Biomolecular Interaction

Specific, reversible interaction between two molecules is a phenomenon from which there is no escape in any field of biology. For example, an understanding of metabolism at the molecular level requires the quantitative consideration of a vast array of interactions between enzymes and
substrates, cofactors, inhibitors and effectors. Antigen-antibody interactions are central to immunology. Control of gene expression and protein synthesis entails a maze of interactions between proteins and nucleic acids. Signal transduction through biomembranes relies upon a cascade of events involving recognition and binding of cellular constituents. Furthermore, the interactions of proteins, hormones, drugs or other ligands with matrix proteins and membrane receptors control widely divergent cellular events such as muscle contraction, blood coagulation,
hormone response and lymphocyte activation. In other words, binding events are the essence of biological control. A feature of these binding phenomena is the non-covalence of the interactions, the equilibrium nature of which allows the extent of complex formation to vary with prevailing reactant concentrations and hence with fluctuations in the metabolic state of the cell. A prerequisite for biological relevance of an interaction is the demonstration of a significant extent of complex formation in mixtures with reactant concentrations that are likely to prevail in the physiological environment. The usual way of establishing such significance is to measure the binding constant governing the equilibrium, thereby allowing the composition of an equilibrium mixture to be predicted for any specified combination of reactant concentrations by applying the law of mass action.

Our two surface plasmon resonance (SPR) based instruments, a Biacore T100 and a ProteOn XPR36, measure the changes in angle of deflection of the evanescent internal absorbance of plane-polarized monochromatic light. This deflection angle is sensitive to picogram levels of analyte that are flowing over a surface with immobilized ligand. Based on surface density variation, the signal differentiates between bound and unbound analyte. The response of the instrument is mass proportionate, and the plot of response versus time (a sensorgram) can be fit to a variety of kinetic models to calculate kinetic rate constants and reaction affinity.

Requirements for an SPR experiment

• Approximately 10ug of analyte, and 5ug of ligand. More material is required to study low affinity interactions.
• Samples are to be introduced at concentrations of 10KD to 0.1KD.
• The ligand must be >95% pure, or it must have a unique tag for capture immobilization.
• A suitable regeneration solution to dissociate ligand-analyte complexes is usually required.

Contacts

Stefan Schauer