[K-35, a fluorescent probe for albumin study: optical properties].

Fluorescent probe N-(carboxyphenyl)imide of 4-(dimethylamino)naphthalic acid, K-35, is used as an indicator of structural changes of human serum albumin molecules in pathology. The probe occupies albumin binding pockets where the probe environment is of very high polarity; probably, the pocket(s) contains protein polar groups and water molecules. At the same time rather small Stokes shift of K-35 fluorescence spectrum shows that the polar group motion is of one-two order of value lower than mobility of polar molecules in polar fluids. K-35 fluorescence decay in HSA can be described as a sum of three exponentials with time constants close to tau1=9 ns; tau2=3.6 ns and tau3=1.0 ns. A difference between excitation maxima of these three decay components shows that environment of these three species of K-35 molecules has been different before excitation. Different r values are probably a consequence of non-identical structure of several binding sites, or a binding site(s) can have a variable conformation.

[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.

[Kinetics of rearrangement of solvation sheath of an excited molecule of the fluorescent probe 4"-dimethylaminochalcone].

Processes accompanying the quenching of the fluorescent probe 4"-dimethylaminochalcone by hydroxyl groups of the proton-donor solvent 1-butanol have been studied. The kinetics of the deactivation of the excited state of 4"-dimethylaminochalcone has been monitored from the transition absorption spectra at a time resolution of 50 fs and fluorescence decay at a time resolution of 30 ps. The data obtained allow thinking that the next picture occurs in 1-butanol. At first stage, the 4"-dimethylaminochalcone molecule in its ground state forms a hydrogen bond with an alcohol molecule. At the second stage, the absorption of light quantum and corresponding rise of the dipole moment of 4"-dimethylaminochalcone take place, the initially existing hydrogen bond is retained. The third stage consists in the rearrangement of the 4"-dimethylaminochalcone solvation shell formed by alcohol dipole molecules due to an increase of the dipole of moment 4"-dimethylaminochalcone; this rearrangement takes an energy of about 24 kJ/mol, the arrangement time constant is close to 40 ps; the initial hydrogen bond is retained. The fourth stage involves processes that lead to fluorescence quenching; their time constant is about 200 ps. Taking into account that the quenching is a much slower process than the relaxation of the solvation shell, it was supposed that the quenching is not a direct consequence of the solvation shell relaxation or the existence of the hydrogen bond formed prior to excitation. Then the fluorescence quenching of 4"-dimethylaminochalcone can be accomplished through some other processes that are observed in other fluorescent molecules: (a) rearrangement of the initial hydrogen bond from a conformation that cannot quench the fluorescence of 4"-dimethylaminochalcone to a more "effective" conformation, (b) charge transfer between the excited of molecule 4"-dimethylaminochalcone and alcohol, or (c) solvent-induced twist of the 4"-dimethylaminochalcone amino group (its withdrawal from the molecule plane) by the action of the solvent.