An improved phenylarsine oxide-affinity method identifies triose phosphate isomerase as a candidate redox receptor protein.
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.
Foley
Timothy D
TD
Department of Chemistry and Program in Biochemistry, Cell, and Molecular Biology, University of Scranton, 800 Linden St, Scranton, PA 18510, USA. foleyt2@scranton.edu
Stredny
Coral M
CM
Coppa
Teresa M
TM
Gubbiotti
Maria A
MA
D000418:Albumins / Q000378:metabolism; D000478:Alkylation; D000818:Animals; D001152:Arsenicals / Q000737:chemistry* / Q000378:metabolism; D001923:Brain Chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000302:isolation & purification; D010084:Oxidation-Reduction; D010720:Phosphines / Q000737:chemistry; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014305:Triose-Phosphate Isomerase / Q000378:metabolism*
D000418:Albumins; D001152:Arsenicals; D009419:Nerve Tissue Proteins; D010720:Phosphines; D013438:Sulfhydryl Compounds; D014305:Triose-Phosphate Isomerase; C029341:oxophenylarsine; C080938:tris(2-carboxyethyl)phosphine