title: Determination of homocysteine thiolactone, reduced homocysteine, homocystine, homocysteine-cysteine mixed disulfide, cysteine and cystine in a reaction mixture by overimposed pressure/voltage capillary electrophoresis.
abstract: 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 15kV 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.
authors.affiliation: Dept Biomedical Sciences and Centre of Excellence for Biotechnology Development and Biodiversity Research, University of Sassari, Sassari, Italy. angelozinellu@libero.it
authors.forename: Angelo
authors.initials: A
authors.lastname: Zinellu
authors.forename: Salvatore
authors.initials: S
authors.lastname: Sotgia
authors.forename: Bastianina
authors.initials: B
authors.lastname: Scanu
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Pisanu
authors.forename: Manuela
authors.initials: M
authors.lastname: Sanna
authors.forename: Satish
authors.initials: S
authors.lastname: Sati
authors.forename: Luca
authors.initials: L
authors.lastname: Deiana
authors.forename: Shantanu
authors.initials: S
authors.lastname: Sengupta
authors.forename: Ciriaco
authors.initials: C
authors.lastname: Carru
mesh_terms: D002138:Calibration; D019075:Electrophoresis, Capillary / Q000379:methods*; D006710:Homocysteine / Q000031:analogs & derivatives* / Q000032:analysis*; D006711:Homocystine / Q000032:analysis*; D011312:Pressure
chemical_list: D006710:Homocysteine; D006711:Homocystine; C007957:homocysteine thiolactone