General question about kinetic

Hi All,
I have a question for you guys
Let's say that my sensorgram fit with the Bivalent analyte model. I would like to know how can i find the Kd for the second binding site since i have the second Kon expressed in RU's instead of M-1s-1.
Can some one help me?
Thanks
Alessandro

Assuming the analyte has two identical binding sites (e.g. antibody), first one site binds to the ligand. The other free site is now in closer contact with other ligand molecules. The second binding will give a stabilization of the ligand-analyte complex without extra response but with shifting the equilibrium constant. The formation of the second binding will depend on the flexibility of the analyte, ligand, the dextran layer and the availability of free ligand (1).

Reaction equation

The factor of two is introduced because the first binding step can occur to one of two unoccupied sites, while the second binding step can only occur to one unoccupied site (3).

A bivalent analyte gives rise to two sets of rate constants, one for each binding step. The meaning of the second set of rate constants is difficult to interpret.

The second association constant k2 has the units RU-1 s-1 because this binding is dependent on the fist binding. How more there is bound (RU) how more the second step kan occur (see also below). In case of an antibody, both binding sites are equal, and consequently the rate constants. But since the second binding is not free (is is to some extent directed) and dependent on the first binding there is no second rate constant with the units M-1 s-1.

In case of a bivalent interaction, the dependence of the concentration on the components of the interaction can be studied. At low analyte concentrations, the AL2 complex is dominating while at high analyte concentrations the AL complex is more abundant.

In case of comparing antibodies it is better to capture the antibody under investigation with an anti Fc immobilized antibody. In this way, the bivalent nature of the antibody will not appear in the sensorgram.
In general, the bivalent model works better for lower affinities when avidity affects are easier to resolve. In order to resolve the kd of extremely stable interactions long dissociation times (hours, overnight) are needed (2).


1. Baumann, S. et al; Indirect immobilization of recombinant proteins to a solid phase using the albumin binding domain of streptococcal protein G and immobilized albumin; J.Immunol.Methods; (221): 95-106; 1998.
2. BIACORE AB; Kinetic and Affinity analysis using BIA - Level 2; 1998.
3. Nelson, R. W. et al; BIA/MS of epitope-tagged peptides directly from E. coli lysate: multiplex detection and protein identification at low-femtomole to subfemtomole levels; Anal.Chem.; (71): 2858-2865; 1999.