Can FIDA measure kinetics?
Yes, Fida Neo can be used to measure kinetics (association kon and dissociation koff rates). It does so in solution, and with no buffer, temperature, detergent or ionic strength constraints.
Understanding the interaction between biomolecules is of great interest within biomolecular research. A key parameter is to understand the affinity between drug and target, but also quantifying the underlying kinetics that define the affinity. Kinetics describes at which rate the drug and target interact. For a 1:1 interaction the interaction is described as follows:
Where t denotes time. Instead of looking at a single time point, one can also look at the change of complex concentration over time
Where kon is the rate constant for the association of the complex and koff is the rate constant for the dissociation of the complex. By looking at a specific time where the rates are equal, we observe:
Where eq means equilibrium, hence there is no net change in the system. At equilibrium the dissociation constant, or the affinity, is described as the following:
Where KD is the dissociation constant and describes the affinity between A and B. In FIDA the kinetics protocol involves two distinct analysis steps:
STEP 1:
By premixing the two interacting molecules we can ensure that the equilibrium has beenestablished prior to measuring the diffusivity, thus eliminating any time components. Doing so we get the following equation:
Where, Dapp is the apparent diffusivity observed at a given concentration of analyte (A) and constant concentration of indicator (I). AT describes the total analyte concentration, whereas DI and DAI are the diffusivity of the indicator and the indicator-analyte complex, respectively. KD is the equilibrium affinity constant. Using a premix protocol at different AT, the equilibrium KD is thus determined.
STEP 2:
In contrast, if the interacting molecules are not allowed to reach equilibrium prior to detection,we need to extend the model to describe the formation of complex with time, which depends onthe kon and the koff as shown below.
Where kon and koff are introduced along with t, describing the time, and the remainingcomponents remain identical as in the equilibrium case. One can express the koff as a product of the KD and kon, hence leaving only 1 fitting parameter, namely kon. Doing so results in the following expression:
The last thing that should be addressed is the time component in the equation above. The FidaNeo instrument is a pressure driven instrument; hence the user is in full control of how much timeis allowed for the reagents to interact in the capillary as the pressure controls the flow rate. By increasing the pressure, less time is allowed for the reaction, hence shifting the reaction away from the equilibrium.
You can continue reading in more detail here.
Watch a webinar about in-solution kinetics here.
