80
Where K
D
is the equilibrium dissociation constant defined in concentration units i.e. moles/litre.
In defining the system this way, a quick quantification of the strength of the binding interaction
can be stated simply by reporting the order of magnitude of the K
D
value. The smaller the K
D
the
stronger the interaction.
OpenSPR™ can be used to determine the kinetic constants, k
a
, k
d
, K
D
and K
eq
(the reciprocal of
K
D
) of a binding system through experimentation. There are two main ways of determine the
kinetic constants of a binding system, steady state affinity experiments and kinetic affinity
experiments.
7.2.1 Steady State Affinity Experiments
The convention for defining the interacting species where one is immobilized, as in the case with
OpenSPR™, the immobilized species is called the ligand (B) and the free species is called the
analyte (A). This convention will be used throughout this manual.
Some manuals call this type of kinetic analysis ‘equilibrium analysis’, using the concepts of steady
state and equilibrium interchangeably. In this manual it is only called steady state analysis.
The steady state equation defined above can be adjusted to incorporate the refractive index
measurements (R) made by OpenSPR™. The amount of AB in the system at any time is measured
directly by R. The maximum change in R (R
max
) is defined by the total amount of B that is
immobilized onto the sensor surface and is constant. Therefore, the rate equation can be written
completely in terms of measureable quantities determined by OpenSPR™:
Where C is the injected analyte concentration
At Steady State (R=R
ss
)
From this equation it can be seen that when R
ss
is equal to
, K
D
is equal to C. Therefore when
R
ss
is plotted against C, K
D
can be easily determined.
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