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

Abstract

An automated fluorescence protein sequencer using 7-methylthio-5-(2,1,3-benzoxadiazolyl) isithiocyanate (MTBD-NCS), a fluorescent Edman reagent, is developed by the modification of a commercial protein sequencer. The generated MTBD-thiohydantoin amino acids fluoresced strongly, whereas the by-products such as MTBD-thiocarbamoyl amino acids and MTBD-carbamoly amino acids did not fluoresce. A few interfering peaks were observed in the chromatogram and amino acid sequence was easily determined. The coupling and cyclization/cleavage reaction conditions and extraction conditions of generated MTBD-thiazolinone amino acids were optimized using an autonalyzer. Finally, the sequence of a synthetic peptide (25 pmol), leucine-enkephalin-Thr-amide, was determined and up to six residues were successively analyzed.

Structural factors influencing the reaction rates of 4-aryloxy-7-nitrobenzofurazans with amino acids

“…Otherwise, the same remarks hold true as for 5, including the similarity of chemical shifts in 13 C-NMR for COOH, CH-5, and C-2'. Our observations on the fairly rapid reaction of compounds 2 with amino acids (and certainly also with polypeptides) have the following important consequences: (i) compounds 2 can have biological effects; (ii) they can be used to tag by fluorescence amino acids, polypeptides and proteins; (iii) they may be also be applied in analytical determinations.…”