New 2:1 Heterogeneous Ligand Model
Now Available for Kinetic Analysis

Krista Witte Ph. D., Sr. Director of Chemistry and Consumables R&D
kwitte@fortebio.com

When performing kinetic analysis it is always preferable to engineer the molecular system to study as simple an interaction as possible. For most protein-protein or small molecule-protein interactions, this means assaying and analyzing an interaction where one molecule in solution binds to one molecule on the biosensor surface, i.e. a 1:1 interaction. In this type of interaction, the reaction can be represented as Equation 1 (where A is the analyte in solution and B is the ligand on the surface).

However, in some cases, the interaction to be studied cannot be reduced to a simple 1:1 interaction model. One such case relevant to label-free kinetics is where two ligand species bind in parallel to an analyte species. This type of interaction can now be fit with the new 2:1 Heterogeneous Ligand (HL) fitting model available in the Octet Data Analysis 6.1 software or later. The 2:1 HL interactions can be represented as Equation 2 (where B1 and B2 are two different ligands on the surface and A is the analyte in solution). This model assumes that the interactions are non-cooperative and that the analyte in solution is a single species.

The 2:1 HL model is appropriate for use in situations when it is known that the ligand is not homogeneous or fully active. A few examples of this include when a ligand preparation is known to have a small percentage of misfolded protein that will bind to the analyte or if the immobilization of the ligand onto the biosensor surface has altered the activity.

When using this model to fit kinetic data, two sets of kinetics parameters (k_on, k_off, K_D) will be calculated. These two sets of parameters correspond to the two interactions occurring in Equation 2. The percentage of these two kinetic interactions in the total binding can be determined by looking at the ratio of the two calculated R_max parameters (R_max1 and R_max2). The R_max will reflect the amount that the interaction contributes to the overall signal at saturation, and thus the predominant interaction will have the largest R_max.

To aid in determining the predominant interaction, the Data Analysis Software tabulates the kinetic parameters with k_on1, k_off1, K_D1 coming from the interaction with the largest R_max and k_on2, k_off2, K_D2 coming from the interaction with the smaller R_max.

In the example shown in Figure 1, the receptor immobilized onto the biosensor surface is known to contain impurities which also bind to the analyte. The 1:1 interaction model does not adequately describe this interaction. The 2:1 HL model fits to the experimental data and provides information on both the predominant interaction (the receptor binding to the analyte) as well as the minor interaction (the impurity binding to the analyte).

The addition of this 2:1 HL model provides even greater flexibility to the Octet platform and allows for a larger breadth of kinetic analysis to be performed.