Xue Christine, Lee Yoon Kyung, Tran Joyce, Chang Dennis, Guo Zhefeng
Department of Neurology, Brain Research Institute, Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.
R Soc Open Sci. 2017 Aug 9;4(8):170325. doi: 10.1098/rsos.170325. eCollection 2017 Aug.
Aggregation of amyloid-β (Aβ) protein plays a central role in Alzheimer's disease. Because protein aggregation is a concentration-dependent process, rigorous investigations require accurate concentration measurements. Owing to the high aggregation propensity of Aβ protein, working solutions of Aβ are typically in the low micromolar range. Therefore, an ideal Aβ quantification method requires high sensitivity without sacrificing speed and accuracy. Absorbance at 280 nm is frequently used to measure Aβ concentration, but the sensitivity is low with only one tyrosine and no tryptophan residues in the Aβ sequence. Here we present a fluorescence method for Aβ quantification using fluorescamine, which gives high fluorescence upon reaction with primary amines. We show that, using hen egg white lysozyme as a standard, fluorescence correlates linearly with primary amine concentration across a wide range of fluorescamine concentrations, from 62.5 to 1000 µM. The maximal sensitivity of detection is achieved at a fluorescamine concentration of 250 µM or higher. The fluorescamine method is compatible with the presence of dimethyl sulfoxide, which is commonly used in the preparation of Aβ oligomers, and limits the use of absorbance at 280 nm due to its high background reading. Using aggregation kinetics, we show that the fluorescamine method gives accurate concentration measurements at low micromolar range and leads to highly consistent aggregation data. We recommend the fluorescamine assay to be used for routine and on-the-fly concentration determination in Aβ oligomerization and fibrillization experiments.
淀粉样β蛋白（Aβ）的聚集在阿尔茨海默病中起着核心作用。由于蛋白质聚集是一个浓度依赖性过程，因此严格的研究需要准确的浓度测量。由于Aβ蛋白具有很高的聚集倾向，Aβ的工作溶液通常处于低微摩尔浓度范围。因此，一种理想的Aβ定量方法需要在不牺牲速度和准确性的前提下具有高灵敏度。280nm处的吸光度经常用于测量Aβ浓度，但由于Aβ序列中只有一个酪氨酸且没有色氨酸残基，其灵敏度较低。在此，我们提出一种使用荧光胺对Aβ进行定量的荧光方法，荧光胺与伯胺反应时会产生高荧光。我们表明，以鸡蛋清溶菌酶为标准，在62.5至1000µM的宽荧光胺浓度范围内，荧光与伯胺浓度呈线性相关。在荧光胺浓度为250µM或更高时可实现最大检测灵敏度。荧光胺方法与常用于制备Aβ寡聚体的二甲基亚砜兼容，并且由于其高背景读数限制了280nm处吸光度的使用。通过聚集动力学，我们表明荧光胺方法在低微摩尔浓度范围内可进行准确的浓度测量，并能得出高度一致的聚集数据。我们建议在Aβ寡聚化和纤维化实验中使用荧光胺测定法进行常规和即时浓度测定。
