Abstract
Publisher Summary This chapter discusses affinity chromatography using an immobilized phenylarsine oxide derivative coupled to Sepharose for the purification of vicinal dithiolcontaining proteins. This methodology uses a phenylarsine oxide (PAO) resin to selectively bind, elute, and identify proteins that might be involved in redox regulation and to test a possible disulfide formation as a regulatory mechanism in oxidative stress. Because such conditions are found in activated polymorphonuclear leukocytes (PMN), a cytosolic fraction from PMN is used to isolate potential target proteins. This way the isolation and identification of six proteins with affinity for PAO can be achieved: thioredoxin (Trx), calcineurin (CAN), glutathione S-transferase (GST), calgranulin, L-plastin, and cofilin. From these candidates, a more detailed characterization of the interaction of PAO and oxidants with the serine-threonine phosphatase CaN (also known as protein phosphatase 2B) can be explored, which strongly suggests a dithiol-disulfide equilibrium in the enzyme as a mechanism for redox regulation.
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