Wyatt Technology's Dynapro DLS Instruments Determine Whether Freezing Alters The Macromolecular Homogeneity Of Proteins

Santa Barbara, CA - A recent application by IBMC-CNRS, Strasbourg, France, has demonstrated that Wyatt Technology's innovative DynaPro Titan range of Dynamic Light Scattering (DLS) instruments can be used to determine whether freezing alters a macromolecule's homogeneity or not. The findings of this analysis are of particular interest for protein characterization applications and are detailed in a new, free of charge application note from Wyatt, the world leader in absolute macromolecular characterization instrumentation and software.

With certain proteins the biological activity disappears after being stored in a frozen state, possibly due to aggregation. IBMC-CBRS used the DynaPro as it is the industry standard in the protein crystallization market, and it enabled verification of the protein quality before reproducible investigations were undertaken on the sample.

For this particular application, two samples – one never subjected to freezing and one kept frozen for two weeks – were available for comparison. The samples were transferred into two 12µL cuvettes and spun for three minutes at 4,000 rpm in a small, tabletop centrifuge to remove dust particles as well as the largest aggregates.

The analysis of the unfrozen protein exhibited a stable baseline (mean ~0.999), a low sum of squares (<5), a diffusion coefficient of 1054 x 10-9 cm2s-1, a hydrodynamic radius of ~2.0nm and was virtually monodisperse (index~0.03). Assuming that the protein has a spherical shape, the Mw was 16-17kDa – close to the one expected from amino acid composition. By contrast, the protein population of the frozen sample was characterized by a far less stable baseline (mean 1.005), and high SOS values (~8). The smaller diffusion coefficient (965 x 10-9 cm2s-1), the greater radius (2.2nm) larger polydispersity index (0.07) and the higher apparent Mw (>20kDa) indicated clearly the presence of aggregates.

Altogether, the results demonstrated that this protein tends to aggregate upon freezing. The DynaPro™ DLS system has proven to offer the most direct and least invasive method to answer the question of whether freezing actually alters the macromolecule's homogeneity or not within just a few minutes.

SOURCE: Wyatt Technology