General method to identify and enrich vicinal thiol proteins present in intact cells in the oxidized, disulfide state.
Some 5% of the soluble proteins of L1210 murine leukemia lymphoblasts contain surface vicinal thiols (Kalef, E., Walfish, P. G., and Gitler, C. (1993) Anal. Biochem. 212, 325-334). Redox dithiol to intraprotein disulfide conversion could regulate the cellular function of these proteins. A general method is presented to identify and enrich vicinal thiol proteins existing in cells in their oxidized, disulfide state. The method is based on the in situ blockage by cell permeable N-ethylmaleimide (NEM) of readily accessible cellular protein sulfhydryls. Following removal of the excess NEM, disulfide-containing proteins were identified by reduction with DTT and specific labeling with N-iodoacetyl-[125I]-3-iodotyrosine. The vicinal thiol proteins formed could also be enriched, prior to labeling with [125I]IAIT, by their selective binding to Sepha-rose-aminohexanoyl-4-aminophenylarsine oxide. Exponentially growing L1210 lymphoblasts contain more than 20 proteins with thiols in the oxidized, disulfide state. The majority derive from vicinal thiol proteins. The fraction oxidized, in some proteins, represents almost the totality of the protein present in the cell. Exposure of lymphoblasts to diamide increases the number and concentration of proteins with intraprotein disulfides. This method allows sensitive direct identification of vicinal thiol proteins that participate in redox regulation and those that are targets to oxidative stress conditions.
Gitler
C
C
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel. bmgitler@weizmann.weizmann.ac.il
Zarmi
B
B
Kalef
E
E
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D002457:Cell Extracts; D002846:Chromatography, Affinity; D003958:Diamide / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004229:Dithiothreitol / Q000737:chemistry; D005033:Ethylmaleimide / Q000737:chemistry; D007942:Leukemia, Experimental / Q000188:drug therapy; D051379:Mice; D007470:Monoiodotyrosine / Q000031:analogs & derivatives / Q000737:chemistry; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D015203:Reproducibility of Results; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002457:Cell Extracts; D004220:Disulfides; D011506:Proteins; D013438:Sulfhydryl Compounds; D003958:Diamide; C087944:N-iodoacetyl-3-iodotyrosine; D007470:Monoiodotyrosine; D005033:Ethylmaleimide; D004229:Dithiothreitol