Fluorescence Decay Time Distribution Analysis Reveals Two Types of Binding Sites for 1,8-Anilinonaphthalene Sulfonate in Native Human Oxyhemoglobin

D. A. Parul¹, S. B. Bokut^1*, P. A. Kisselev¹, A. A. Milyutin¹, E. P. Petrov², N. A. Nemkovich², A. N. Sobchuk², and B. M. Dzhagarov³

¹Sakharov International Ecological University, ul. Dolgobrodskaya 23, Minsk, 220009 Belarus; fax: (+ 375 17) 230-6897; E-mail: bokut@isir.minsk.by

²Stepanov Institute of Physics, National Academy of Sciences of Belarus, pr. Skaryna 68, Minsk, 220072 Belarus; fax: (+ 375 17) 284-0879; E-mail: petrov@ifanbel.bas-net.by

³Institute of Molecular and Atomic Physics, National Academy of Sciences of Belarus, pr. Skaryna 70, Minsk, 220072 Belarus; fax: (+ 375 17) 284-0030; E-mail: bmd@imaph.bas-net.by

^* To whom correspondence should be addressed.

Received June 7, 2000; Revision received November 8, 2000

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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 lambda_em 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.

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KEY WORDS: oxyhemoglobin, 1,8-anilinonaphthalene sulfonate, fluorescence

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