Tessema Mathewos, Simons Peter C, Cimino Daniel F, Sanchez Lilliana, Waller Anna, Posner Richard G, Wandinger-Ness Angela, Prossnitz Eric R, Sklar Larry A
Department of Pathology, Cancer Research and Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA.
Cytometry A. 2006 May;69(5):326-34. doi: 10.1002/cyto.a.20259.
Glutathione, a ubiquitous tripeptide, is an important cellular constituent, and measurement of reduced and oxidized glutathione is a measure of the redox state of cells. Glutathione-S-transferase (GST) fusion proteins bind naturally to beads derivatized with glutathione, and elution of such bead-bound fusion proteins with buffer containing millimolar glutathione is a commonly used method of protein purification. Many protein-protein interactions have been established by using GST fusion proteins and measuring binding of fusion protein binding partners by GST pulldown assays, usually monitored by Western blot methodology.
Dextran beads suitable for flow cytometry were derivatized with glutathione. A fusion protein of GST and green fluorescent protein was used to define kinetic and equilibrium binding characteristics of GST fusion proteins to glutathione beads. Free glutathione competes with this binding, and this competition was used to measure free glutathione concentration.
A 10 microl assay can measure 5 microl of 20 microM glutathione (100 pmol glutathione) in 2 h by flow cytometry. This concentration is two orders of magnitude lower than cellular glutathione concentrations, and three orders of magnitude lower than affinity chromatography eluates. One important result is that by generating high site density, the GST fusion proteins can be constrained to the surface of one bead without hopping to the next bead in multiplex assays.
Glutathione in cellular lysates and GST-fusion protein affinity chromatography eluates can be measured by flow cytometry. Many interactions between GST fusion proteins and their fluorescent binding partners should be quantifiable by flow cytometry. Although a system may have the disadvantage that it has a low affinity and a correspondingly quick off-rate in solution, it may remain on beads if the site density can be increased to offer a slow apparent off rate.
谷胱甘肽是一种普遍存在的三肽，是重要的细胞成分，还原型和氧化型谷胱甘肽的测量是细胞氧化还原状态的一种衡量方法。谷胱甘肽 - S - 转移酶（GST）融合蛋白能自然地与用谷胱甘肽衍生化的珠子结合，用含有毫摩尔浓度谷胱甘肽的缓冲液洗脱这种与珠子结合的融合蛋白是一种常用的蛋白质纯化方法。许多蛋白质 - 蛋白质相互作用已通过使用GST融合蛋白并通过GST下拉实验测量融合蛋白结合伴侣的结合来确定，通常通过蛋白质印迹法进行监测。
用谷胱甘肽对适用于流式细胞术的葡聚糖珠子进行衍生化。使用GST和绿色荧光蛋白的融合蛋白来确定GST融合蛋白与谷胱甘肽珠子的动力学和平衡结合特性。游离谷胱甘肽会竞争这种结合，利用这种竞争来测量游离谷胱甘肽浓度。
一个10微升的检测可在2小时内通过流式细胞术测量5微升20微摩尔的谷胱甘肽（100皮摩尔谷胱甘肽）。该浓度比细胞内谷胱甘肽浓度低两个数量级，比亲和色谱洗脱液低三个数量级。一个重要的结果是，通过产生高位点密度，GST融合蛋白可以被限制在一个珠子的表面，而不会在多重检测中跳到下一个珠子上。
细胞裂解物中的谷胱甘肽和GST融合蛋白亲和色谱洗脱液可以通过流式细胞术进行测量。许多GST融合蛋白与其荧光结合伴侣之间的相互作用应该可以通过流式细胞术进行定量。尽管一个系统可能存在亲和力低且在溶液中相应解离速率快的缺点，但如果位点密度可以增加以提供缓慢的表观解离速率，它可能会保留在珠子上。
