Question

How can BIAcore sensorgram be used for measuring kinetic (k_on and k_off) and thermodynamic parameters (Kd) of non-covalent molecular interactions?

Answer 1

Measure kinetics in Biacore :
There are three ways to obtain kinetic and affinity data in Biacore:

Track association and dissociation rates Analyzing association and dissociation levels for a series of analyte concentrations offers kinetic information that can also be expressed as affinity and steady state is not needed.

Track steady state levels Analyzing steady state levels as a function of analyte concentration provides affinity but not kinetics. It is useful for off on-fast interactions where kinetics can not be calculated. Limited use for slower reactions due to the time taken to reach steady state.

Test free analyte in solution Tests affinity in solution: allows interactive mixtures to be balanced in solution and uses Biacore to test the concentration of free analyte in mixtures.It can be extended to conditions where direct measurement can be difficult (small molecules, interactions that are disrupted by ligand immobilization, etc.) and to obtain solution values for comparison with surface bound values.

Thermodynamic research with Biacore T100 showed how mutations in variable fragments of a monoclonal antibody affected the kinetics of their interaction with hen egg lysozyme.

Analyzing a series of temperatures permitted thermodynamic characterization, showing that the interaction was enthalpically induced and entropically unfavorable for both wild and mutant antibodies.

Interactions have been resolved into association and dissociation stages, making it possible to thermodynamically describe the transition state.The mutations affected only the dissociation rate, suggesting that the variations in binding energies were due to different free energy barriers for dissociation, while the activation of the wild-type and mutant antibodies were identical.