Considering a protein in fast exchange, the position of the peak will be concentration dependent. If the peaks are not well separated by SEC due to close retention times, careful analysis of the molecular weight distribution across a peak may reveal the presence of multiple species ( Margarit et al., 2003). At slow exchange, distinct oligomeric species may elute as separate peaks in SEC, and their molecular weights can be determined (e.g., in the case of nucleotide-free WspR see below) ( De et al., 2008). The experimental approach is amenable to interacting systems, self-associating macromolecules or heterocomplexes. The new generation of detectors have a large dynamic range that allows for the analysis of protein samples at concentrations ranging from ∼ 1 μg/ml to ∼ 20 mg/ml. In addition, the method is amenable to transmembrane or peripheral membrane proteins since samples can be analyzed in the presence of detergent or lipids. Buffers may contain ligands such as nucleotides or phosphomimetic compounds that can stabilize certain conformations or molecular assemblies. Similar to stand-alone SEC experiments, SEC-coupled MALS provides flexibility regarding the choice of mobile phase. Batch mode measurements at multiple protein concentrations provide the second virial coefficient, a measure of nonspecific interactions, as well as stoichiometry and equilibrium constants for reversible, specific interactions ( Attri and Minton, 2005 Kameyama and Minton, 2006 Valente et al., 2005). While fractionation via SEC in-line with MALS yields accurate measurements for various species that may be present in the sample, experiments can be also carried out in batch mode. In addition, polydispersity indices can be calculated that provide an estimate for the mass distribution in the sample ( Mogridge, 2004 Sondermann et al., 2005 Wyatt, 1997). The measurements are independent of the shape of a protein and its retention time in SEC. While MALS experiments can yield the radius of gyration ( R g) of larger proteins (with an R g > 10 nm), scattering from smaller proteins (with an R g < 10 nm) shows little angular dependence, and Eq. The second virial coefficient term is negligible due to the low solute concentration in SEC peaks. For experiments at a single concentration, as it is the case for SEC-coupled MALS approaches, only the molecular weight can be determined. Double extrapolation to zero angle and zero concentration from multiangle measurements provides the molecular weight and second virial coefficient for the solute particles. K is the constant 4π 2 n 2(d n/d c) 2/(λ 4 N A), where n is the refractive index of the solvent, d n/d c is the refractive index increment of the solution, λ is the wavelength of the light source, and N A is Avogadro's number. Where c is the solute concentration, R(θ) is the Rayleigh ratio of the solution at scattering angle θ and concentration c and is directly proportional to the light scattering intensity, M w is the weight-averaged molecular mass, P(θ) is a descriptor for the angular dependence of the scattered light, and A 2 is the second virial coefficient.
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