| Abstract
| - Bovine β-lactoglobulin, genetic variant B, has been labeled with 2-(4‘-maleimidylanilino)naphthalene-6-sulfonic acid through covalent attachment through the Cys-121 thiol group for the study of stepwisepressure denaturation of this whey protein by fluorescence spectroscopy. The labeling was performedunder nondenaturing conditions with a factor of 5 excess of the fluorophore in dimethylformamide/water (1:10) to yield the whey protein highly labeled after chromatographic separation. MALDI-TOF mass spectroscopy confirmed labeling. The emission from the fluorophore, which is sensitiveto the microenvironment, has been characterized for the labeled protein (aqueous pH 7.4 solution,25 °C) and has a λem,max = 410 nm (λex,max = 318 nm) with a fluorescence lifetime of 6.1 ± 0.2 ns.Fluorescence anisotropy increases and fluorescence quantum yield (Φf = 0.103 at 320 nm) decreaseswith increasing excitation wavelength. For increasing hydrostatic pressure, fluorescence quantumyield showed a minimum at ∼50 MPa, corresponding to the pre-denatured “pressure-melted” statein which thiol reactivity previously was found to increase prior to reversible protein unfolding. Keywords: β-Lactoglobulin; fluorescence labeling; 2-(4‘-maleimidylanilino)naphthalene-6-sulfonicacid
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