Time-resolved fluorescence reveals two binding sites of 1,8-ANS in intact human oxyhemoglobin

Time-resolved fluorescence of 1,8-anilinonaphthalene sulfonate (1,P-ANS) fl uorescent probe bound to intact human oxyhemoglobin (HbO(2)) is investigate d. Fluorescence emission spectra of 1,8-ANS in a potassium buffer solution (pH 7.4) of HbO(2) undergo a substantial blue shift during first 6 ns after pulsed optical excitation at 337.1 nm. Nonexponential fluorescence kinetic s of 1,8-ANS in the HbO(2) solution are studied by the decay time distribut ion and conventional multiexponential analyses for a set of emission wavele ngth range of lambda (3m) = 455-600 nm. These fluorescence decays contain c omponents with mean decay times of <0.5 ns, 3.1-5.5 ns, and 12.4-15.1 ns wi th spectrally-dependent relative contributions. The shortest decay componen t is assigned to free 1,8-ANS molecules in the bulk buffer environment, whe reas the two longer decay components are assigned to two types of binding s ites of 1,8-ANS in the HbO(2) molecule presumably differing by polarity and accessibility to water molecules. The results represent the first experime ntal evidence of heterogeneous binding of 1,8-ANS to intact human oxyhemogl obin. (C) 2000 Elsevier Science B.V. All rights reserved.