Sensitivity and selectivity of the electrochemical detection of the copper(II) complexes of bioactive peptides, and comparison to model studies by rotating ring-disc electrode

Abstract

Post-column reaction of peptides with Cu(II) can be used for the electrochemical detection of peptides as their biuret complexes. Understanding of the behavior (sensitivity at the anode and cathode in the dual-series electrochemical detector) of the system is facilitated through the observation of the rotating ring disc voltammetry of some model compounds. In operation, the anodic signal from the oxidation of the Cu(II)-peptide to the Cu(III) form can be used to detect peptides, or the downstream cathode can be used to detect the Cu(III) form. The signals appear at about 0.4 V (anode) for tetra- and longer peptides, 0.65 V for tripeptides. The anode signal is augmented by tyrosine (oxidation at 0.4–0.5 V) and tryptophan (0.5–0.6 V). If the cathode is used as the detector in a two working electrode cell, the sensitivity depends on the stability of the Cu(III) product. This is peptide dependent, but the signal is significant and useful analytically. Twenty-three bioactive peptides in two groups, naturally electrochemically active and naturally electrochemically silent, and several model compounds have been studied. Both naturally electrochemically active peptides (contain tyrosine and/or tryptophan) and naturally electrochemically silent peptides have been studied. Chromatography with an acetonitrile gradient has been used to separate the peptides in each group. Detection limits are for non-electroactive peptides in the range of 16–100 fmol (10- μl injection 1.6–10 n M, 100 μl injection 0.16–1.0 n M), and for electroactive peptides in the range of 6–40 fmol (0.6–4.0 n M for a 10- μl injection and 60–400 p M for a 100- μl injection). A tryptic digest of bovine cytochrome c is easily seen at 100 n M.