High-sensitivity sequence analysis of peptides and proteins by 4-NN-dimethylaminoazobenzene 4′-isothiocyanate

Abstract

A manual high-sensitivity sequencing method is described, in which 4-NN-dimethylaminoazobenzene 4′-isothiocyanate is used for the stepwise degradation of amino acid residues from the peptides. The 4-NN-dimethylaminoazobenzene 4′-thiazolinones of amino acids that were released, after conversion into their thiohydantoin derivatives, were identified by t.l.c. on polyamide sheets. This new method is simple and sensitive, and requires only 2-10nmol of peptides or proteins for extended sequence analysis. The method was tested on the sequence analysis of a hexapeptide (Leu-Trp-Met-Arg-Phe-Ala), bradykinin, glucagon and native lysozyme. Results show that the proposed procedure is a sensitive method for the sequence determination of short peptides as well as for the partial sequence determination of intact proteins.