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

Zinellu, Angelo and Sotgia, Salvatore and Scanu, Bastianina and Pisanu, Elisabetta and Sanna, Manuela and Sati, Satish and Deiana, Luca and Sengupta, Shantanu and Carru, Ciriaco (2010) Determination of homocysteine thiolactone, reduced homocysteine, homocystine, homocysteine–cysteine mixed disulfide, cysteine and cystine in a reaction mixture by overimposed pressure/voltage capillary electrophoresis. Talanta, Vol. 82 (4), p. 1281-1285. eISSN 1873-3573. Article.

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DOI: 10.1016/j.talanta.2010.06.054

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 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.

Item Type:Article
ID Code:5582
Status:Published
Refereed:Yes
Uncontrolled Keywords:Protein homocysteinylation, homocysteine, capillary electrophoresis
Subjects:Area 05 - Scienze biologiche > BIO/12 Biochimica clinica e biologia molecolare clinica
Divisions:001 Università di Sassari > 02 Centri > Centro di eccellenza interdisc. sviluppo ricerca biotecnologica e studio biodiversità della Sardegna e dell'area mediterranea
001 Università di Sassari > 01 Dipartimenti > Scienze biomediche
002 Altri enti e centri di ricerca del Nord Sardegna > Porto Conte Ricerche, Alghero
002 Altri enti e centri di ricerca del Nord Sardegna > National laboratory of the national institute of biostructures and biosystems, Osilo
Publisher:Elsevier
eISSN:1873-3573
Deposited On:11 Mar 2011 12:20

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