Capillary Electrophoretic Screening for the Inhibition of Homocysteine Thiolactone-Induced Protein Oligomerization

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• 作者：Arther T. Gates ; Mark Lowry ; Kristin A. Fletcher ; Abitha Murugeshu ; Oleksandr Rusin ; James W. Robinson ; Robert M. Strongin ; Isiah M. Warner
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：November 1, 2007
• 年：2007
• 卷：79
• 期：21
• 页码：8249 - 8256
• 全文大小：255K
• 年卷期：v.79,no.21(November 1, 2007)
• ISSN：1520-6882

文摘

We report the first demonstration of rapid electrophoreticmonitoring of homocysteine thiolactone-induced proteinoligomerization (HTPO), a unique type of post-translational protein modification that may have clinical significance as an indicator of cardiovascular and neurovasculardiseases. HTPO of the model protein bovine cytochromec was initiated in vitro. The relative monomer and aggregate levels of the resultant protein mixtures weredetermined following separation using capillaries coatedwith the cationic polymer, poly(diallyldimethylammoniumchloride). UV detection provided adequate sensitivity forthe monitoring of higher order species, which exist atrelatively low concentrations in the protein reactionmixture as compared to the monomeric species. Separations performed under standard injection conditions wereoptimized on the basis of applied voltage and sampledenaturation conditions. Separations performed usingshort-end injection allowed for more rapid analyses,typically in less than 70 s. Relative errors for run-to-runmigration times were less than 0.5%. This novel oligomeric system provides a rapid and straightforward in vitromethod to screen therapeutic agents for their ability toinhibit HTPO. Changes in peak area for monomer andaggregate species were used to assess HTPO inhibitionas a function of pyridoxal 5-phosphate (PLP) concentration. PLP was shown to effectively inhibit HTPO in vitro.Rapid analysis times of ~1.5 min were achieved forinhibition screening.