block which triggers the acquisition to begin.  Reactions can then be monitored from the point of the stop therefore, a small part of the reaction is lost.  This lost time is called the instrument dead time (2 – 3 ms), which is the time it takes the solution to reach the observation from the initial mixing.  This technique takes its name from this action, hence stopped-flow. 

 

Double mixing experiments can also be carried out.  This method allows two solutions to be mixed and after a chosen time, the mixed solution is mixed once more.  This method allows the observation of the formation of short-lived intermediates followed by their degradation.

 

 

(A) Single Mix system. Reactants A and B are rapidly mixed and the flow is stopped using the stopper plate.  The fluorescence of the mixed solution is measured in the cell.  See Figure 2.5 for details.  (B)  Double-mix system.  Reactants A and B are rapidly mixed (Mixer 1).  After a chosen time period a second mix occurs (Mixer 2).  The fluorescence following this mix is observed in the Cell.  

 

  

Fluorescence Anisotropy

 

Fluorescence anisotropy can also be measured in real time using the stopped-flow technique.  Fluorescence anisotropy requires the emission to be measured through parallel and perpendicular polarisers, with respect to the excitation plane.  In order to achieve this, the stopped-flow can be equipped with two photomultipliers in a ‘T-format’ to enable the parallel and perpendicular emission to be measured simultaneously.  Click here for more information on Fluorescence Anisotropy.

 

(A) Fluorescence setup. White light from a Xenon –Mercury lamp passes through a monochromator, exciting the mixed solution at a chosen wavelength. Emission is measured at 90° through an emission cut off filter, at the appropriate wavelength, and the intensity measured using a photomultiplier tube (PMT). An analogue to digital converter is used to transfer the data to the computer.  The response of the analogue to digital converter (in percent) is used as the units of fluorescence by the software.  (B) Polarisation setup. White light from a Xenon–Mercury lamp passes through a monochromator, and a vertical polarizer (//) exciting the mixed solution with vertically polarized light at a chosen wavelength.  Emission is measured using two channels simultaneously, both at 90° to the excitation plane.  Each channel has either a parallel (//), or perpendicular polarizer (-), which polarize the emitted light before going through an emission cut off filter, at the appropriate wavelength.  The intensity is measured using a photomultiplier tube (PMT) for each channel. The signal is processed as before and the software converts the signal to anisotropy.

 

The majority of the measurements we perform in the stopped-flow apparatus concern protein-ligand interactions.  In order to fully understand the measurements, it is important to have an idea of the conditions used and how this relates to the kinetics.  This is applicable in the reactions carried out under pseudo-first order conditions.  The basis of these conditions is defined below using adaptations from Eccleston et al.  Click here for more information.

 

Read More:

 

Toseland, C.P.Fluorescence to study the ATPase mechanism of motor proteins”. Experientia supplemetum. 2014 105:67-86

Toseland, C.P. & Geeves, M. “Rapid reaction kinetic techniques”. Experientia supplemetum. 2014 105:49-65.

Toseland, C.P. & Webb, M.R.  Fluorescence tools to measure helicase activity in real-time”. 2010 Methods 51:259.