Welder Frank, Paul Beverly, Nakazumi Hiroyuki, Yagi Shigeyuki, Colyer Christa L
Department of Chemistry, PO Box 7486, Wake Forest University, Winston-Salem, NC 27109, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Aug 5;793(1):93-105. doi: 10.1016/s1570-0232(03)00367-2.
Noncovalent interactions between two squarylium dyes and various model proteins have been explored. NN127 and SQ-3 are symmetric and asymmetric squarylium dyes, respectively, the fluorescence emissions of which have been shown to be enhanced upon complexation with proteins such as bovine serum albumin (BSA), human serum albumin (HSA), beta-lactoglobulin A, and trypsinogen. Although these dyes are poorly soluble in aqueous solution, they can be dissolved first in methanol followed by dilution with aqueous buffer without precipitation, and are then suitable for use as fluorescent labels in protein determination studies. The nature of interactions between these dyes and proteins was studied using a variety of buffer systems, and it was found that electrostatic interactions are involved but not dominant. Dye/protein stoichiometries in the noncovalent complexes were found to be 1:1 for SQ-3, although various possible stoichiometries were found for NN127 depending upon pH and protein. Association constants on the order of 10(5) and 10(7) were found for noncovalent complexes of SQ-3 and NN127, respectively, with HSA, indicating stronger interactions of the symmetric dye with proteins. Finally, HSA complexes with NN127 were determined by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). In particular, NN127 shows promise as a reagent capable of fluorescently labeling analyte proteins for analysis by CE-LIF without itself being significantly fluorescent under the aqueous solution conditions studied herein.
已对两种方酸染料与各种模型蛋白之间的非共价相互作用进行了探索。NN127和SQ - 3分别是对称和不对称方酸染料，已证明它们与牛血清白蛋白（BSA）、人血清白蛋白（HSA）、β - 乳球蛋白A和胰蛋白酶原等蛋白质络合时荧光发射增强。尽管这些染料在水溶液中溶解度很差，但它们可先溶于甲醇，然后用水性缓冲液稀释而不沉淀，因此适合用作蛋白质测定研究中的荧光标记物。使用多种缓冲系统研究了这些染料与蛋白质之间相互作用的性质，发现存在静电相互作用但并非主导作用。发现非共价复合物中SQ - 3的染料/蛋白质化学计量比为1:1，而NN127的化学计量比因pH和蛋白质而异。发现SQ - 3和NN127与HSA的非共价复合物的缔合常数分别约为10⁵和10⁷，表明对称染料与蛋白质的相互作用更强。最后，通过毛细管电泳 - 激光诱导荧光检测（CE-LIF）测定了HSA与NN127的复合物。特别是，NN127有望作为一种试剂，能够对分析物蛋白进行荧光标记，以便通过CE-LIF进行分析，而在本文研究的水溶液条件下其本身荧光不显著。
