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

Abstract

We report the first demonstration of rapid electrophoretic monitoring of homocysteine thiolactoneinduced 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 ~1.5 min were achieved for inhibition screening.Hyperhomocysteinemia is a disorder characterized by elevated plasma levels of the naturally occurring amino acid homocysteine (Hcy), which has been identified as an independent risk factor for cardiovascular disease. 1,2 Although the exact function of Hcy in the pathogenesis of cardiovascular disease remains unknown, there is a growing body of evidence that suggests Hcy thiolactone (HTL), the thioester metabolite of Hcy, is a contributing agent. 3-6 HTL is produced in vivo via enzymatic aminoacyl-tRNA synthase pathways at a rate proportional to extracellular Hcy concentration. 7 Thus, it is likely that HTL production is enhanced to some degree in individuals with hyperhomocysteinemia.Post-translational protein modification by HTL involves the site-specific acylation of lysine residues at ε-amino moieties to yield free thiols in the form of protein homocystamide (Scheme 1). 8,9 If left untreated, protein homocystamide can undergo spontaneous in vitro intermolecular disulfide bonding to yield oligomeric species. 10 Protein oligomerization can give rise to a range of adverse physiological affects. For example, oligomeric proteins often exhibit decreased biological activity, decreased solubility, 8 and increased immunogenicity. 11 As one would expect, the extent of physiological dysfunction caused by oligomerization *To whom correspondence should be addressed. strongin@pdx.edu. Tel: (225) In Vitro Protein ModificationProtein reaction mixtures were prepared by dissolving HTL (2.5 mM), and cyt c (10 mg/mL) in pH 7.4, 100 mM so...

Homocysteine in Protein Structure/Function and Human Disease

“…N-homocysteinylation of ferricytochrome c results in its conversion to a ferrous form, which is manifested as a change in the color of the solution from red to green. Multimers of N-Hcy-cytochrome c can also be separated by capillary electrophoresis [325]. 5.10 formation of multimeric forms of N-Hcy-cytochrome c (Fig.…”

Isolation and Self-Association Studies of Beta-Lactoglobulin

“…Capillary electrophoresis (CE) is one of such techniques and provides a rapid and efficient protein analysis with a low sample and buffer consumption [ 35 , 36 ]. The technique is typically applied for protein separation [ 37 , 38 , 39 ], but recently the use of CE as a screening method for the protein oligomers determinations is attracting attention [ 40 , 41 ]. Therefore, the potential use of CE for rapidly monitoring of β-LG forms at different pH conditions was studied in this paper.…”

Gold Nanoparticle Sensor for Homocysteine Thiolactone-Induced Protein Modification

“…For example, we reported the development a rapid capillary electrophoresis (CE) method for separating mixtures containing oligomeric protein homocystamide derived from in vitro modified cytochrome c . This CE method was successfully applied to monitoring the inhibition of HTL-induced protein oligomerization via in situ pyridoxal tetrahydrothazine formation …”