通过平衡沉降技术对大分子异质性进行表征。

Characterization of macromolecular heterogeneity by equilibrium sedimentation techniques.

作者信息

Xu Yujia

机构信息

Department of Chemistry, Rutgers University, Piscataway, NJ 08854, USA.

出版信息

Biophys Chem. 2004 Mar 1;108(1-3):141-63. doi: 10.1016/j.bpc.2003.10.015.

DOI:10.1016/j.bpc.2003.10.015

PMID:15043927

Abstract

New graphical procedures have been developed to investigate the heterogeneity of protein preparations using sedimentation equilibrium. The heterogeneous systems that can be studied include self-associating systems contaminated by incompetent monomer, self-associating systems contaminated by non-dissociating oligomer and simple non-interacting monomer-oligmer disperse systems. The new procedures are based on the concentration dependence of the apparent association constants estimated by a non-linear least square fitting program (NONLIN), on the assumption of conservation of mass during sedimentation and on the applications of several standard techniques for statistical inferences of NONLIN estimations. The procedures outlined here can detect various types of heterogeneity, discriminate amongst different types of heterogeneity, estimate the amount of contaminant causing heterogeneity and determine the true equilibrium constant of the self-associating components. The procedures appear to be sensitive, accurate and easily applicable when tested using both protein samples and computer simulated data.

摘要

已开发出新的图形程序，用于利用沉降平衡研究蛋白质制剂的异质性。可研究的异质系统包括被无活性单体污染的自缔合系统、被非解离寡聚体污染的自缔合系统以及简单的非相互作用单体 - 寡聚体分散系统。新程序基于通过非线性最小二乘拟合程序（NONLIN）估计的表观缔合常数对浓度的依赖性、沉降过程中质量守恒的假设以及几种用于NONLIN估计统计推断的标准技术的应用。这里概述的程序可以检测各种类型的异质性，区分不同类型的异质性，估计导致异质性的污染物量，并确定自缔合组分的真实平衡常数。当使用蛋白质样品和计算机模拟数据进行测试时，这些程序似乎灵敏、准确且易于应用。