摘要
在pH 3.00的柠檬酸-Na2HPO4缓冲溶液中,偶氮胂(III)与La3+发生结合反应生成蓝色螯合物,此时溶液的pH恰恰低于蛋白质的等电点。因此,血清蛋白带有正电荷,并且由于静电作用力的存在,2 min之内即可结合若干La3+-偶氮胂(III)螯合物生成高分子化合物。该反应能引起La3+-偶氮胂(III)螯合物在650 nm处吸收的减少,减少的吸收直接正比于血清蛋白的浓度。同时,高分子化合物的生成使得蛋白分子尺寸增大,更利于血清蛋白从水相中分离出来。据此,建立了基于上述结合反应修饰血清蛋白的浊点萃取-激光热透镜光谱法测定人血清白蛋白和牛血清白蛋白的新方法。在室温条件下,Triton X-114和十二烷基苯磺酸钠共同萃取并富集了痕量的非电中性的血清蛋白。最后,剩余的表面活性剂相被稀释,并且以热透镜光谱法测定。各种影响萃取效率及热透镜信号变化的因素也被考察及优化。方法已用于人血清样品中蛋白质总量的测定。
The anionic dye of arsenazo Ⅲ can react with La^3＋ to form a blue coloured complex in the citric acidNa2HPO4buffer solution （pH 3.00）. Here, the pH was just lower than the isoelectric point of protein, and accordingly bore a positive charge in the medium. So, it can combine with umpty Arsenazo Ⅲ-La^3＋ complexes to form a macromolecule complex by virtue of electrostatic interaction forces within 2 rain. The binding interaction only caused a decrease in absorbance of the complex at 650 nm, and the decreased absorbance was directly proportional to the concentration of protein. Due to the formation of the macromolecule complex, the bulk of protein molecule became much bigger than before available for precipitation from aqueous phase. Hereby, a new thermal lens spectrometry （TLS） combined with cloud point extraction （CPE） technique utilizing the above described reaction for determining human serum albumin （HSA） and bovine serum albumin （BSA） was proposed. And, the preconcentration of trace non-electrically neutral albumin at room temperature was achieved by CPE with two mixed surfactants, sodium dodecylbenzenesulfonic acid （SDBSA） and Triton X-114. Finally, the retained surfactant-rich phase was diluted and directly determined by TLS. The effect of several experimental variables on the method sensitivity and stability was investigated in detail. The method has been applied to the determination of total amounts of proteins in human serum samples with satisfactory results.
出处
《分析试验室》
CAS
CSCD
北大核心
2014年第4期382-387,共6页
Chinese Journal of Analysis Laboratory
基金
西安理工大学博士启动基金(108-211103)
国家自然科学基金项目(21304074)资助
