Fluorescence decay time distribution analysis reveals two types of binding sites for 1,8-anilinonaphthalene sulfonate in native human oxyhemoglobin.
Binding of 1,8-anilinonaphthalene sulfonate (1,8-ANS) with native human oxyhemoglobin (Hb) in 50 mM potassium phosphate buffer (pH 7.4) was studied by steady-state fluorescence spectroscopy and by laser spectrofluorimetry with subnanosecond time resolution. The distribution of fluorescence decay times and parameters of two- and three-exponential deconvolution of the fluorescence kinetics of 1,8-ANS in Hb solution demonstrate that the emission at wavelengths lambdaem 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 amplitudes depending on the emission wavelength. Analysis of time-resolved fluorescence spectra shows that the shortest-lived component should be assigned to 1,8-ANS molecules in the aqueous medium, whereas the two longer-lived components are assigned to two types of binding sites for 1,8-ANS in the Hb molecule characterized by different polarity and accessibility to water molecules.
Parul
D A
DA
Sakharov International Ecological University, Minsk, 220009, Belarus.
Bokut
S B
SB
Kisselev
P A
PA
Milyutin
A A
AA
Petrov
E P
EP
Nemkovich
N A
NA
Sobchuk
A N
AN
Dzhagarov
B M
BM
D000817:Anilino Naphthalenesulfonates / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D010108:Oxyhemoglobins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D010108:Oxyhemoglobins; C027132:1-anilino-8-naphthalenesulfonate