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 hydrolysed with acid and their qualitative amino acid compositions were confirmed by reacting with 4-NN-dimethylaminoazobenzene-4'-sulphonylchloride and 4-NN-dimethylaminoazobenzene 4'-isothiocyanate. Sequence determination of 20-200 nmol of peptide was then performed by the combined use of phenyl isothiocyanate and 4-NN-dimethylaminoazobenzene 4'-isothiocyanate, a new procedure that is analogous to the dansyl-Edman method with the replacement of dansyl chloride by 4-NN-dimethylaminoazobenzene 4'-isothiocyanate as the N-terminal residue determination reagent. On t.l.c. this new N-terminal reagent gave brightly coloured 4-NN-dimethylaminoazobenzene-4-thiohydantoins of amino acids and showed the following advantages: (1) the detection sensitivity is in the pmol range; (2) u.v. observation is not required; (3) there is no destruction of acid-labile amino acids; (4) two-dimensional t.l.c. separation is adequate to identify 24 amino acids, except leucine and isoleucine (this pair of amino acids can be resolved by using 4-NN-dimethylaminoazobenzene-4'-sulphonyl chloride); (5) the determination of a new N-terminal residue (from coupling to t.l.c. identification) takes only 3 h; (6) the colour difference beteen isothiocyanate, thiocarbamoyl and thiohydantoin derivatives facilitates the identifications.

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