Problem: Macromolecular interactions enact vital functions necessary for life, including DNA replication, transcription, mRNA translation, protein degradation, and signal transduction. It is essential to characterize the intermolecular affinity and stoichiometry of these interactions for a variety of biochemical applications, such as understanding in vivo processes and studying disease pathways. In addition, the development of therapeutic antibodies and other biopharmaceuticals brings the characterization of complex, multivalent interactions to the forefront of biotechnology.

An array of techniques exists for analyzing macromolecular interactions, but many cannot provide a complete picture of a macromolecular interaction. Methods that require tagging or immobilization, such as surface plasmon resonance (SPR) and enzyme-linked immunosorbent assays (ELISA), can potentially influence the interaction of interest since the molecules are no longer free in solution or in their native state. Isothermal titration calorimetry (ITC ) and analytical ultracentrifugation (AUC ) are both label-free methods that provide quantification of affinity and stoichiometry. However, ITC can only provide stoichiometric ratio— not absolute stoichiometry—and has a limited affinity range, and AUC experiments require many hours to days to perform, which may degrade sensitive molecules. Thus, several complementary techniques are often required to describe macromolecular interactions fully.

Multi-angle light scattering (MALS) has long been coupled with separation techniques, such as size exclusion chromatography (SEC-MALS) and field flow fractionation (FFF-MALS) to provide an absolute measure of the molar mass of macromolecules and define their molecular weight distribution in solution. These techniques allow the analyst to determine the stoichiometry of macromolecular complexes through the combination of information from multiple detectors (MALS with UV absorption, differential refractometry, and/or viscometry). However, these techniques are limited in their study of interactions, due in part to the process of dilution and fractionation.

Calypso II connected in series to light scattering and concentration detector.

Solution: Composition-gradient multiangle light scattering (CG-MALS) enables characterization of macromolecular interactions in solution without immobilization or tagging. This batch technique involves delivery of different compositions of one or more macromolecular species to a light scattering and concentration detector. For each composition, the flow is stopped to allow for slow kinetics and ensure that the solution has reached equilibrium. The presence of interactions is observed as a change in the apparent molar mass of the solution, measured by MALS, as a function of composition.

CG-MALS offers the unique capability of determining both the binding affinity and stoichiometry of all interactions present in solution with no need for immobilization or tagging. This method is capable of quantifying equilibrium binding affinity of self- and heteroassociating proteins with KD from ~100 pM to ~1 mM, kinetics of reversible or irreversible aggregation, and complex stoichiometries including infinite selfassembly. Furthermore, CG-MALS can account for nonspecific intermolecular attraction and repulsion via virial coefficients. The data from CG-MALS experiments complement results obtained from other techniques and can provide additional information about the equilibrium association state that is not accessible to traditional methods.

A new technology was recently developed to automate CG-MALS measurements and provide the analyst with rapid, reproducible results. The Calypso system (Wyatt Technology) prepares a sample of the appropriate composition and delivers it to a MALS detector flow cell. All experimental parameters, including injection volume, flow rate, and amount of time the flow is stopped, are easily controlled by the user, and data analysis is facilitated via a graphical software interface. Compared with traditional batch measurements, the Calypso provides a robust platform for achieving fast, reliable results.

For more information, visit www.wyatt.com/calypso