Chromogenic groupings in the Lowry protein determination

Abstract

In the Lowry protein determination the phenol reagent is reduced to yield a blue color. The color yield of a completely hydrolyzed protein is due to its content of tyrosine, tryptophan and cysteine, the only amino acids that react significantly. Preliminary treatment with alkaline copper solution leads to a great increase in the color yield of proteins. Simple peptides as well as chain B of insulin show this large color increment to be largely attributable to sequences of amino acids containing functional side chains. Particularly chromogenic are dipeptides containing a histidine, arginine or glutamic acid residue, the nature of the second residue being relatively unimportant. The behavior of a compound obtained in small yield and presumed to be the oxidized product of histidine suggests that electron removal from the dipeptide to the phenol reagent involves the amino nitrogen and imidazole ring of the N-terminal residue. Efficient copper catalysis of electron transfer from a chromogenic grouping in protein to the phenol reagent is believed to require formation of a coplanar quadridentate chelate. The color yield depends critically upon the rate of electron transfer since the phenol reagent is rapidly destroyed in the alkaline solution.