Molecular crowding reduces to a similar extent the diffusion of small solutes and macromolecules: measurement by fluorescence correlation spectroscopy

Aqueous environments in living cells are crowded, with up to >50 wt% small and macromolecule-size solutes. We investigated quantitatively one important consequence of molecular crowding-reduced diffusion of biologically important solutes. Fluorescence correlation spectroscopy (FCS) was used to measure the diffusion of a series of fluorescent small solutes and macromolecules. In water, diffusion coefficients (D-w(o)) were (in cm(2)/s X 10(-8)): rhodamine green (270), albumin (52), dextrans (75, 10 kDa; 10, 500 kDa), double-stranded DNAs (96, 20 bp; 10, 1 kb; 3.4, 4.5 kb) and polystyrene nanospheres (5.4, 20 nm w diameter; 2.3, 100 nm). Aqueous-phase diffusion (D-w) in solutions crowded with Ficoll-70 (0-60 wt%) was reduced by up to 650-fold in an exponential manner: D-w=D(w)(o)exp(-[C]/[C](exp)), where [C](exp) is the W concentration (in wt%) of crowding agent reducing Do by 63%. FCS data for all solutes and Ficoll-70 W concentrations fitted well to a model of single-component, simple (non-anomalous) diffusion. Interestingly [C](exp) were nearly identical (11 +/- 2 wt%, SD) for diffusion of the very different types of macromolecules in Ficoll-70 solutions. However, [C](exp) was dependent on the nature of the crowding agent: for example, [C](exp) for diffusion of rhodamine green was 30 wt% for glycerol and 16 wt% for 500 kDa dextran. Our results indicate that molecular crowding can greatly reduce aqueous-phase diffusion of biologically important macromolecules, and demonstrate a previously unrecognized insensitivity of crowding effects on the size and characteristics of the diffusing species. Copyright (C) 2004 John Wiley Sons, Ltd.