Our current understanding of molecular interactions is based mostly on research conducted in dilute solutions. In vivo biological systems differ significantly from dilute solutions. The excluded volume theory has been developed to describe such solutions. The following will present evidence as to whether this theoretical framework is sufficient to explain experimental data from solutions with macromolecular concentrations up to 70 grams per liter.Sedimentation velocity and equilibrium experiments were conducted on green fluorescent protein as a tracer in high concentrations of 3 crowding molecules, hen egg-white lysozyme, dextran, and soybean trypsin inhibitor in the FDS optical system. All solutions were made in phosphate buffered saline pH 7.4.In the equilibrium experiments, GFP in HEL was found to have a high degree of attractive nonideality, due to charge-charge attraction between GFP and HEL. GFP in STI and GFP in dextran were found to have a similar degree of repulsive nonideality. In the interference experiments, dextran was found to have an extremely high effective volume in solution, explaining the similarity in repulsive nonideality between GFP in STI and GFP in dextran. Additionally, dextran alone had a greater degree of repulsive nonideality than did GFP in dextran, pointing towards attraction between GFP and dextran.In the sedimentation velocity experiments, GFP was found to have significant interactions with both HEL and dextran. A variety of excipients was added to determine the nature of the interaction. The only excipient seen to eliminate the second peak was NaCl. This points to charge-charge attraction between GFP and dextran.Such unexpected results point towards additional complications in high concentration solutions that excluded volume theory does not account for. Additional study and the development of a new model are necessary to fully understand nonideality in high concentration solutions.

Nonideality in high concentration solutions

243rd National Meeting (2012)

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