Constant brain potentials as neurophysiological markers of autoimmune process]

Neuroimmune interactions in systemic rheumatic diseases were studied. The state of the central nervous system was assessed from the parameters of constant brain potentials, and the state of the immune system, from a complex of immunobiochemical parameters. The highest multiple correlation coefficients were revealed between the immunobiochemical parameters and the parameters of the constant brain potential, which characterize linear and standard deviations of potentials in temporal zones from potentials at other points of recording. The results are discussed in terms of structural and functional integration of the immune and nervous systems.     

Activity of the autoimmune process during systemic rheumatic diseases and distribution of constant brain potential]

The data obtained upon examination of patients with systemic rheumatic diseases were analyzed by the methods of multiple regression and step-by-step discriminant analysis. Is was shown that the activity of autoimmune process correlates with the parameters of distribution of constant potential level in the brain of each patient, irrespective of diagnosis. The scatter of potential values was the most important predictor of activity. An increase in the activity of the autoimmune process correlated with a decrease in the scatter of potential values at recording points; the cerebral cortex became equipotential.     

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.     

Determination with a microelectrode of membrane potential of giant human erythrocytes obtained by La(3+)-induced fusion

The effect of La3+ on the fusion of erythrocytes of blood stored for a week at +4 degrees C was studied. It was shown that the fusion of erythrocytes begins after one day of storage of blood. The most intensive fusion of erythrocytes was observed on day 4 of blood storage. As a result, giant cells with a size of 100 microns and more arise. The electrical potential of giant cells was measured using a microelectrode and was -6.6 +/- 1.5 mV.     

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

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 · 10⁴ M⁻¹. The τ₂ of the second type of site is close to 3.6 ns and K ₂ = 1 · 10⁴ 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 τ₃ is 1 ns, which is significantly lower than τ₂. 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 τ₄ ≪ 1, i.e., are virtually nonfluorescent, and K ₄ = 1 · 10⁴ 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.     

Size of a human serum albumin molecule in solution]

The size of a human serum albumin molecule in aqueous solution containing 150 mM NaCl was studied using small-angle neutron scattering. The molecular radius of gyration was estimated to be 27.4 +/- 0.35 A. The compact sphere should have a smaller radius of gyration, whereas the popular human serum albumin model, a "cigar" 136 A long, should correspond to a greater radius of gyration. Possible shapes of the human serum albumin molecule which are in accordance with the results obtained, are the following: an extended ellipsoid less than 110 A of length or a nonsymmetrical oblate ellipsoid with a diameter of 85 A. The oblate ellipsoid might be close to the heart"-shaped structure of the crystalline human serum albumin molecule. The size of the albumin molecule does not change significantly as pH increases to 8.9. The possibility of the dynamic coexistence of various human serum albumin conformers in solution is discussed.     

Binding of fatty acids facilitates oxidation of cysteine-34 and converts copper-albumin complexes from antioxidants to prooxidants

As a transition metal capable of undergoing one-electron oxidation-reduction conversions, copper (Cu) is essential for life and fulfills important catalytic functions. Paradoxically, the same redox properties of copper can make it extremely dangerous because it can catalyze production of free radical intermediates from molecular oxygen. Factors involved in regulation of redox activity of albumin-bound copper have not been well characterized. In the present study, effects of modification of the albumin cysteine-34 (Cys-34) and binding of nonesterified fatty acids on the redox-cycling activity of the complex of copper with human serum albumin (Cu/HSA) were studied. Because ascorbate is the most abundant natural reductant/scavenger of free radicals in blood plasma, the electron paramagnetic resonance assay of ascorbate radical formation was used as a method to monitor Cu/HSA redox-cycling activity. At Cu/HSA ratios below 1:1, the bound Cu was virtually redox inactive, as long as Cys-34 was in reduced state (Cu/HSA-SH). Alkylation, nitrosylation, or oxidation of Cu/HSA resulted in the appearance of redox-cycling activity. Experiments with ultrafiltration of Cu/HSA alkylated with N-ethylmaleimide (Cu/HSA-NEM) showed that at Cu/HSA-NEM ratios below 1:1, the ascorbate radicals were produced by Cu tightly bound to HSA rather than by Cu released in solution. The rate of ascorbate radical production in HSA-NEM and S-nitrosylated HSA (HSA-NO) was, however, more than one order of magnitude lower than that in a solution containing equivalent concentration of free copper ions. While Cu/HSA-SH was redox inactive, binding of oleic or linoleic acids induced Cu-dependent redox-cycling with maximal activity reached at a fatty acid to protein molar ratio of 3:1 for oleic acid and 2:1 for linoleic acid. Binding of fatty acids caused profound conformational changes and facilitated oxidation of the Cys-34 SH-group at essentially the same ratios as those that caused redox-cycling activity of Cu/HSA. We conclude that fatty acids regulate anti-/prooxidant properties of Cu-albumin via controlling redox status of Cys-34.     

A study of ligand binding to protein using resonance energy transfer from the isoindole fluorescent label to ligand

A method for studing the binging of ligands absorbing the light in the region of 350-550 nm to protein is described. The method is based on resonance energy transfer between the fluorescent label covalently bound to the protein and the ligand. The isoindole label, a product of the reaction of the protein with o-phthalaldehyde in the presence of 2-mercaptoethanol, was used as a fluorescent donor. The method was used to determine the binding parameters of a fluorescent probe (a naphthalimide derivative) with human serum albumin. A comparison of the results obtained by the resonance energy and transfer by equilibrium dialysis showed a high accuracy of the resonance energy transfer method.     

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 pockets contain protein polar groups and also water molecules. At the same time the rather small Stokes shift of K-35 fluorescence spectrum shows that the polar group motion is one-two orders of magnitude lower than the 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 τ₁ = 9 ns; τ₂ = 3.6 ns and τ₃ = 1.0 ns. The difference between excitation maxima of these three decay components shows that the environment of these three species of K-35 molecules has been different before excitation. Different τ values are probably a consequence of nonidentical structure of several binding sites, or the binding site(s) can have variable conformation.