4-NN-Dimethylaminoazobenzene 4‘-isothiocyanate was synthesized for the purpose of improving the ease and sensitivity of peptide sequence analysis. The method of 4-NN-dimethylaminoazobenzene 4′-isothiocyanate synthesis, the preparation of 24 4-NN-dimethylaminoazobenzene-4′-thiohydantoins of amino acids and their t.l.c. separation are described. All the thiohydantoins, except those of leucine and isoleucine, could be satisfactorily separated by chromatography on a two-dimensional polyamide sheet. The sensitive azo group permits the detection of 4-NN-dimethylaminoazobenzene-4′-thiohydantoins of amino acids as red spots down to pmol amounts directly on the sheet. A simple sensitive method for sequencing dipeptides and the first two or three N-terminal amino acids of proteins is also reported. The colour change of the spots from purple to blue to red after being exposed to HCl vapour, corresponding to the chemical change from 4-NN-dimethylaminoazobenzene-4′ ’ isothiocyanate to the 4-NN-dimethylaminoazobenzene-4′-thiocarbamoyl amino acid derivative to the 4-NN-dimethylaminoazobenzene-4′-thiohydantoin amino acid derivative, reveals a very interesting and valuable feature of this reagent.

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