LeVine Harry
Department of Molecular and Cellular Biochemistry, Chandler School of Medicine and the Center on Aging, University of Kentucky, Lexington, KY 40536-0230, USA.
Anal Biochem. 2004 Dec 1;335(1):81-90. doi: 10.1016/j.ab.2004.08.014.
When diluted from dimethyl sulfoxide or 1,1,1,3,3,3-hexafluoro-2-propanol, synthetic human Abeta(1-42) readily forms oligomeric structures at near physiologic concentrations (1-20 nM). Oligomers 40 kDa are detected in a sandwich enzyme-linked immunosorbant assay where the capture and detection antibodies recognize the same primary sequence epitope. Monomeric peptide with a single epitope does not react in this format. Abeta(1-40) peptide does not oligomerize readily under these conditions. The rate of oligomer formation has a steep linear temperature dependence but is weakly affected by ionic strength up to 0.5M NaCl or KCl. Oligomer formation is inhibited by concentrations of Tween 20 and several other detergents well below their critical micelle concentrations. Once formed, high-molecular-weight oligomers are stabilized by Tween 20. Gel permeation chromatography of an oligomer preparation formed at nanomolar concentrations indicates that the majority of the Abeta(1-42) peptide chromatographs as monomers/dimers of apparent mw approximately 10 kDa. The most abundant oligomers have apparent mobilities corresponding to 220 kDa (48-mer) and higher multiples of this without detectable concentrations of intermediate low-molecular-weight species. Very little immunoreactive peptide appears in the void volume (>1.5 MDa) of a Superose 12 column. The oligomers are stable, rechromatographing at their original position. Abeta(1-42) oligomer formation at physiologic concentrations is a reproducible process that is amenable to kinetic analysis and inhibition.
当从二甲基亚砜或1,1,1,3,3,3 - 六氟 - 2 - 丙醇中稀释时，合成的人β淀粉样蛋白（1 - 42）在接近生理浓度（1 - 20 nM）时很容易形成寡聚体结构。在夹心酶联免疫吸附测定中检测到40 kDa的寡聚体，其中捕获抗体和检测抗体识别相同的一级序列表位。具有单个表位的单体肽在此检测形式中不发生反应。在这些条件下，β淀粉样蛋白（1 - 40）肽不容易寡聚化。寡聚体形成速率具有陡峭的线性温度依赖性，但在高达0.5M NaCl或KCl的离子强度下受到的影响较弱。吐温20和其他几种洗涤剂在远低于其临界胶束浓度的情况下就能抑制寡聚体的形成。一旦形成，高分子量寡聚体可通过吐温20来稳定。对以纳摩尔浓度形成的寡聚体制剂进行凝胶渗透色谱分析表明，大多数β淀粉样蛋白（1 - 42）肽以表观分子量约为10 kDa的单体/二聚体形式进行色谱分离。最丰富的寡聚体的表观迁移率对应于220 kDa（48聚体）及更高倍数，且未检测到中间低分子量物种的浓度。在Superose 12柱的空体积（> 1.5 MDa）中出现的免疫反应性肽非常少。这些寡聚体很稳定，在其原始位置重新进行色谱分离。在生理浓度下β淀粉样蛋白（1 - 42）寡聚体的形成是一个可重复的过程，适合进行动力学分析和抑制研究。
