Capillary electrophoretic screening for the inhibition of homocysteine thiolactone-induced protein oligomerization.
We report the first demonstration of rapid electrophoretic monitoring of homocysteine thiolactone-induced protein oligomerization (HTPO), a unique type of post-translational protein modification that may have clinical significance as an indicator of cardiovascular and neurovascular diseases. HTPO of the model protein bovine cytochrome c was initiated in vitro. The relative monomer and aggregate levels of the resultant protein mixtures were determined following separation using capillaries coated with the cationic polymer, poly(diallyldimethylammonium chloride). UV detection provided adequate sensitivity for the monitoring of higher order species, which exist at relatively low concentrations in the protein reaction mixture as compared to the monomeric species. Separations performed under standard injection conditions were optimized on the basis of applied voltage and sample denaturation conditions. Separations performed using short-end injection allowed for more rapid analyses, typically in less than 70 s. Relative errors for run-to-run migration times were less than 0.5%. This novel oligomeric system provides a rapid and straightforward in vitro method to screen therapeutic agents for their ability to inhibit HTPO. Changes in peak area for monomer and aggregate species were used to assess HTPO inhibition as a function of pyridoxal 5-phosphate (PLP) concentration. PLP was shown to effectively inhibit HTPO in vitro. Rapid analysis times of approximately 1.5 min were achieved for inhibition screening.
Gates
Arther T
AT
Department of Chemistry and College of Basic Sciences, Louisiana State University, Baton Rouge, Louisiana 70803, USA.
Lowry
Mark
M
Fletcher
Kristin A
KA
Murugeshu
Abitha
A
Rusin
Oleksandr
O
Robinson
James W
JW
Strongin
Robert M
RM
Warner
Isiah M
IM
D000818:Animals; D002412:Cations / Q000737:chemistry; D002417:Cattle; D045304:Cytochromes c / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D019075:Electrophoresis, Capillary / Q000379:methods; D006710:Homocysteine / Q000031:analogs & derivatives* / Q000032:analysis / Q000037:antagonists & inhibitors / Q000737:chemistry; D011095:Polyethylenes / Q000737:chemistry; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors
D002412:Cations; D011095:Polyethylenes; D000644:Quaternary Ammonium Compounds; D006710:Homocysteine; C041004:poly-N,N-dimethyl-N,N-diallylammonium chloride; D011732:Pyridoxal Phosphate; D045304:Cytochromes c; C007957:homocysteine thiolactone