Development of an Efficient Amino Acid Sequencing Method Using Fluorescent Edman Reagent 7-[(N,N-Dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl Isothiocyanate

Abstract

In this paper, a new method is described for N-terminal amino acid sequencing of peptides using the fluorescent reagent 7-[(N,N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate (DBD-NCS). Sequence determination is carried out by identifying thiazolinone (TZ) amino acids, which are generally unstable and difficult to detect. The employed system can easily and quickly derive TZ amino acids using the Edman reaction with DBD-NCS; these amino acids are also stable enough to be efficiently detected by high-performance liquid chromatography. Resultant detection limits for DBD-TZ amino acids range from 50 fmol to a sub-picomole level (S/N = 3). This system successfully analyzed sequences of Leu5-enkephalin (25 pmol) and angiotensin I (100 pmol) using fluorometric detection at 524 nm with excitation at 387 nm.

Fluorogenic and fluorescent labeling reagents with a benzofurazan skeleton

“…The DBD-TZ derivatives were separated using HPLC and the detection limits were 50 fmol to sub pmol (S/N = 3). The sequencing of Leu 5 -enkephalin (25 pmol) and angiotensin I (100 pmol) were achieved (Matsunaga et al, 1995). Recently, the peptides or proteins containing D-amino acids were found, and therefore the method for the amino acid sequence and D/L-configuration determination were studied .…”

An automated fluorescence protein sequencer using 7‐methylthio‐4‐(2,1,3‐benzoxadiazolyl) isothiocyanate (MTBD‐NCS) as an Edman reagent

“…To overcome these disadvantages, we have reported a modified procedure using the following fluorescent benzofurazan Edman reagents: 7-(N,N-dimethylamino)-sulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS; Matsunaga et al, 1995) and 7-methylsulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (MSBD-NCS; Toriba et al, 1999). From these agents, the TZ amino acids generated by cyclization/cleavage reaction can be detected fluorimetrically, since the TZ-amino acids of these reagents were much more stable than those generated from PITC.…”

Amino acid sequence and D/L‐configuration determination methods for D‐amino acid‐containing peptides in living organisms

“…It should be noted that amino acid residues derived from 7-(N,N-dimethylamino)sulphonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS; Matsunaga et al, 1995) and 7-methylsulphonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (MSBD-NCS; Toriba et al, 1999) were detected as TZ derivatives. TZ-amino acids of these reagents were stable compared with ATZ-amino acids generated from PITC and were separated by reversed-phase HPLC.…”