Glutathione‐S‐transferase‐green fluorescent protein fusion protein reveals slow dissociation from high site density beads and measures free GSH
Open Access
- 18 April 2006
- journal article
- research article
- Published by Wiley in Cytometry Part A
- Vol. 69A (5), 326-334
- https://doi.org/10.1002/cyto.a.20259
Abstract
Background: 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. Methods: 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. Results: A 10 μl assay can measure 5 μl of 20 μM 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. Conclusions: 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. © 2006 International Society for Analytical CytologyKeywords
This publication has 13 references indexed in Scilit:
- Oxidative stress, redox, and the tumor microenvironmentSeminars in Radiation Oncology, 2004
- Glutathione Metabolism during Aging and in Alzheimer DiseaseAnnals of the New York Academy of Sciences, 2004
- Real-time Analysis of Ternary Complex on ParticlesJournal of Biological Chemistry, 2004
- Glutathione Metabolism and Its Implications for HealthJournal of Nutrition, 2004
- Free Radicals in Cell BiologyInternational Review of Cytology, 2004
- The contributions of excitotoxicity, glutathione depletion and DNA repair in chemically induced injury to neurones: exemplified with toxic effects on cerebellar granule cellsJournal of Neurochemistry, 2003
- Ligand-Receptor-G-Protein Molecular Assemblies on Beads for Mechanistic Studies and Screening by Flow CytometryMolecular Pharmacology, 2003
- Determination of intracellular glutathione and thiols by high performance liquid chromatography with a gold electrode at the femtomole level: comparison with a spectroscopic assayBiochimica et Biophysica Acta (BBA) - General Subjects, 2002
- Competition between solution and cell surface receptors for ligand. Dissociation of hapten bound to surface antibody in the presence of solution antibodyBiophysical Journal, 1989
- Purification of glutathione S-transferases from human liver by glutathione-affinity chromatographyAnalytical Biochemistry, 1977