Phenylarsine Oxide‐Affinity Isolation of Candidate Redox Receptor Proteins from the Brain

Abstract

Proteins containing vicinal dithiols, capable of reversible oxidation to form intraprotein disulfide bonds, may play important roles in redox signaling. Affinity chromatography using immobilized phenylarsine oxide (PAO), which binds vicinal dithiol‐containing proteins has been used in limited studies to isolate proteins containing vicinal thiols that undergo reversible oxidation. In this method, the oxidized proteins are captured selectively by reduction of the thiols to enable binding to PAO following oxidative stress and alkylation to block non‐oxidized thiols. The PAO‐bound proteins are removed by the addition of low‐molecular‐weight thiols which compete for binding to PAO. A major limitation of this method has been the lack of a disulfide reducing agent that would promote the reduction of reversibly‐oxidized thiols but not also compete with proteins for binding to PAO. The present study demonstrates using a Triton X‐100‐soluble fraction from rat brain as a source of proteins that the efficiency of PAO‐affinity chromatography as a means to capture selectively vicinal dithiol proteins as well as the subset of these that undergo reversible oxidation in response to mild oxidative stress can be improved greatly by using the reducing agent tris(2‐carboxyethyl)phosphine (TCEP) which does not contain a thiol group. The results show that (i) a large fraction of brain proteins bind PAO in a thiol‐sensitive manner and (ii) a small number of these undergo reversible oxidation in response to mild oxidative stress in vitro. In addition, the known vicinal thiol protein SNAP‐25, which plays a critical role in neurotransmitter release, is identified by Western blotting as a candidate "redox receptor" protein in the brain.