Improved peak capacity for CE separations of enzyme inhibitors with activity-based detection using magnetic bead microreactors.
Improved peak capacity for CE separations of enzyme inhibitors with activity-based detection using magnetic bead microreactors.
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DOI：
10.1002/elps.200900055
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发表时间：
2010-01
期刊：
影响因子：
2.9
通讯作者：
Gilman, S. Douglass
中科院分区：
文献类型：
--
作者：
Yan, Xiaoyan;Gilman, S. Douglass
建立了一种利用毛细管电泳酶微反应器分离检测酶抑制剂的方法。在柱式酶微反应器中，利用S(NdFeB)磁体在检测窗口前将包被碱性磷酸酶的超顺磁珠(直径2.8μm)固定在毛细管中。酶抑制试验是通过将一塞抑制剂注入充满底物AttoPhos的毛细管中进行的。用激光诱导荧光法对酶微反应器中产生的产物进行检测。抑制物带通过毛细管，通过酶微反应器，由于产物形成减少，观察到负峰。这项研究的目的是提高抑制剂分离的峰容量，相对于以前的工作，以前的工作结合了连续参与电泳介导的微量分析(EMMA)和瞬时参与EMMA来研究酶抑制。考察了电场强度、微珠注入时间和缓蚀剂浓度对峰容量和峰宽的影响。在最佳电场强度和最佳进珠时间条件下，用砷酸盐和茶碱进行抑制试验，峰容量提高到≥20。分离和检测了5种碱性磷酸酶的可逆抑制剂(茶碱、钒酸、砷酸、L-色氨酸和钨酸盐)，以验证该技术分析复杂缓蚀剂混合物的能力。
A technique for separating and detecting enzyme inhibitors was developed using capillary electrophoresis with an enzyme microreactor. The on-column enzyme microreactor was constructed using NdFeB magnet(s) to immobilize alkaline phosphatase-coated superparamagnetic beads (2.8 μm diameter) inside a capillary before the detection window. Enzyme inhibition assays were performed by injecting a plug of inhibitor into a capillary filled with the substrate, AttoPhos. Product generated in the enzyme microreactor was detected by laser-induced fluorescence. Inhibitor zones electrophoresed through the capillary, passed through the enzyme microreactor, and were observed as negative peaks due to decreased product formation. The goal of this study was to improve peak capacities for inhibitor separations relative to previous work, which combined continuous engagement electrophoretically mediated microanalysis (EMMA) and transient engagement EMMA to study enzyme inhibition. The effects of electric field strength, bead injection time and inhibitor concentrations on peak capacity and peak width were investigated. Peak capacities were increased to ≥20 under optimal conditions of electric field strength and bead injection time for inhibition assays with arsenate and theophylline. Five reversible inhibitors of alkaline phosphatase (theophylline, vanadate, arsenate, L-tryptophan and tungstate) were separated and detected to demonstrate the ability of this technique to analyze complex inhibitor mixtures.
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影响因子：
7.4
作者：
Hadd, AG;Jacobson, SC;Ramsey, JM
通讯作者：
Ramsey, JM
DOI：
10.1111/j.1432-1033.1993.tb18234.x
发表时间：
1993-10-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
BUBLITZ, R;ARMESTO, J;FISCHER, W
通讯作者：
FISCHER, W
影响因子：
4.1
作者：
SHIRAZI, SP;BEECHEY, RB;BUTTERWORTH, PJ
通讯作者：
BUTTERWORTH, PJ
影响因子：
2.9
作者：
Whisnant, AR;Gilman, SD
通讯作者：
Gilman, SD
影响因子：
2.9
作者：
STANKIEWICZ, PJ;GRESSER, MJ
通讯作者：
GRESSER, MJ