Light scattering goes beyond molar mass and size – it is also a great tool for studying macromolecular interactions in solution without recourse to labelling or immobilization. Composition-Gradient Multi-Angle Static Light Scattering (CG-MALS or CG-SLS) employs the same detectors used in chromatography mode: a multi-angle static light scattering photometer, often in combination with a differential refractometer or UV/Vis absorption spectrophotometer. Instead of a continuously flowing fractionation apparatus to provide size-separated samples, CG-MALS utilizes the Calypso multi-syringe pump system, programmed to provide stop-flow injections at a series of well-defined concentrations or compositions. Data acquired at each step, after equilibration, is analyzed to determine dynamic equilibrium properties: the stoichiometry and binding affinity (KA) of specific complexes, and/or thermodynamic parameters such as virial coefficients in the case of non-specific interactions. The reaction rates at each composition may also be analyzed to determine kinetic parameters such as the Michaelis constant (kM) or association/dissociation rate constants (kon, koff).
Conceptually, CG-MALS is an ‘absolute’ technique based entirely on first principles and a rigorous thermodynamic foundation. Since static light scattering measures weight-average molar mass, CG-MALS provides not only the stoichiometric ratio, but full complex stoichiometry. Systems undergoing self-interaction, hetero-association, or even simultaneous reversible self- and hetero-association may be quantified. A particular strength of CG-MALS is the analysis of metacomplexes, i.e. the self-association of simple complexes such as the reaction A+B+A+B↔AB+AB↔(AB)2. Interaction analysis is not limited to macromolecules but may be extended to the behaviour of larger objects such as liposomes or microgel particles (including macromolecules adhering to such objects), and the same instrumentation may serve to automate the study of additional phenomena such as nanoparticle degradation kinetics.
These sections contain a concise overview of the theory behind CG-MALS:
Additional details on the theory and practice of CG-MALS, as well as a variety of examples, may be found in “Characterization of Protein-Protein Interactions via Static and Dynamic Light Scattering”, in Protein Interactions, ed. Jianfeng Cai and Rongsheng Wang, InTech , 2012. The publication is freely downloadable here.