Glutathione-S-transferase-green fluorescent protein fusion protein reveals slow dissociation from high site density beads and measures free GSH.
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 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.


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.
Tessema
Mathewos
M
Department of Pathology, Cancer Research and Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA.
Simons
Peter C
PC
Cimino
Daniel F
DF
Sanchez
Lilliana
L
Waller
Anna
A
Posner
Richard G
RG
Wandinger-Ness
Angela
A
Prossnitz
Eric R
ER
Sklar
Larry A
LA
D001665:Binding Sites; D015153:Blotting, Western; D002846:Chromatography, Affinity; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods; D005978:Glutathione / Q000032:analysis* / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008863:Microspheres; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D005978:Glutathione