title: Hindered diffusion in polymeric solutions studied by fluorescence correlation spectroscopy.
abstract: Diffusion of molecules in the crowded and charged interior of the cell has long been of interest for understanding cellular processes. Here, we introduce a model system of hindered diffusion that includes both crowding and binding. In particular, we obtained the diffusivity of the positively charged protein, ribonuclease A (RNase), in solutions of dextrans of various charges (binding) and concentrations (crowding), as well as combinations of both, in a buffer of physiological ionic strength. Using fluorescence correlation spectroscopy, we observed that the diffusivity of RNase was unaffected by the presence of positively charged or neutral dextrans in the dilute regime but was affected by crowding at higher polymer concentrations. Conversely, protein diffusivity was significantly reduced by negatively charged dextrans, even at 0.4 μM (0.02% w/v) dextran. The diffusivity of RNase decreased with increasing concentrations of negative dextran, and the amount of bound RNase increased until it reached a plateau of ∼80% bound RNase. High salt concentrations were used to establish the electrostatic nature of the binding. Binding of RNase to the negatively charged dextrans was further confirmed by ultrafiltration.
authors: Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Development, National Institutes of Health, Bethesda, Maryland, USA. zustiaksp@mail.nih.gov
authors: Silviya P
authors: SP
authors: Zustiak
authors: Ralph
authors: R
authors: Nossal
authors: Dan L
authors: DL
authors: Sackett
mesh_terms: D000818:Animals; D002417:Cattle; D003911:Dextrans / Q000737:chemistry*; D004058:Diffusion; D009994:Osmolar Concentration; D011485:Protein Binding; D015203:Reproducibility of Results; D012260:Ribonucleases / Q000737:chemistry*; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods*; D055672:Static Electricity; D019802:Succinic Acid / Q000378:metabolism; D013997:Time Factors; D014462:Ultrafiltration
chemical_list: D003911:Dextrans; D012996:Solutions; D019802:Succinic Acid; D012260:Ribonucleases