Use of the Ninhydrin Reaction for Quantitative Estimation of Amino Groups in Insoluble Specimens

Abstract

IT is well known that the development of a purple colour (Ruhemann's purple) on reaction of ninhydrin with primary amino (NH₂) groups of proteins occurs even when the protein is not in solution. Thus, Pearse¹ describes attempts to use the ninhydrin reaction for histochemical staining of protein, which failed due to the ease of diffusion of the coloured product. In fact, the soluble purple compound, to which formula (I) is usually assigned, is not in any way attached to the protein; it results from deamination of the protein (left otherwise unchanged), the abstracted nitrogen being incorporated in (I)². Under suitable conditions, the coloured product is released completely from the solid into the solution, and the quantitative ninhydrin estimation, usually carried out on dissolved amino-acids and proteins, then becomes equally applicable to insoluble material. All primary NH₂ groups react, ɛ-NH₂ groups (lysine residues) giving, for reasons which are not certain, 65–70 per cent of the colour yield of α-NH₂ groups, in almost all cases³. Where the former far outnumber the latter, as in tissue preparations and very many proteins, comparison with a standard gives the actual or relative amino content with fair accuracy. This appears to be the simplest method at present available for obtaining such information.