Griess G A, Guiseley K B, Miller M M, Harris R A, Serwer P
Department of Biochemistry, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78284-7760, USA.
J Struct Biol. 1998 Oct;123(2):134-42. doi: 10.1006/jsbi.1998.4021.
Previous studies have shown that, during the formation of an underivatized agarose gel, agarose molecules laterally aggregate to form thicker fibers called suprafibers; the suprafibers branch to form a gelled network. In the present study, electron microscopy of thin sections is used to investigate both the thickness and the spacing of the fibers of gels formed by agarose chemically derivatized with carboxymethyl (negatively charged) groups. For carboxymethyl agarose, electron microscopy reveals that gels cast in water consist of both fibers narrower and pores smaller than those observed for water-cast underivatized agarose gels at the same concentration. This result is confirmed by using the electrophoretic sieving of spheres to determine the radius (PE) of the effective pore of the gel. At a given concentration of gel less than 1%, the PE for a water-cast carboxymethyl agarose gel is 0.25-0.30x the PE for a water-cast underivatized agarose gel. The value of PE predicts the extent of the electrophoretic sieving that is observed when double-stranded DNA is subjected to electrophoresis through a water-cast carboxymethyl agarose gel; DNA bands formed in a water-cast carboxymethyl agarose gel are comparable in quality to DNA bands formed in a water-cast underivatized agarose gel of equal PE. The following observation supports the hypothesis that electrical charge-charge repulsion among carboxymethyl agarose molecules inhibits the formation of suprafibers in water-cast carboxymethyl agarose gels: Increased content of suprafibers in carboxymethyl agarose gels is observed when the ionic strength is raised by the presence of NaCl, MgCl2, or any of several buffers during gelation of carboxymethyl agarose.
先前的研究表明，在未衍生化琼脂糖凝胶形成过程中，琼脂糖分子横向聚集形成称为超纤维的更粗纤维；超纤维分支形成凝胶网络。在本研究中，使用薄切片电子显微镜来研究由羧甲基（带负电荷）基团化学衍生化的琼脂糖形成的凝胶纤维的厚度和间距。对于羧甲基琼脂糖，电子显微镜显示，在水中浇铸的凝胶由比相同浓度下在水中浇铸的未衍生化琼脂糖凝胶中观察到的更窄的纤维和更小的孔组成。通过使用球体的电泳筛分来确定凝胶有效孔的半径（PE），这一结果得到了证实。在凝胶浓度小于1%的给定条件下，在水中浇铸的羧甲基琼脂糖凝胶的PE是在水中浇铸的未衍生化琼脂糖凝胶的PE的0.25 - 0.30倍。PE值预测了双链DNA通过在水中浇铸的羧甲基琼脂糖凝胶进行电泳时观察到的电泳筛分程度；在水中浇铸的羧甲基琼脂糖凝胶中形成的DNA条带在质量上与在具有相同PE的水中浇铸的未衍生化琼脂糖凝胶中形成的DNA条带相当。以下观察结果支持了这样的假设，即羧甲基琼脂糖分子之间的电荷 - 电荷排斥抑制了在水中浇铸的羧甲基琼脂糖凝胶中超纤维的形成：当在羧甲基琼脂糖凝胶化过程中通过存在NaCl、MgCl2或几种缓冲液中的任何一种来提高离子强度时，观察到羧甲基琼脂糖凝胶中超纤维的含量增加。
