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

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.

SNAP-25 Contains Non-Acylated Thiol Pairs that can Form Intrachain Disulfide Bonds: Possible Sites for Redox Modulation of Neurotransmission

“…Preparation of the Immobilized Phenylarsine Oxide p-Aminophenylarsine oxide (amino-PAO) was prepared by reduction of arsanilic acid (Acros Organics) and coupled via the amine group to an N-hydroxysuccinimide ester derivative of agarose (Affi-Gel 10; Bio-Rad), as described by us previously (Foley et al 2010). Any unreacted esters of the Affi-Gel 10 were blocked by the addition of excess ethanolamine.…”

“…PAO-affinity chromatography was performed as described by us previously (Foley et al 2010) with minor modifications. Briefly, immobilized PAO (0.3 ml packed) in microspin columns was resuspendend with the non-alkylated or alkylated protein fractions (0.3 mg in a volume of 0.6 ml) and incubated in the absence or presence of TCEP (5 mM) for 1 h at room temperature.…”

“…Following the alkylation step, only proteins containing disulfide bonds are able to bind PAO and they are able to do so only in the presence of TCEP, which reduces the disulfides to the PAO-binding dithiols (Foley et al 2010). Thus, oxidation to disulfides of PAO-binding vicinal thiols is operationally defined by the TCEP-dependent and DTTsensitive binding of proteins to the immobilized PAO.…”

“…Recent work in this laboratory updated an immobilized PAO-affinity method to isolate, from tissues, proteins containing PAObinding vicinal thiols and, following protein thiol alkylation and disulfide reduction steps, the subfraction of proteins containing vicinal thiols that had been oxidized reversibly to disulfide bonds (Foley et al 2010). The key to the improved method is the use of the disulfide reducing agent tris(2-carboxyethyl)-phosphine (TCEP) (Getz et al 1999) which, unlike traditional disulfide reducing agents, does not contain thiol groups.…”

Proteomic Characterization of Reversible Thiol Oxidations in Proteomes and Proteins

“…Dithiols can occur through proximity in the primary sequence, typically a -CXnC-motif where "n" is usually in the range of 2-6 residues (34) or close in the tertiary structure of the protein. Dually-Cys reacting probes such as phenylarsine oxide (PAO) have been used to identify vicinal dithiol groups in native proteins (28,77). A proteomic approach to study vicinal dithiols through MS (Fig.…”