PURIFICATION OF VICINAL DITHIOL-CONTAINING PROTEINS BY ARSENICAL-BASED AFFINITY CHROMATOGRAPHY

Publisher Summary This chapter presents methodology that allows selective enrichment of vicinal dithiol-containing proteins (VDPs) based on the affinity of VDPs for trivalent arsenicals to form dithioarsine derivatives. The chapter discusses the nature of vicinal dithiol-containing proteins and presents methodology to label them selectively and to determine the fraction of a given protein that is present as the protein-(SH) 2 versus protein-S 2 (redox state). 4-aminophenylarsine oxide is attached directly to Sepharose 4B and used it to separate small molecular weight monothiols from dithiols. Binding of monothiols required two molecules to form the stable dithioarsine derivatives. The dithiols formed dithioarsines with different affinities depending on the nature of the ring formed. The seven-membered dithioarsine rings formed by the 1,4-dithiols are less stable than the five- or six-member rings formed by the 1,2- and 1,3-dithiols, respectively. Thus, 1,2- and 1-3-dithiols bound with much higher affinities than 1,4-dithiols. This agrees with observations that 1,2-dimercaptoepropanol (BAL) and lipoid acid (a 1,3-dithiol) are more effective than dithiothreitol (DTT) or dithioerythritol (1,4-dithiols) in reactivating enzymes inhibited by the addition of trivalent arsenicals, such as phenylarsine oxide.