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

The binding of the fluorescent probe K-35 (CAPIDAB, N-(carboxyphenyl)imide of 4-(dimethylamino)naphthalic acid), which is used as an indicator of albumin structural changes in pathology, to human serum albumin has been studied. Based on the data on the fluorescence decay of the probe, four types of sites of binding of K-35 to albumin have been recoonized, which differ by fluorescence decay time (tau) and binding constants (K). Probe molecules bound to the first type of sites have a decay time close to 8-10 ns; this value corresponds to a high fluorescence quantum yield of about 0.7. These sites have a maximal binding constant, K1 = 5 x 10(4) M(-1). Tau2 of the second type of sites is close to 3.6 ns and K2 = 1 x 10(4) M(-1), which is much lower than K1; however, the number of the 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 tau3 is equal to 1 ns, which is significantly lower than tau2. 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 2 per one HSA molecule. There are one to two sites of the fourth type where bound K-35 molecules have a very low decay time tau4 << 1; i.e. they are virtually nonfluorescent, and K4 = 1 x 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, this metabolite or the probe in these experiments, is distributed between different sites in accordance with their K(i)n(i) values (n(i) is the number of sites of the ith type per albumin molecule). It was shown that the low occupation of the sites leads to 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 near the drug site I, while the sites of the second and third types are close to it.