High-sensitivity fluorescence anisotropy detection of protein-folding events: application to alpha-lactalbumin.
An experimental procedure has been devised to record simultaneously fluorescence intensity and fluorescence anisotropy. A photoelastic modulator on the excitation beam enables the anisotropy signal to be recorded in one pass using a single photomultiplier tube and eliminates the need for a polarizer on the emission path. In conjunction with a stopped-flow mixer, providing a time-resolved capability, this procedure was used to study the refolding of apo alpha-lactalbumin following dilution from guanidinium chloride. Although the fluorescence intensity does not change detectably, the fluorescence anisotropy was found to resolve the conformational changes occurring between the initial unfolded state and the molten globule state formed either kinetically during refolding at pH 7.0 or at equilibrium at pH 2.0 (A-state). This result provides further evidence that fluorescence anisotropy is a valuable probe of protein structural transitions and that the information it provides concerning the rotational mobility of a fluorophore can be complementary to the information about the local environment provided by fluorescence intensity.
Oxford Centre for Molecular Sciences, University of Oxford, New Chemistry Laboratory, Oxford OX1 3QT, United Kingdom. denis.canet@ogs.co.uk
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Canet
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Doering
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Dobson
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Dupont
D000818:Animals; D016880:Anisotropy; D001671:Biochemistry / Q000379:methods*; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry*; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013997:Time Factors
D007768:Lactalbumin; D019791:Guanidine