[1][2][3] The method combines the reactions of copper ions with the peptide bonds under alkaline conditions (the Biuret test) with the oxidation of aromatic protein residues.
The reaction mechanism is not well understood, but involves reduction of the Folin–Ciocalteu reagent and oxidation of aromatic residues (mainly tryptophan, also tyrosine).
Proper caution must be taken when dealing with the Folin's reagent, which is only active in acidic conditions.
Mixing the protein solution as the Folin's reagent is simultaneously added will ensure that the reaction occurs in the desired manner.
[6] The concentration of the reduced Folin reagent (heteropolymolybdenum Blue) is measured by absorbance at 660 nm.