Two-site ionic labeling with pyranine: implications for structural dynamics studies of polymers and polypeptides by time-resolved fluorescence anisotropy

Journal of the American Chemical Society
Jai SharmaJohn D Brennan

Abstract

Time-resolved fluorescence anisotropy (TRFA) is widely used to study dynamic motions of biomolecules in a variety of environments. However, depolarization due to rapid side chain motions often complicates the interpretation of anisotropy decay data and interferes with the accurate observation of segmental motions. Here, we demonstrate a new method for two-point ionic labeling of polymers and biomolecules that have appropriately spaced amino groups using the fluorescent probe 8-hydroxyl-1,3,6-trisulfonated pyrene (pyranine). TRFA analysis shows that such labeling provides a more rigid attachment of the fluorophore to the macromolecule than the covalent or single-point ionic labeling of amino groups, leading to time-resolved anisotropy decays that better reflect the backbone motion of the labeled polymer segment. Optimal coupling of pyranine to biomolecule dynamics is shown to be obtained for appropriately spaced Arg groups, and in such cases the ionic binding is stable up to 150 mM ionic strength. TRFA was used to monitor the behavior of pyranine-labeled poly(allylamine) (PAM) and poly-d-lysine (PL) in sodium silicate derived sol-gel materials and revealed significant restriction of backbone motion upon entrapment for both polym...Continue Reading

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Citations

Jun 29, 2014·Analytical and Bioanalytical Chemistry·Clémence SicardJohn D Brennan
Jan 1, 2014·Angewandte Chemie·Jesús MosqueraJonathan R Nitschke
Nov 30, 2011·Chemical Communications : Chem Comm·Arlin Jose AmaliRohit Kumar Rana
May 23, 2008·Analytical Chemistry·Mark LowryIsiah M Warner
Jan 30, 2009·The Journal of Physical Chemistry. B·Ramon Barnadas-Rodríguez, Joan Estelrich

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