Features of the binding of the fluorescent probe K-35 to albumin

• Published in: Biophysics (Oxford) 2010-04, Vol.55 (2), p.182-187
• Main Authors: Dobretsov, G. E., Syreishchikova, T. I., Gryzunov, Yu. A., Smolina, N. V., Komar, A. A.
• Format: Article
• Language: English
• Subjects: Binding sites; Biological and Medical Physics; Biophysics; Fluorescence; Molecular biology; Molecular Biophysics; Physics; Physics and Astronomy; Plasma; Proteins
• ISSN: 0006-3509; 1555-6654
• DOI: 10.1134/S000635091002003X

A study was made of the binding of a fluorescent probe K-35 (N-(carboxyphenyl)imide of 4-(dimethylamino)naphthalic acid), used as an indicator of albumin structural changes in pathology, to human serum albumin (HSA). Based on the data on the fluorescence decay of the probe, four types of site of K-35 binding to HSA have been recognized, which differ in fluorescence decay time (τ) and binding constant ( K ). Probe molecules bound to the first type of site have a decay time of 8–10 ns; this value corresponds to a high fluorescence quantum yield of about 0.7. These sites have a maximal binding constant, K 1 = 5 · 10 4 M −1 . The τ 2 of the second type of site is close to 3.6 ns and K 2 = 1 · 10 4 M −1 , which is much lower than K 1 ; however, the number of these sites is several times greater. The number of sites of the third type and the binding constant are close to those of the second type, but the decay time τ 3 is 1 ns, which is significantly lower than τ 2 . The binding of K-35 to sites of the second and the third types is characterized by a positive cooperativity. Their properties are similar but not completely identical. The total number of sites of these three types is about two per one HSA molecule. There are also one-two sites of the fourth type where bound K-35 molecules have a very short decay time τ 4 ≪ 1, i.e., are virtually nonfluorescent, and K 4 = 1 · 10 4 M −1 . The major contribution to the steady-state fluorescence is made by probe molecules bound to sites of the first and second types. As a rule, the concentration of albumin binding sites in blood is significantly higher than the concentration of metabolites and xenobiotics transferred by albumin. Therefore, the metabolite—or the probe in these experiments—is distributed among different sites in accordance with their K i n i values ( n i is the number of sites of the i -th type per albumin molecule). The low occupancy of the sites results in an approximately equal number of K-35 molecules bound to different sites of types 1, 2, and 3. The competition of K-35 with phenylbutazone, a marker of the albumin drug-binding site I, allows one to suggest that the K-35 site of the first type is localized exactly in the drug site I region, while the sites of the second and third types are close to it.