When investigating molecular interactions, equilibrium dissociation constants (KD) are commonly used as a measure of two molecules’ affinity for each other. In other words, the KD is a direct measure of the strength of an interaction. Determining the KD is very important to evaluate the biological relevance of an interaction such as in study of fusion proteins, DNA-protein binding and for standard protein analysis.

However, obtaining only a KD value does not independently provide information on the kinetics of the molecular interaction. In other words, KD does not tell you how quickly the two molecules bind (i.e., the association rate constant or “on rate” of the interaction), or how quickly the molecules dissociate (i.e., the dissociation rate constant or “off rate” of the interaction). Indeed, molecular interactions that have the same KD can have widely different on and off rates.

The kinetics of a molecular interaction are very important to drug discovery and development. The off rate is especially important because it tells you how long the compound binds to the ligand or target. A long off rate would mean the drug could be formulated for less frequent dosing, whereas a short off rate might require multiples doses per day. Given how important patient compliance is for treating many diseases—such as chronic diseases and acute infections—ensuring a compound can be used in a convenient dosage regimen is vital to drug development. Many studies have shown that patients are less likely to miss a dose when they are taking a drug once a day compared with twice a day, and a once-weekly dose results in even better compliance.

Kinetics data also can help reveal a compound’s mechanism of action by determining if a compound is interacting with one or multiple binding sites on the ligand or target. This information is important for a number of reasons. For example, while most enzyme targets have a unique active site, they typically share other structural features with other enzymes. Interacting with such an allosteric site could lead to unwanted reactions with other enzymes. Alternatively, a researcher might be looking for an allosteric interaction in order to activate or modulate the target. Surface Plasmon Resonance (SPR) can be used to determine both kinetic and equilibrium data, as well as concentration data, including protein concentration.

We hope you found this post helpful and informative. You can read more about SPR elsewhere on Reichert’s website, including our first blog post and information on the technology of SPR.

If you would like advice on how to best determine equilibrium constants and association and dissociation rate constants, please do not hesitate to contact us. Also, please contact us if you would like to suggest topics for future blog posts or if you have any suggestions to make this column even better.