Richards M P, Beattie J H
USDA, Agricultural Research Service, Growth Biology Laboratory, Beltsville, MD 20705-2350, USA.
J Chromatogr B Biomed Appl. 1995 Jul 7;669(1):27-37. doi: 10.1016/0378-4347(95)00008-7.
We have investigated free-solution capillary electrophoresis (FSCE) and micellar electrokinetic capillary chromatography (MECC) separations of metallothionein (MT) isoforms conducted in uncoated and surface-modified fused-silica capillaries. At alkaline pH, FSCE rapidly resolves isoforms belonging to the MT-1 and MT-2 charge classes. At acidic pH, additional resolution of MT isoforms is achieved. The use of high-ionic-strength (0.5 M) phosphate buffers can result in high peak efficiencies and increased resolution for some MT isoforms. Interior capillary surface coatings such as polyamine and linear polyacrylamide polymers permit separation of MT isoforms with enhanced resolution through their effects on electroosmotic flow (EOF) and protein-wall interactions. Improvements in MT isoform resolution can also be achieved by MECC using 100 mM borate buffer pH 8.4 containing 75 mM SDS. Deproteinization of tissue cytosol samples with acetonitrile (60-80%) or perchloric acid (7%) produces extracts that can be subjected to direct analysis of MT by FSCE or MECC. We conclude that optimal separation of MT isoforms by capillary electrophoresis (CE) can be achieved with the appropriate combination of different capillaries, buffers and sample preparation techniques.
我们研究了在未涂层和表面改性熔融石英毛细管中进行的金属硫蛋白（MT）亚型的自由溶液毛细管电泳（FSCE）和胶束电动毛细管色谱（MECC）分离。在碱性pH条件下，FSCE能快速分离属于MT-1和MT-2电荷类别的亚型。在酸性pH条件下，可实现MT亚型的进一步分离。使用高离子强度（0.5M）的磷酸盐缓冲液可使某些MT亚型获得较高的峰效率和更高的分辨率。内部毛细管表面涂层，如多胺和线性聚丙烯酰胺聚合物，通过其对电渗流（EOF）和蛋白质-壁相互作用的影响，可实现MT亚型的分离，分辨率更高。使用含有75mM SDS的100mM pH 8.4硼酸盐缓冲液的MECC也可提高MT亚型的分辨率。用乙腈（60-80%）或高氯酸（7%）对组织细胞质样品进行脱蛋白处理，可得到提取物，可直接通过FSCE或MECC对MT进行分析。我们得出结论，通过不同毛细管、缓冲液和样品制备技术的适当组合，可实现毛细管电泳（CE）对MT亚型的最佳分离。
