A novel manual method for protein-sequence analysis

Abstract

A novel manual method for protein-sequence analysis is described. Three peptides, the hexapeptide (Leu-Trp-Met-Arg-Phe-Ala), insulin A chain and glucagon were used to test this technique. Peptides (1 or 2 nmol) were hydrolyzed with acid and their qualitative amino acid compositions were confirmed by reacting with 4-N,N-dimethylaminoazobenzene-4''-sulfonyl chloride and 4-N,N-dimethylaminoazobenzene 4''-isothiocyanate. Sequence determination of 20-200 nmol of peptide was then performed by the combined use of phenyl isothiocyanate and 4-N,N-dimethylaminoazobenzene 4''-isothiocyanate, a new procedure that is analogous to the dansyl-Edman method with the replacement of dansyl chloride by 4-N,N-dimethylaminoazobenzene 4''-isothiocyanate as the N-terminal residue determination reagent. On TLC, this new N-terminal reagent gave brightly colored 4-N,N-dimethylaminoazobenzene-4-thiohydantoins of amino acids and showed the following advantages: the detection sensitivity is in the pmol range; UV observation is not required; there is no destruction of acid-labile amino acids; 2-dimensional TLC separation is adequate to identify 24 amino acids, except leucine and isoleucine (this pair of amino acids can be resolved by using 4-N,N-dimethylaminoazobenzene-4''-sulfonyl chloride); the determination of a new N-terminal residue (from coupling to TLC identification) takes only 3 h; the color difference between isothiocyanate, thiocarbamoyl and thiohydantoin derivatives facilitates the identifications.