What is Binding Affinity?
Binding affinity is the strength of the binding interaction between a single biomolecule (e.g. protein or DNA) to its ligand/binding partner (e.g. drug or inhibitor). Binding affinity is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluate and rank order strengths of bimolecular interactions. The smaller the KD value, the greater the binding affinity of the ligand for its target. The larger the KD value, the more weakly the target molecule and ligand are attracted to and bind to one another.
Binding affinity is influenced by non-covalent intermolecular interactions such as hydrogen bonding, electrostatic interactions, hydrophobic and Van der Waals forces between the two molecules. In addition, binding affinity between a ligand and its target molecule may be affected by the presence of other molecules.
Why should I measure binding affinity for my application?
Whenever you are characterizing proteins, nucleic acids, and any biomolecule, understanding the binding affinity to substrates, inhibitors, and cofactors is key to the appreciation of the intermolecular interactions, relevant in studying for example enzymatic reactions, protein complexes or receptor binding. For drug discovery, binding affinity helps design drugs that bind their targets selectively and specifically.
There are many methods to measure binding. Qualitative methods (i.e. binding: yes/no) such as ELISA, gel-shift assays and quantitative methods (i.e. binding affinity) such as spectroscopic assays, optical biosensors (such as GCI) and isothermal titration calorimetry.
What binding affinity solutions does Malvern Panalytical offer?
Malvern Panalytical offers both Grating Coupled Interferometry (GCI) and Isothermal Titration Calorimetry (ITC). Both are label-free quantification techniques, allowing the use of native molecules. Highly quantitative affinity (KD values) can be derived from both for a broad range of interactions.
GCI is an optical method that measures the change in refractive index in an evanescent field caused by the binding event and is used to study the affinity and kinetics of an interaction. GCI measures KD values in the millimolar to picomolar range and additionally determines the kinetics of an interaction, more specifically, the on (ka)and off (kd) rates.
ITC measures the heat change associated with the binding event. ITC measures KD values in the millimolar to nanomolar range and determines the binding stoichiometry and binding thermodynamics of the interaction. Both kinetics and thermodynamics are important in the characterization of intermolecular interactions.
Is the GCI or ITC most suitable for my application?
The affinity from both devices is orthogonal and taken together can provide confidence when a highly quantitative KD is required for instance in lead optimization applications amongst others.
The GCI benefits from a higher sensitivity, higher throughput and lower sample consumption and performs well with crude samples. If these factors and the kinetic information are the most important to your application, then this is clearly the instrument of choice.
If the thermodynamic data (enthalpy and entropy) and stoichiometry are most important, then the ITC is the best solution. The ITC also benefits from minimal assay development and therefore can be quicker to a result if only a small number of measurements of a given interaction pair are anticipated. The technique is also non-destructive and the sample can be recovered post experiment.
The Creoptix WAVEsystem (GCI) and MicroCal PEAQ-ITC are both designed with the user in mind and have reputations for ease of use in their respective instrument classes.