New methods for measuring macromolecular interactions in solution via static light scattering: basic methodolog, and application to nonassociating and self-associating proteins
New methods for measuring macromolecular interactions in solution via static light scattering: basic methodolog, and application to nonassociating and self-associating proteins
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DOI：
10.1016/j.ab.2004.09.045
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发表时间：
2005-02-01
影响因子：
2.9
通讯作者：
Minton, AP
中科院分区：
文献类型：
--
作者：
Attri, AK;Minton, AP
提出了一种快速检测和表征溶液中大分子可逆缔合的方法。可编程双注射器输液泵用于将时变成分的溶液引入平行流动池中，以同时测量多个角度的激光散射和紫外-可见光吸光度。持续不到 15 分钟的实验会产生大量信息丰富的数据，其中包含数千个瑞利比值，作为溶质浓度和散射角的函数。使用新颖的数据处理方法，可以在自关联模型的背景下同样快速地分析整个数据集。对先前表征的非缔合和自缔合蛋白质进行的验证实验产生了 10-330 kDa 范围内的分子量的稳健值以及 2 x 10(3)-6 x 10(5) M-1 范围内二聚体形成的平衡缔合常数。 (C) 2004 Elsevier Inc. 保留所有权利。
A method for rapid detection and characterization of reversible associations of macromolecules in solution is presented. A programmable dual-syringe infusion pump is used to introduce a solution of time-varying composition into parallel flow cells for concurrent measurement of laser light scattering at multiple angles and ultraviolet-visible absorbance. An experiment lasting less than 15 min produces a large and information-rich set of data, consisting of several thousand values of the Rayleigh ratio as a function of solute concentration(s) and scattering angle. Using a novel treatment of the data, the entire data set may be equally rapidly analyzed in the context of models for self-association. Validation experiments conducted on previously characterized nonassociating and self-associating proteins yielded robust values for molecular weights in the range 10-330 kDa and equilibrium association constants for dimer formation in the range 2 x 10(3)-6 x 10(5) M-1. (C) 2004 Elsevier Inc. All rights reserved.