Isolation and characterization of polypeptide at the picomole level. Pre-column formation of peptide derivatives with dimethylaminoazobenzene isothiocyanate

Abstract

Polypeptides coupled with dimethylaminoazobenzene isothiocyanate through their amino groups to form dimethylaminoazobenzenethiocarbamoyl- (DABTC-)peptides can be separated by reversed-phase high-pressure liquid chromatography and detected in the visible region (436 nm). As little as 1 ng (2 pmol) of a DABTC-pentapeptide can be identified against a stable base-line with the signal-to-noise ratio of 10. The DABTC-peptides can also be recovered from the column, and their N-terminal amino acids (obtained by direct treatment with aqueous acid) and amino acid compositions and sequences can be all analysed at the picomole level. The power of this method is demonstrated by the complete separation and characterization of model peptides, peptide hormones and peptides derived from enzymic fragmentation of proteins. This new technique should provide a sensitive and efficient tool for peptide analysis at the nanogram level.