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

✍ Scribed by Emmanuel Dauty; A. S. Verkman

• Publisher: John Wiley and Sons
• Year: 2004
• Tongue: English
• Weight: 246 KB
• Volume: 17
• Category: Article
• ISSN: 0952-3499
• DOI: 10.1002/jmr.709

✦ Synopsis

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 cm^2^/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 (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 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.