Badly Behaving BSA

For the charge model, we would like to know the surface charge density and size of our experimental molecules.  I’ve attempted to use the Zetasizer for both measurements, but as mentioned in my diffusion paper, DLS gives sizes that deviate from the expected or crystal sizes.  Since zeta potential measurements require large amounts of highly concentrated samples, I can’t easily perform this measurement on my more expensive proteins.  I have however attempted to get this data for BSA.

Zeta potential results:

BSA has a higher zeta potential at lower salt concentration (-20mV as compared to -10mV).  Generally zeta potential will be higher, or closer to the surface potential, at lower salt concentration because you don’t have as many adsorbed ions within the stern layer.  Whether or not this holds for proteins, I do not know.

Size results:

BSA becomes smaller in size at lower salt concentrations.  As many of the interactions that determine the shape and size of a protein are electrostatic in nature, less shielding by the salt could mean stronger attractive interactions and a more compact size.

What does this mean in terms of the model?  Well right now it breaks any agreement between my model and the results we have seen all along.  The major cuprit is this size difference – it causes the free diffusion and membrane diffusion coefficients to be higher at the smaller size.  The faster diffusion results in higher sieving coefficients for the molecule at the lower salt concentration as shown in the following chart showing sieving coefficients over time.

Note that experimentally, the proteins always seem to diffuse through easier in the higher salt.  The model would predict faster diffusion for the higher salt if the size didn’t change.  Maybe we need to work with nanoparticles (QDs?) that won’t change size for the best model comparison.