Electrochemical probing into cytochrome c modification with homocysteine-thiolactone

Homocysteine thiolactone modification is a unique process of posttranslational protein modification as well as a significant clinical indicator of cardiovascular and neurovascular diseases, so we report a new method in this paper to sensitively monitor such a modification using horse heart cytochrome c as a model protein. After the modification has been confirmed by UV–vis spectroscopy and ESI-MS, N-linked cytochrome c is then covalently assembled onto the surface of a gold electrode via the resulted homocysteine thiol group, thus electrochemical techniques, especially differential pulse voltammetry, have been employed and proven to provide an efficient way to probe into the modification of the protein. While the immobilized protein can exhibit well-defined voltammetric response, the signal of the modified cytochrome c is positively correlated to the concentration of homocysteine-thiolactone. The detectable electrochemical signal can be attained with the minimum concentration of 5 × 10⁻⁵ M homocysteine-thiolactone. Furthermore, screening of N-homocysteinylation inhibitors can be also feasible since the electrochemical waves linearly decrease with the concentration of an inhibitor pyridoxal 5-phosphate. The limit of detection for the inhibitors can be about 1 × 10⁻⁵ M.