REMOVAL OF THE FLUORESCENT 4-(AMINOSULFONYL)-2,1,3-BENZOXADIAZOLE LABEL FROM CYSTEINE-CONTAINING PEPTIDES

The complete removal of the fluorescent cysteine derivative 4-(aminosu lfonyl)-7-fluoro-2, 1,3-benzoxadiazole (ABD-F) from an intact protein has not been demonstrated even after extended treatment with a reducin g agent. It has been suggested that this may be due to incomplete dena turation under the conditions employed. We were interested in investig ating this phenomenon utilizing small peptides containing individual A BD-labeled cysteine residues. After incubating the fluorescent peptide s in the presence of a reductant, it was shown that the ABD label coul d be completely removed from all of the cysteine-containing peptides i nvestigated. Therefore, delabeling irreversibility is due to residual structure in proteins. Electrospray ionization mass spectrometry (ESI- MS) was used to determine the molecular mass of each peptide after rem oval of the ABD label. The ESI-MS data were consistent with the genera tion of a free sulfhydryl. The generation of the free sulfhydryl was f urther substantiated when a delabeled peptide was completely relabeled with ABD-F in the absence of reductant. (C) 1998 Elsevier Science B.V .