title: Potential of capillary electrophoresis with wavelength-resolved fluorescence detection for protein unfolding studies using β-lactoglobulin B as a test compound.
abstract: Capillary electrophoresis (CE) with wavelength-resolved fluorescence detection (wrFlu) was evaluated for the study of protein unfolding using non-reduced and reduced β-lactoglobulin B (β-LGB) as model compounds. Protein unfolding was achieved by incubation in sodium phosphate (pH 3.0) containing increasing concentrations of urea (0-7.1 M). CE-wrFlu was performed using the incubation media as background electrolytes (BGEs). At low urea concentrations (0-3.1 M), CE-wrFlu analysis of non-reduced β-LGB showed a single peak with a maximum emission wavelength (λmax) of 333 nm, indicating the protein was in its folded state. When β-LGB was exposed to 3.6 and 4.1 M urea, CE-wrFlu revealed an additional peak with a λmax of 353 nm and a reduced migration time, suggesting the formation of fully unfolded species. Upon further raising the urea concentration up to 6.5 M, the peak intensity of the unfolded protein increased. At the same time, the later-migrating peak became wider and lower, showing a decrease of migration time and a shift of λmax (333-353 nm), indicating gradual unfolding. Construction of a λmax-based transition curve for the later-migrating β-LGB species provided a denaturant-concentration midpoint of unfolding (cm) of 5.39 M, which was similar to the cm determined by plotting the corrected effective electrophoretic mobility (μeff,c) vs. urea concentration. Stand-alone fluorescence spectroscopy of the same β-LGB solutions revealed a lower cm (4.97 M), most probably because the respective β-LGB species were not separated, yielding ensemble average data. For reduced β-LGB, at all tested urea concentrations one protein peak was observed, whereas λmax and μeff,c indicated protein unfolding at much lower urea concentrations (cm of 1.2 M). We conclude that CE-wrFlu can distinguish protein conformational states and thus may provide useful additional information on unfolding pathways.
authors.affiliation: Biomolecular Analysis, Utrecht University, The Netherlands.
authors.forename: Bregje J
authors.initials: BJ
authors.lastname: de Kort
authors.forename: Gerhardus J
authors.initials: GJ
authors.lastname: de Jong
authors.forename: Govert W
authors.initials: GW
authors.lastname: Somsen
mesh_terms: D000818:Animals; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000379:methods; D007782:Lactoglobulins / Q000032:analysis* / Q000737:chemistry*; D011489:Protein Denaturation; D058767:Protein Unfolding; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D007782:Lactoglobulins