Macromolecular Interactions: Light Scattering

Abstract

Static light scattering (SLS) is a spectroscopic technique for determination of molar masses (molecular weights) and radii of gyration for macromolecules in solution. Dynamic light scattering (DLS) measurement allows determination of translational diffusion coefficient and hydrodynamic radius. Measurement of molar mass from SLS experiment determines the oligomeric state and thus provides association stoichiometry for biological macromolecules such as native and modified proteins and their complexes, nucleic acids as well as phospholipid vesicles, viruses or drug delivering nanoparticles. Both static and DLS are routinely used to assess monodispersity (homogeneity with respect to molar mass or size) of protein stocks prepared for structural studies. Both homo‐ and hetero‐association of proteins can be detected and quantitatively characterized from light scattering measurements performed at various concentrations.

Keywords: light scattering; molar mass; radius of gyration; hydrodynamic radius; molecular weight

Figure 1.

Schematic light scattering system. Light scattering photometer with a laser light source, temperature‐controlled samples cell and photodetector that records scattered light at the angle Θ.

Figure 2.

Zimm plot: K*c/R(Θ) versus sin2(Θ/2); R(Θ) is the excess intensity of scattered light at an angle Θ, c the w/v concentration and K* an optical parameter equal to 4π2n2 (dn/dc)2/(λ4NA). The radius of gyration, Rg, is extracted from the slope of the angular dependence extrapolated to infinite dilution, c=0 (red line); second virial coefficient can be determined from the limiting slope at zero angle, i.e. Θ=0 (green line). Weight‐average molar mass, Mw, is determined from a common intercept of 1/Mw. (a) Zimm plot for a 500 kDa dextran (courtesy of Wyatt Technology Corporation): Mw= 499 700 g mol−1; Rg=21.9 nm, A2=1.608×10−4 mol mL g−2. (b) Zimm plot for a monomer of bovine serum albumin (BSA): Mw=66 220 g mol−1; Rg <10 nm and A2=1.149×10−4 mol mL g−2.

Figure 3.

Molar mass distribution plots from ASTRA analysis of SEC‐UV/LS/RI data. Calculated molecular weight values for LL‐GltTBc (polypeptide:140 kDa; 1.1 g of lipids/gram of polypeptide; open triangles), maltoporin (LamB, polypeptide: 149 kDa; 1.2 g of lipids/gram of polypeptide; closed squares) and α‐HL (polypeptide: 226 kDa; 0.3 g of lipids/gram of polypeptide; open squares) are plotted. Solid lines correspond to UV traces collected at 280 nm of the proteins eluting from the SEC column. Reprinted with permission from Yernool et al.. Copyright (2003) American Chemical Society.

Figure 4.

Static light scattering data for recombinant human interleukin‐1 receptor antagonist (rhIL‐1ra) at 25°C and pH 6.5 over a protein concentration range 1–100 mg mL−1. Debye plots (K*c/R(Θ) versus concentration) at various ionic strengths (and respective buffers): ○, 0.184 m (46.5 mM citrate);♦, 0.111 m (83.3 mM phosphate); ▪, 0.091 (20.8 mM citrate);⋄, 0.060 (42.7 mM phosphate) and Δ, 0.031 (16.0 mM phosphate); •, 0.025 (4.5 mM citrate). Error bars from the standard deviation are less than the symbol size. Reprinted from Alford et al. with permission from John Wiley & Sons, Inc.

Figure 5.

AFFF‐LS/UV/RI analysis of the final fraction of DEAE‐purified virus‐like particles (VLP) for DNA delivery. (a) Analysis using AFFF; the radii of gyration, Rg, and hydrodynamic radii, Rh, were determined using multiangle SLS, dynamic light scattering (DLS), respectively. (b) Same run as (a); the molecular mass was determined using SLS and refractive index (RI) signals. (c) Analysis using SDS–PAGE: lane 1, MW marker; lane 2, sample purified by PEG precipitation from 5 days culture; lane 3, DEAE‐purified VLP reduced with DTT; lane 4, blank; lane 5, same sample as in lane 3 not reduced. (d) UV/Vis spectrum collected during AFFF‐LS/UV/RI analysis of DEAE‐purified VLP analysed by photodiode array detector (PDA). Reprinted with permission from Citkowicz et al.. Copyright (2008) Elsevier.

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Zimm BH (1948b) The scattering of light and the radial distribution function of high polymer solutions. Journal of Chemical Physics 16: 1093–1099.

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Folta‐Stogniew, Ewa J(Sep 2009) Macromolecular Interactions: Light Scattering. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0003143]