Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Surface Plasmon Resonance of CaBP 109
9. Once the dissociation rate constant has been established, the association rate constant
may be evaluated. Determine the exact time of injection t 0. Delete the first
few minutes of the association phase because they are influenced by the bulk
concentration change. Fit the rest of the association phase to the following equation
in the 1:1 complex case (seethe manual for other stochiometry):
R(t) = R 0 + R eq(1 – exp[–([A]k on + k off)·(t – t 0)])
k on is the association rate constant and R 0 is the SPR value at time 0. R 0 is generally
not the same as the baseline value, because of the sensitivity of the SPR to
changes in solvent composition. It is assumed that the analyte concentration [A]
is held constant in the flow cell during the entire association phase of a certain
SPR experiment because the flow of the mobile phase is fast compared to the
association and dissociation reactions. The SPR value approaches R eq when t
approaches infinity. R eq is equal to
R eq = R max · [A] ·k on / [A] ·k on + k off
R max is the maximum SPR response, which would be observed if there was analyte
bound to all available immobilized molecules (some molecules may be immobilized,
but not available to binding, e.g., because of unsuitable geometry of the
immobilization). R max would, in principle, be constant for all association experiments
run on a particular immobilization surface. Alas, instabilities in the instrument
and the deterioration of the immobilized molecule with time and number of
experiments may cause variations in R max. If the values of k on from the six different
experiments vary within a factor of 2, they can be considered constant.
4. Notes
1. The most versatile matrix for immobilization of macromolecules is the
carboxymethylated dextran matrix, which allows immobilization via native -NH2, -SH, -CHO, and -COOH groups. Other immobilization matrices for use in the
BIAcore instruments are:
a. Streptavidin-coated matrix for binding of biotinylated ligand;
b. Flat hydrophobic surface for lipid coating and binding of membrane binding
macromolecules;
c. NTA-coated matrix for nickel chelation and binding of hisitdine-tagged
ligand. Other matrices are under development by the vendors.
2. When it comes to commercial SPR instruments, the BIAcore instruments are the
most widespread (www.biacore.com). They all use a continuous flow technique,
where the analyte is dissolved in the flow buffer and this is the technique
described in this chapter. A newcomer to the field is the KI1 instrument by BioTul
(www.biotul.com). This is a cuvet-based instrument; i.e., does not support the
continuous flow technique. KI1 will be released after the preparation of this
manuscript (August 1999). A technique similar to SPR, called resonant mirror