title: Capillary electrophoresis with lamp-based wavelength-resolved fluorescence detection for the probing of protein conformational changes.
abstract: Native protein fluorescence spectra encompass information on protein conformation. In this study, capillary electrophoresis (CE) combined with lamp-based wavelength-resolved fluorescence detection (wrFlu) is presented as a novel tool for the analysis of protein mixtures and the monitoring of protein unfolding. The CE-wrFlu system provides three-dimensional data (time, emission wavelength, intensity) from which electropherograms and accurate emission spectra of separated proteins can be extracted. For model proteins, linear detector responses (peak height vs concentration) were obtained (R(2) > 0.96) with detection limits (LODs) in the 6-32 nM range. The minimum protein concentration required for precise determination of the maximum emission wavelength by CE-wrFlu was about 15 times the LOD. Unfolding of various model proteins was induced by protein incubation and analysis in background electrolyte (BGE) containing 7.0 M urea. CE-wrFlu of the unfolded species revealed peaks with clear red-shifted spectra, which adequately corresponded to reference spectra obtained on a standard spectrophotometer. Moreover, unfolded proteins showed a significant decrease in effective electrophoretic mobility (after correction for BGE viscosity) due to the increase of their molecular hydrodynamic radii. It is concluded that the CE-wrFlu system provides two independent indicators for changes in protein folding and will allow the simultaneous assessment of protein purity and conformation.
authors.affiliation: Biomolecular Analysis, Department of Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands. B.J.deKort@uu.nl
authors.forename: Bregje J
authors.initials: BJ
authors.lastname: de Kort
authors.forename: Geert A
authors.initials: GA
authors.lastname: ten Kate
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; D024402:Carbonic Anhydrase II / Q000737:chemistry; D002417:Cattle; D002919:Chymotrypsinogen / Q000737:chemistry; D004573:Electrolytes / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D007782:Lactoglobulins / Q000737:chemistry; D008027:Light; D009113:Muramidase / Q000737:chemistry; D058767:Protein Unfolding; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D004573:Electrolytes; D007782:Lactoglobulins; D011506:Proteins; D002919:Chymotrypsinogen; D009113:Muramidase; D024402:Carbonic Anhydrase II