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 sulhydryl 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 ADB-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. To determine the pairing of Cys residues in disulfide bonds, the individual peptides from a nonreduced protein sample, separated and collected by IIPLC, were treated simultaneously with ADB-F and Bu3P, and the resulting single or paired fluorescent peptides, separated and collected after rechromatography, were identified by sequencing. The ability to reduce disulfides and block the resulting thiols simultaneously in a single reaction, the stability of ABD-Cys to acid hydrolysis, and the recovery, albeit in low yield, of PTH-(ABD-)Cys on sequencing, along with the characteristic fluorescence of ABD-thiol derivatives, are all very favorable features of this methodology. The apparently strong effect of environmental factors on the relative fluorescence intensity of ABD-Cys peptides represents a minor disadvantage; Cys-containing peptides with very low fluorescence may be encountered and must be anticipated in the analysis of unknown proteins.