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 cation… Show more
“…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.…”
“…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.…”
“…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.…”
The aim of this study was to investigate isolated β-lactoglobulin (β-LG) from the whey protein isolate (WPI) solution using the column chromatography with SP Sephadex. The physicochemical characterization (self-association, the pH stability in various salt solutions, the identification of oligomeric forms) of the protein obtained have been carried out. The electrophoretically pure β-LG fraction was obtained at pH 4.8. The fraction was characterized by the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/TOF MS) technique. The use of the HCCA matrix indicated the presence of oligomeric β-LG forms, while the SA and DHB matrices enabled the differentiation of A and B isoforms in the sample. The impact of sodium chloride, potassium chloride, ammonium sulfate, and sodium citrate in dispersion medium on β-LG electrophoretic stability in solution was also studied. Type of the dispersion medium led to the changes in the isoelectric point of protein. Sodium citrate stabilizes protein in comparison to ammonium sulfate. Additionally, the potential of capillary electrophoresis (CE) with UV detection using bare fused capillary to monitor β-LG oligomerization was discussed. Obtained CE data were further compared by the asymmetric flow field flow fractionation coupled with the multi-angle light scattering detector (AF4-MALS). It was shown that the β-LG is a monomer at pH 3.0, dimer at pH 7.0. At pH 5.0 (near the isoelectric point), oligomers with structures from dimeric to octameric are formed. However, the appearance of the oligomers equilibrium is dependent on the concentration of protein. The higher quantity of protein leads to the formation of the octamer. The far UV circular dichroism (CD) spectra carried out at pH 3.0, 5.0, and 7.0 confirmed that β-sheet conformation is dominant at pH 3.0, 5.0, while at pH 7.0, this conformation is approximately in the same quantity as α-helix and random structures.
“…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 …”
Homocysteine thiolactone-induced protein modification (HTPM) is a unique post-translational protein modification that is recognized as an emergent biomarker for cardiovascular disease. HTPM involves the site-specific acylation of proteins at lysine residues by homocysteine thiolactone (HTL) to produce protein homocystamide, which has been found at elevated levels in patients with coronary heart disease. Herein, we report the development of a novel gold nanoparticle (GNP) biochemical sensor for detection of protein homocystamide in an in vitro serum protein-based model system. Human serum albumin (HSA) and human sera were subjected to HTPM in vitro to produce HSA-homocystamide or serum protein homocystamide, respectively, which was subsequently treated with citrate-capped GNPs. This GNP sensor typically provided instantaneous visual confirmation of HTPM in the protein model systems. Transmission electron microscopy images of the GNPs in the presence of HSA-homocystamide suggest that modification-directed nanoparticle assembly is the mechanism by which the biochemical sensor produces a colorimetric signal. The resultant nanoparticle-protein assembly exhibited excellent thermal and dilutional stability, which is expected for a system stabilized by chemisorption and intermolecular disulfide bonding. The sensor typically provided a linear response for modified human sera concentrations greater than approximately 5 mg/mL. The calculated limit of detection and calibration sensitivity for the method in human sera were 5.2 mg/mL and 13.6 AU . (microg/mL)-1, respectively.
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