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

• Published in: Biochemistry (Moscow) 2001-04, Vol.66 (4), p.390-396
• Main Authors: Parul, D A, Bokut, S B, Kisselev, P A, Milyutin, A A, Petrov, E P, Nemkovich, N A, Sobchuk, A N, Dzhagarov, B M
• Format: Article
• Language: English
• Subjects: Anilino Naphthalenesulfonates - analysis; Anilino Naphthalenesulfonates - chemistry; Anilino Naphthalenesulfonates - metabolism; Binding sites; Binding Sites - physiology; Decay; Emissions; Fluorescence; Fluorescent Dyes - analysis; Fluorescent Dyes - chemistry; Humans; Kinetics; Oxyhemoglobins - analysis; Oxyhemoglobins - chemistry; Oxyhemoglobins - metabolism; Spectrometry, Fluorescence - methods; Spectrum analysis; Sulfonates; Wavelengths
• ISSN: 0006-2979; 1608-3040
• DOI: 10.1023/A:1010293211370

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.