Molecular crowding reduces to a similar extent the diffusion of small solutes and macromolecules: measurement by fluorescence correlation spectroscopy
Molecular crowding reduces to a similar extent the diffusion of small solutes and macromolecules: measurement by fluorescence correlation spectroscopy
复制标题
DOI：
10.1002/jmr.709
复制
发表时间：
2004-09-01
影响因子：
2.7
通讯作者：
Verkman, AS
中科院分区：
文献类型：
--
作者：
Dauty, E;Verkman, AS
活细胞中的水性环境是拥挤的，具有高达>50重量%的小分子和大分子尺寸溶质。我们定量研究了分子拥挤的一个重要后果，减少生物重要溶质的扩散。采用荧光相关光谱法（FCS）测量了一系列荧光小分子和大分子的扩散。在水中，扩散系数（D-w（o））为（cm（2）/s × 10（-8））：罗丹明绿色（270）、白蛋白（52）、葡聚糖（100）、（75，10 kDa; 10，500 kDa）、双链DNA（96，20 bp; 10，1 kb; 3.4，4.5 kb）和聚苯乙烯纳米球（5.4，20 nm w直径; 2.3，100 nm）。用Ficoll-70（0-60重量%）拥挤的溶液中的水相扩散（D-w）以指数方式降低高达650倍：D-w=D（w）（o）exp（-[C]/[C]（exp）），其中[C]（exp）是使Do降低63%的拥挤剂的W浓度（以重量%计）。所有溶质和Ficoll-70 W浓度的FCS数据与单组分简单（非异常）扩散模型拟合良好。有趣的是，对于Ficoll-70溶液中非常不同类型的大分子的扩散，[C]（exp）几乎相同（11 +/-2 wt%，SD）。然而，[C]（exp）取决于拥挤剂的性质：例如，若丹明绿色扩散的[C]（exp）对于甘油为30重量%，对于500 kDa葡聚糖为16重量%。我们的研究结果表明，分子拥挤可以大大减少生物学重要大分子的水相扩散，并证明拥挤效应对扩散物质的大小和特征具有以前未认识到的不敏感性。版权所有（C）2004约翰威利父子有限公司。
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.