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Updated: Jul 23, 2026

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions
Published on: October 21, 2016
Time-resolved fluorescence reveals two binding sites of 1,8-ANS in intact human oxyhemoglobin
D A Parul1, S B Bokut, A A Milyutin
1A.D. Sakharov International Environmental University, Minsk, Belarus.
Abstract:
Time-resolved fluorescence of 1,8-anilinonaphthalene sulfonate (1,8-ANS) fluorescent probe bound to intact human oxyhemoglobin (HbO2) is investigated. Fluorescence emission spectra of 1,8-ANS in a potassium buffer solution (pH 7.4) of HbO2 undergo a substantial blue shift during first 6 ns after pulsed optical excitation at 337.1 nm. Nonexponential fluorescence kinetics of 1,8-ANS in the HbO2 solution are studied by the decay time distribution and conventional multiexponential analyses for a set of emission wavelength range of lambdaem = 455-600 nm. These fluorescence decays contain components with mean decay times of <0.5 ns, 3.1-5.5 ns, and 12.4-15.1 ns with spectrally-dependent relative contributions. The shortest decay component is assigned to free 1,8-ANS molecules in the bulk buffer environment, whereas the two longer decay components are assigned to two types of binding sites of 1,8-ANS in the HbO2 molecule presumably differing by polarity and accessibility to water molecules. The results represent the first experimental evidence of heterogeneous binding of 1,8-ANS to intact human oxyhemoglobin.
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