The copper-catalyzed oxidation of peptides and proteins by phosphomolybdic/phosphotungstic acid (Folin phenol reagent) was studied with respect to tedox stoichiometry of color formation and nature of the oxidation products. From peptides without reducing side chains two reducing equivalents were transferred under ideal conditions to Mo6+w
for each unit of tetradentate copper complex with concomitant formation of an imino peptide. Tyrosine and tryptophan side chains contributed four additional reducing equivalents. Oxidation of praline-containing peptides was greatly impaired as judged from color formation due to the interference of the imino acid with complex formation. Reaction of the oxidized peptides with 2,4dinitrophenyl (DNP)-hydmzine gave a peptide amide and the DNP-hydrazone of a 2-oxoacyl peptide. The oxidation products from tetraalanine were identified as dialanine amide and pyruvoylalanine DNP-hydrazone. From the time course of the development of the blue color on reduction of Folin reagent with tetraalanine it was inferred that the reaction consisted of an initial (~5 s) oxidation to a Cu3+ peptide complex followed by slow changes in absorbance, especially above 0.2 mM. Due to these complications the two-clcctron stoichiometry has to be considered only as a limiting case for pcptide concentrations below 0.02 mM.