LABELING OF MITOCHONDRIAL PROTEINS IN LIVING CELLS BY THE THIOL PROBETHIOBUTYLTRIPHENYLPHOSPHONIUM BROMIDE

Alterations to the redox state of mitochondrial thiols is a particular ly important response to oxidative stress, because mitochondria are a major source of reactive oxygen species within cells, To investigate t hese changes we designed and synthesized a novel probe for mitochondri al thiols, thiobutyltriphenylphosphonium bromide [TBTP; R, J, Burns, R ,A. J. Smith, and M. P. Murphy (1995) Arch. Biochem. Biophys. 322, 60- 68]. This lipophilic cation was accumulated several hundred-fold into the negatively charged matrix of isolated mitochondria, where it equil ibrated with endogenous thiols and during oxidative stress formed disu lfide bonds to exposed protein thiols, In this paper we show that TBTP can also selectively react with mitochondrial protein thiols in livin g cells. Since TBTP localizes specifically to mitochondria and forms d isulfide bonds selectively with mitochondrial proteins during oxidativ e stress, we conclude that TBTP has utility for investigating changes in mitochondrial thiols, independently of alterations occurring in the cytoplasm. (C) 1997 Academic Press.