Distinctive test with copper(II)-ninhydrin reagent for small α-peptides separated by paper chromatography

Abstract

A Cu²⁺-ninhydrin reagent was used to distinguish qualitatively small α-peptides and α-amino acid amides from free amino acids on paper after chromatography. With the exception of peptides containing N-terminal L-tryptophan, all the peptides gave a yellow chromophore with a λ_max at 395 nm. Protein and non-protein amino acids and polyamines gave different chromophores which varied from one compound to another. Peptides with N-terminal L-proline gave a very faint yellow chromophore or no coloured product at all, while glutathione, a γ-glutamyl peptide, gave a red colour. Polypeptides and proteins did not produce a yellow chromophore. Evidence is provided for a reaction sequence in which peptides first react with Cu²⁺ to form a complex which then reacts with ninhydrin to give the yellow chromophore. These results and also studies with several peptides having N- and C-terminal substitutions suggest that the minimum structural requirement around the α-peptide linkage for the formation of the yellow chromophore with the Cu²⁺-ninhydrin reagent is as follows: