Fluorescence decay time distribution analysis reveals two types of bindingsites for 1,8-anilinonaphthalene sulfonate in native human oxyhemoglobin

Binding of 1,8-anilinonaphthalene sulfonate ( 1,I-ANS) with native human ox yhemoglobin (Hb) in 50 mM potassium phosphate buffer (pH 7.4) was studied b y steady-state fluorescence spectroscopy and by laser spectrofluorimetry wi th subnanosecond time resolution. The distribution of fluorescence decay ti mes and parameters of two- and three-exponential deconvolution of the fluor escence kinetics of 1,3-ANS in Hb solution demonstrate that the emission at wavelengths h,, of 455-600 nm is not single-exponential and has components with mean decay times <0.5, 3.1-5.5, and 12.4-15.1 nsec with the amplitude s depending on the emission wavelength. Analysis of time-resolved fluoresce nce spectra shows that the shortest-lived component should be assigned to 1 ,8-ANS molecules in the aqueous medium, whereas the two longer-lived compon ents are assigned to two types of binding sites for I,I-ANS in the Hb molec ule characterized by different polarity and accessibility to water molecule s.