DNA拓扑结构、温度和溶剂粘度对回转色谱中DNA阻滞的影响。

Hirabayashi J, Kasai K I

Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikvo University, Sagamiko, Kanagawa. Japan.

J Chromatogr A. 2000 Sep 29;893(1):115-22. doi: 10.1016/s0021-9673(00)00693-2.

Slalom chromatography is a unique size-fractionation method applicable to large DNA molecules [>5 kilobase pairs (kbp)]. The method was first developed by using columns packed with microbeads (diameter, <20 microm) used for high-performance liquid chromatography and by applying a relatively fast flow-rate (>0.3 ml/min). Previous studies suggested that the separation is attributed to a hydrodynamic rather than to an equilibrium phenomenon (J. Hirabayashi and K. Kasai, Anal. Biochem. 178 (1989) 336; J. Hirabayashi, N. Itoh, K. Noguchi and K. Kasai, Biochemistry, 29 (1990) 9515). In the present report, the results of a systematic study on the effects of DNA topology, temperature, and solvent viscosity on DNA retardation are described. Firstly, the behaviour of circular (super-coiled) and linearized forms of charomid DNAs (20-42 kbp) was studied. Circular-form DNA molecules were found to be fractionated size-dependently similarly to linear forms in a flow-rate dependent manner. However, the extent of retardation of the circular form DNA was apparently less than that of the corresponding linear forms. Circular DNAs showed almost the same retardation (e.g., 42 kbp) as DNA fragments (e.g., 20 kbp) having approximately half of the size of the former. This observation indicates that DNA retardation is basically related to physical length, not to mass. Secondly, to study the effect of temperature with special reference to solvent viscosity, we carried out chromatographic analysis at various temperatures ranging from 6 to 65 degrees C in both the absence and presence of sucrose (10 or 20%, w/v). The results showed that it is the solvent viscosity that determines the extent of retardation. Taken together, all of physicochemical parameters that define hydrodynamic properties, i.e., particle size, flow-rate and solvent viscosity, proved to be critical in slalom chromatography as well as the potential physical length of the DNA, thus supporting the concept that slalom chromatography is based on a hydrodynamic principle.

蛇形色谱法是一种独特的尺寸分级方法，适用于大的DNA分子[>5千碱基对（kbp）]。该方法最初是通过使用填充有用于高效液相色谱的微珠（直径<20微米）的柱子并应用相对较快的流速（>0.3毫升/分钟）而开发的。先前的研究表明，分离归因于流体动力学现象而非平衡现象（J. Hirabayashi和K. Kasai，《分析生物化学》178 (1989) 336；J. Hirabayashi、N. Itoh、K. Noguchi和K. Kasai，《生物化学》，29 (1990) 9515）。在本报告中，描述了关于DNA拓扑结构、温度和溶剂粘度对DNA滞留影响的系统研究结果。首先，研究了charomid DNA（20 - 42 kbp）的环状（超螺旋）和线性化形式的行为。发现环状DNA分子与线性形式一样，以流速依赖的方式按尺寸进行分级分离。然而，环状形式DNA的滞留程度明显小于相应的线性形式。环状DNA显示出与大小约为前者一半的DNA片段（例如20 kbp）几乎相同的滞留（例如42 kbp）。这一观察结果表明，DNA滞留基本上与物理长度有关，而不是与质量有关。其次，为了特别参照溶剂粘度研究温度的影响，我们在6至65摄氏度的各种温度下，在有无蔗糖（10%或20%，w/v）的情况下进行了色谱分析。结果表明，决定滞留程度的是溶剂粘度。综上所述，所有定义流体动力学性质的物理化学参数，即颗粒大小、流速和溶剂粘度，在蛇形色谱法中以及DNA的潜在物理长度方面都被证明是至关重要的，从而支持了蛇形色谱法基于流体动力学原理的概念。