The use of tributylphosphine and 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole in the study of protein sulfhydryls and disulfides.
The use of the reagent tributyl phosphine (Bu3P) to reduce disulfides (Ruegg, U.T., and Rudinger, J., Methods Enzymol. 47, 111-116, 1977) and of 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) to block free sulphydryl groups (Toyo'oka, T., and Imai, K. Anal. Chem. 56, 2461-2464, 1984) is well established in the literature. Since the two reagents apparently do not react with each other, their combination offers a convenient and quite general method for the complete characterization of free Cys (SH) and crosslinked Cys (S-S) in proteins (Kirley, T.L., J. Biol. Chem. 264, 7185-7192, 1989). We review some of the characteristics of the reaction of these reagents with Cys in peptides and proteins and some of the properties of the ABD-Cys derivatives. The review includes reactions with model compounds (e.g., Cys and glutathione), proteins such as enolase from yeast and rabbit muscle, containing only free Cys, a protein, fetuin, containing only crosslinked Cys, and a protein, superoxide dismutase, containing both free and crosslinked Cys. In all cases the direct comparison of the tryptic or chymotryptic peptides derived from the products of parallel reactions of the protein with ABD-F alone and with ABD-F together with Bu3P permitted the determination of both free and total Cys in the protein. Sequencing the fluorescent ABD-peptides established the position of the Cys residues in the primary sequence.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004220:Disulfides / Q000032:analysis*; D004798:Enzymes / Q000737:chemistry; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010069:Oxadiazoles; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D010720:Phosphines; D011506:Proteins / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000032:analysis*; D014357:Trypsin
D004220:Disulfides; D004798:Enzymes; D007202:Indicators and Reagents; D010069:Oxadiazoles; D010446:Peptide Fragments; D010720:Phosphines; D011506:Proteins; D013438:Sulfhydryl Compounds; C043255:tri-n-butylphosphine; C046364:4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole; D014357:Trypsin