(17f) Diffusion and Binding of RNase A in Dextran Polymeric Solutions Studied by Fluorescence Correlation Spectroscopy

The diffusion of molecules in the cytoplasm and nucleoplasm of cells has long been of interest in targeted drug delivery as well as in understanding basic cellular processes. Oftentimes one is concerned with the movement of charged molecules through a sea of much larger molecules and supramolecular assemblies, which is possibly affected by molecular crowding and charge-mediated binding. For small molecules such as certain metabolites and small proteins, an important parameter that describes cytoplasmic rheology is the translational diffusion coefficient. In this study we developed a model system in which the effects of both molecular crowding and charge-mediated binding on the movement of the molecules were addressed independently in a controlled manner. Using Fluorescence Correlation Spectroscopy (FCS), we measured the translational diffusion coefficient of the positively charged protein, RNase A, in polymeric solutions of dextrans of various charges. We observed that the diffusion of RNase A in a medium containing physiological strength, pH 7.0 phosphate-buffered saline, was unaffected by the presence of positively charged or neutral dextrans up to 10 mg/ml dextran. Above this concentration, the movement was affected by crowding. On the other hand, the presence of negatively charged dextrans slowed the protein significantly, even at 0.1 mg/ml dextran. The rate of translational diffusion of RNase A decreased with an increase in the concentration of negatively-charged dextran. The amount of bound RNase A also increased until it reached equilibrium binding of ~90% bound RNase A at 5 mg/ml dextran. Binding of RNase to the negatively charged dextrans was further confirmed by ultrafiltration. The exact equilibrium dissociation constants also were determined.