Potty Ajish S R, Fu Joseph Y, Balan Sindhu, Haymore Barry L, Hill David J, Fox George E, Willson Richard C
Department of Chemical Engineering, University of Houston, 4800 Calhoun, Houston, TX 77204-4004, USA.
J Chromatogr A. 2006 May 19;1115(1-2):88-92. doi: 10.1016/j.chroma.2006.02.077. Epub 2006 Apr 4.
Immobilized metal-chelate affinity chromatography has been widely used in the purification of proteins, and we have recently found that it can also be applied to purification of nucleic acids through interactions involving exposed bases, especially purines. Here we report that the inclusion of moderate quantities of neutral solutes in the buffer substantially enhances the binding affinity of nucleic acids for immobilized metal-chelate affinity adsorbents. Addition of 20% (v/v) of solutes such as ethanol, methanol, isopropanol, n-propanol, and dimethyl sulfoxide enhances the initial affinity of binding of total yeast RNA by 4.4-, 3.8-, 3.7-, 3.0-, and 2.8-fold, respectively for Cu(II)-iminodiacetic acid (IDA) agarose adsorbent, and the weaker adsorption by Cu(II)-nitrilotriacetic acid (NTA) agarose was even more strongly enhanced. The adsorption affinities of the smaller oligodeoxynucleotide molecules A20, G20, C20 and T20 also increase with the addition of ethanol, suggesting that the effect is not significantly mediated by conformational changes. Binding enhancement generally correlates with reduction of water activity by the various solutes, as predicted by several models of solution thermodynamics, consistent with an entropic contribution by displacement of waters from the metal-chelate. Interestingly, the enhancement was not seen with the proteins bovine serum albumin and lysozyme.
固定化金属螯合亲和色谱已广泛应用于蛋白质的纯化，并且我们最近发现它还可通过涉及暴露碱基（尤其是嘌呤）的相互作用应用于核酸的纯化。在此我们报告，在缓冲液中加入适量的中性溶质可显著增强核酸与固定化金属螯合亲和吸附剂的结合亲和力。加入20%（v/v）的乙醇、甲醇、异丙醇、正丙醇和二甲基亚砜等溶质，可使总酵母RNA与铜（II）-亚氨基二乙酸（IDA）琼脂糖吸附剂的初始结合亲和力分别提高4.4倍、3.8倍、3.7倍、3.0倍和2.8倍，而铜（II）-次氮基三乙酸（NTA）琼脂糖较弱的吸附作用增强得甚至更明显。较小的寡脱氧核苷酸分子A20、G20、C20和T20的吸附亲和力也随着乙醇的加入而增加，这表明该效应并非由构象变化显著介导。如几种溶液热力学模型所预测的，结合增强通常与各种溶质引起的水活性降低相关，这与水从金属螯合物中被取代的熵贡献一致。有趣的是，牛血清白蛋白和溶菌酶这两种蛋白质未观察到这种增强作用。
