Sedimentation of Reversibly Interacting Macromolecules with Changes in Fluorescence Quantum Yield.
作者： Sumit K. Chaturvedi , Huaying Zhao , Peter Schuck
DOI: 10.1016/J.BPJ.2017.02.020
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摘要: Abstract Sedimentation velocity analytical ultracentrifugation with fluorescence detection has emerged as a powerful method for the study of interacting systems macromolecules. It combines picomolar sensitivity high hydrodynamic resolution, and can be carried out photoswitchable fluorophores multicomponent discrimination, to determine stoichiometry, affinity, shape macromolecular complexes dissociation equilibrium constants from micromolar. A popular approach data interpretation is determination binding affinity by isotherms weight-average sedimentation coefficients s w . prevailing dogma in analysis that coefficient transport corresponds signal- population-weighted average all species. We show this does not always hold true exhibit significant signal changes complex formation—properties may readily encountered practice, e.g., change quantum yield. Coupled reaction boundary rapidly reversible make contributions observed migration way cannot accounted standard population-based average. Effective particle theory provides simple physical picture reaction-coupled process. On basis, we develop more general model converges well-known form constant signals, but account simultaneously cotransport presence believe will useful when studying exhibiting quenching, enhancement, or Forster resonance energy transfer methods.
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