THE MICROESTIMATION OF THREONINE

Abstract

The isolation and synthesis of the indispensable amino acid threonine, ar-amino+hydroxybutyric acid, by Professor W. C. Rose and his collaborators (cf.(1)) opened a new era in protein and amino acid research.The development of a purely chemical method besides that of isolation or that of Higasi et al. (2) for the estimation of threonine is desirable both from the view-point of nutrition and the formulation of protein structure.Examination of the structural formula of threonine, CH&HOHCHNH&OOH, suggested that it should yield acetaldehyde on oxidation with lead tetraacetate, for Criegee, Kraft, and Rank (3) have shown that glycols and related compounds are split by this reagent to yield aldehydes or ketones.They propose the mechanism shown in Reaction I for the oxidation of glycol.It appeared from these experiments that if threonine were oxidized with sufficient lead tetraacetate, acetaldehyde should be formed in an amount which would be directly proportional to the quantity of threonine used.Although this method appeared to be satisfactory for the estimation of threonine in pure solutions, the possible formation of other volatile aldehydes from the amino acids present in a protein hydrolysate excluded the use of non-specific aldehyde methods such as the determination with bisulfite, dinitrophenylhydrazine, dimethyldihydroresorcinol, etc.However, Eegriwe (4) described a highly sensitive color reaction for the qualitative estimation of lactic acid.This method was based on the conversion of lactic acid to acetaldehyde with warm concentrated HzS04 and the condensation of the aldehyde with p-hydroxydiphenyl.The condensation, which was carried out at room temperature, resulted in the formation of an' intense red-violet color with minute