| Abstract: |
A recent project involved using SEC-MALS-SAXS for the solution structural analysis of a highly elongated, folded (not disordered) protein composed of five and a half B-repeats (Brpt5.5). During data collection at relatively modest loading concentrations (3-10 mg/ml), significant peak broadening and distortion was observed by SEC-MALS for a homogeneous sample. This phenomenon is known as “viscous fingering” (VF) in the liquid chromatography field and has been attributed to the very high viscosity at the leading edge of the sample as particles move unevenly into the less viscous eluent. While this phenomenon is commonly observed at concentrations above 50 mg/ml for globular proteins like BSA, we observed a similar degree of VF with Brtp5.5 at 10-fold lower concentration. A systematic analysis of hydrodynamic nonideality by sedimentation velocity AUC and of VF by SEC for the two proteins was performed. It was found that Brpt5.5 had a ks value about 8-fold higher than BSA – indicating a strong correlation between hydrodynamic nonideality and VF. To further explore this relationship and the impact of shape, a Brpt1.5 construct (with similar surface electrostatics and global conformation to Brpt5.5, but fewer repeats) was also analyzed. A similar correlation was observed, and from these three datasets, we propose a semi-quantitative approach to predicting VF based on ks. With this presentation, we hope to encourage fruitful discussion regarding the interplay between hydrodynamic nonideality and viscosity, with special consideration of the impact of particle shape. |
|---|
