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Neurochemical Research

, Volume 35, Issue 2, pp 306–314 | Cite as

An Improved Phenylarsine Oxide-Affinity Method Identifies Triose Phosphate Isomerase as a Candidate Redox Receptor Protein

  • Timothy D. Foley
  • Coral M. Stredny
  • Teresa M. Coppa
  • Maria A. Gubbiotti
Original Paper

Abstract

Reversible oxidation on proteins of vicinal thiols to form intraprotein disulfides is believed to be an important means by which redox sensitivity is conferred on cellular signaling and metabolism. Affinity chromatography using immobilized phenylarsine oxide (PAO), which binds preferentially to vicinal thiols over monothiols, has been used in very limited studies to isolate the fraction of cellular proteins that exhibit reversible oxidation of vicinal thiols to presumed disulfide bonds. A challenge to the use of PAO-affinity chromatography for isolation of readily oxidizable vicinal thiol proteins (VTPs) has been the lack of a disulfide reducing agent that reverses oxidation of the PAO-binding protein thiols and maintains these in the reduced state necessary to bind PAO but does not also compete with the VTPs for binding to the immobilized PAO. The present study demonstrates that the capture from a detergent-soluble rat brain extract of VTPs by PAO-affinity chromatography was improved greatly by use of the reducing agent tris(2-carboxyethyl)-phosphine which, unlike more traditional disulfide-reducing agents, does not contain a thiol group. Moreover, we show that, while a substantial fraction of total brain proteins contain PAO-binding thiols, only a fraction of these were readily and reversibly oxidized. The two most abundant of these redox-active proteins were identified as albumin and triose phosphate isomerase (TPI). We propose that TPI is a candidate intracellular redox receptor protein. The improved PAO-affinity method detailed here should enable the discovery of lower abundance novel redox-active regulatory proteins.

Keywords

Vicinal thiols Phenylarsine oxide Redox signaling Oxidative stress Triose phosphate isomerase Albumin 

Abbreviations

FT

Flow-through

DTT

Dithiothreitol

LW

Last wash

MALDI-TOF MS

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

ME

2-Mercaptoethanol

PAO

Phenylarsine oxide

TCEP

Tris(2-carboxyethyl)-phosphine

TPI

Triose phosphate isomerase

TSF

TRITON-soluble fraction

RNS

Reactive nitrogen species

ROS

Reactive oxygen species

RSNO

Nitrosothiol

RSOH

Sulfenic acid

RSSR

Disulfide

VTPs

Vicinal thiol proteins

Notes

Acknowledgments

This work was supported in part by NIH Grant AG022357 from the National Institute on Aging. The authors wish to thank Dr. Michel Eschenbrenner of the Institute for Molecular Biology and Medicine at the University of Scranton for his guidance with the mass spectrometry and use of the MASCOT search engine and Dr. Paul Gershon of the University of California at Irvine for his helpful insight into the in-gel digestion process.

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Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Timothy D. Foley
    • 1
  • Coral M. Stredny
    • 1
  • Teresa M. Coppa
    • 1
  • Maria A. Gubbiotti
    • 1
  1. 1.Department of Chemistry and Program in Biochemistry, Cell, and Molecular BiologyUniversity of ScrantonScrantonUSA

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