Reversibility of Cysteine Labeling by 4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole

✍ Scribed by M.J. Treuheit; T.L. Kirley

• Book ID: 102561371
• Publisher: Elsevier Science
• Year: 1993
• Tongue: English
• Weight: 495 KB
• Volume: 212
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1993.1303

✦ Synopsis

The fluorescent cysteine derivative formed upon alkylation of proteins with 4 -(aminosulfonyl)-7-fluoro2,1,3-benzoxadiazole (ABD-F) is shown to be unstable under certain conditions. Although previously thought to be an irreversible reaction, the fluorescence and absorbance associated with the ABD-cysteine can be released from the protein by heating at basic (\mathbf{p H}) in the presence of reductants. Reversal of labeling is accelerated by increasing the temperature, (\mathrm{pH}), and concentration of reductant. Upon reversal of labeling, a free sulfhydryl is regenerated, since the treated protein can subsequently be realkylated (without reductant) to approximately the same level with only ABD-F. The findings reported in this work are relevant to the use of this alkylating reagent prior to any procedure involving heating or long incubation under reducing conditions, including reelectrophoresis or enzymatic deglycosylation. Investigators using ABD-F or structurally similar reagents should be aware that the cysteine derivative formed is not stable under these conditions. The reversal of cysteine derivatization may also be useful as a method for replacing the ABD-cysteine adduct with a label more suitable for subsequent specialized procedures. 1993 Academic Press, Inc.