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

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Abstract
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) were (in cm2/s × 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 diameter; 2.3, 100 nm). Aqueous-phase diffusion (Dw) in solutions crowded with Ficoll-70 (0–60 wt%) was reduced by up to 650-fold in an exponential manner: Dw = D exp (-[C]/[C]exp), where [C]exp is the concentration (in wt%) of crowding agent reducing D by 63%. FCS data for all solutes and Ficoll-70 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 © 2004 John Wiley & Sons, Ltd.