Time-resolved fluorescence reveals two binding sites of 1,8-ANS in intact human oxyhemoglobin.
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.
A.D. Sakharov International Environmental University, Minsk, Belarus.
D A
DA
Parul
S B
SB
Bokut
A A
AA
Milyutin
E P
EP
Petrov
N A
NA
Nemkovich
A N
AN
Sobchuk
B M
BM
Dzhagarov
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D001665:Binding Sites; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D010108:Oxyhemoglobins / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D010777:Photochemistry; D012996:Solutions; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D010108:Oxyhemoglobins; D012996:Solutions; C027132:1-anilino-8-naphthalenesulfonate