Measurement of the Translational Diffusion Coefficient and Hydrodynamic Radius of Proteins by Dynamic Light...

Diffusion is a fundamental process in biological systems that governs the molecular collisions driving biochemical reactions and membrane and transport. Measurement of the diffusion coefficient and application of the Stokes-Einstein equation produces the hydrodynamic radius, which is a commonly used gauge of particle size. Additionally, measurement of the diffusion coefficient and the sedimentation coefficient, and application of the Svedberg equation, yields the molecular weight, which is particularly useful in the characterization of very large macromolecules. Dynamic light scattering (DLS) is the most common method to measure the diffusion coefficient of macromolecules. We describe a procedure to perform DLS measurements on monomeric bovine serum albumin (BSA) purified by size-exclusion chromatography using the Zetasizer Nano S particle size analyzer. We compare several analytical methods in existing software programs to estimate the diffusion coefficient of BSA (extrapolated to water at 20°C at infinite dilution, $D_{20,w}^0$ ) and describe a statistical method to obtain 95% confidence limits of the precision of the estimates. We compare $D_{20,w}^0$ estimates to literature values obtained by diffusiometry, sedimentation velocity analytical ultracentrifugation, and other DLS instruments. The method of cumulant analysis in the program SEDFIT (www.analyticalultracentrifugation.com) produced the most precise estimate, $D_{20,w}^0$ 6.06 ± 0.07 F (1 F = 10^-7 cm² s^-1), which was within the range of estimates obtained by diffusiometry or sedimentation velocity. This protocol is useful for DLS method validation and quality control.