Introduction to Light Scattering
Light scattering (LS) provides the absolute molecular weight (MW) and size (radius of gyration <rg2>) of macromolecules in solution. The amount of light scattered is directly proportional to the product of the weight-average molar mass and the concentration of the macromolecule, i.e., LS ~ Mw·c (for more information about light scattering see Theory). Laser light scattering is a "non-invasive" technique that can be performed in either the batch or chromatography mode. In either instance the sample may be recovered at the end of the study. Unlike molecular weights that are estimated from gel size exclusion chromatography data alone, the MW obtained from light scattering is not dependent upon either the Stokes radius of the protein (or other macromolecule) nor a calibration curve that depends upon running several standard proteins. Since laser light scattering provides the weight average MW for all molecules in solution it is generally more useful to utilize the chromatography mode. Hence, if a weak dimer:tetramer interaction were being studied under conditions where there were equal weights of both species, the batch mode study would provide a MW that would correspond to a trimer while the chromatography study would provide the MWs for both the (separated) dimer and tetramer as well as quantify the amounts of each species.
Although molecular weights can be determined also via mass spectrometry and analytical centrifugation, only light scattering and analytical centrifugation monitor the properties of macromolecules in solution and provide information about the oligomeric state of the protein. While a sedimentation equilibrium run may require 72 hours, a size exclusion chromatography/LS study can be completed in about an hour and a batch mode analysis can be completed in a few minutes. The comparatively short LS run times greatly facilitates carrying out the multiple studies that may be needed to determine the impact of protein concentration, ligands, pH, ionic strength and other variables on protein:protein interactions.
Batch Mode LS Study
A batch mode experiment is carried out by injecting the sample directly into the flow cell and then measuring the amount of scattered light at several angles. The data collected for each sample are processed simultaneously using the Zimm formalism. The experiment may be repeated at various protein concentrations to give the weight-average molar mass (Mw), radius of gyration <rg2> and the second virial coefficient A2, which is a measure of macromolecule-solvent interactions. When performed in micro-batch mode, a typical experiment requires several (at least four) samples of macromolecule at different concentrations (in the range of mg/ml) in a volume of at least 1 ml each (the actual range of concentrations needed depends upon the expected MW, with higher MW macromolecules requiring lower concentrations). Although we recommend using the chromatography mode (see above), the batch mode may be useful in those instances where the protein or other macromolecule of interest cannot be subjected to size exclusion chromatography.
Chromatography mode LS study
During a chromatography mode LS study the laser light scattering detector is coupled with HPLC size exclusion chromatography (SEC) or other separation technique (e.g., reverse-phase HPLC). Coupling LS to a fractionation step avoids the ambiguity that can otherwise result from a batch mode experiment, in particular, the fact that light scattering provides the weight average MW of all species present in solution. Additionally, the chromatographic step eliminates the need for careful buffer exchange. Typically, the SEC-LS study uses three detectors (light-scattering (LS), UV absorbance (UV) and refractive index (RI)) that are connected in series after an HPLC SEC column. The molecular weight determination depends only upon the "downstream" LS and RI detector, with the RI detector being used to determine the concentration of the macromolecule at each "slice" of the chromatogram that is analyzed. Since the MW determination is completely independent of elution position, non-globular shape and/or interaction with the SEC support have no impact on the molecular weights determined by LS. More importantly, the molecules can be segregated by any separation/chromatographic technique (not necessary related to their sizes) and the MW of each separated species can be easily determined. As the system is currently configured, a typical experiment requires 120 - 300 mg protein. Generally, proteins with expected MW > 40 kDa require 120-200 mg while proteins with expected MW < 40 kDa require 200-300 mg for each SEC run. In both instances the sample should be made up to a volume of 250 ml.