18] Derivatives of hemoglobin prepared by reaction with aryl or alkyl isothiocyanates

Abstract

The reaction of protein amino groups with aryl isothiocyanates to form thiourea derivatives, called the “Edman reaction,” has frequently been exploited in the sequencing of proteins. The basis for this application lies in the reaction of aryl isothiocyanates with the amino terminus of the protein. Reaction with the reagent depends on the nucleophilic character of the protein amino group, and as a result, the protonated form is unreactive. Thus, the specificity of the reaction can be controlled to some extent by manipulation of the pH, example, reaction at the amino termini is favored by maintenance of the pH near 7.3, because at this pH most of the ɛ-amino groups of the protein are protonated. Maintenance of the pH near neutrality can also minimize competing hydrolysis reactions of the reagent, which is subject to both acid and base catalysis. Another competing reaction in the case of alkyl isothiocyanates is alkylation of nucleophilic sulfhydryl groups through displacement of the —NCS group. This reaction is unlikely for aryl isothiocyanates because nucleophilic aromatic substitution is extremely slow unless the —NCS group is activated by the presence of electron-withdrawing groups in the 2- and 4-positions.