Determination of homocysteine thiolactone, reduced homocysteine, homocystine, homocysteine–cysteine mixed disulfide, cysteine and cystine in a reaction mixture by overimposed pressure/voltage capillary electrophoresis

An elevated level of thiol amino acid homocysteine is associated with several complex disorders. Homocysteine ability to bind proteins, thereby modulating their structure and function, is proposed to be one of the mechanisms of homocysteine induced pathogenecity. Homocysteine and homocysteine thiolactone bind to protein cysteine and lysine residues respectively. A major hurdle in studying protein homocysteinylation is the lack of suitable analytical techniques to determine simultaneously the concentrations of reduced and oxidized forms of homocysteine and cysteine (especially homocysteine–cysteine mixed disulfide) together with thiolactone formed during the reaction of homocysteine or thiolactone with proteins. Herein we report a capillary electrophoresis method to determine simultaneously the levels of these intermediates. For this 40 mmol/L Tris phosphate buffer at (pH 1.60) was used as running electrolyte, and the separation was performed by the simultaneous application of a CE voltage of 15 kV and an overimposed pressure of 0.1 psi. A rapid separation of these intermediates in less than 6 min with a good reproducibility of both peak areas (CV < 2%) and migration time (CV < 0.2%) was obtained. The applicability of our method was validated by incubating reduced homocysteine and albumin and measuring the reaction intermediates in the solution mixture.