Photoinduced formation of thiols in human hair.

Raman, scanning electron, and optical microscopy of hair and spectrophotometry of soluble hair proteins are used to study the effect of UV-vis radiation on white hair. The samples of a healthy subject are irradiated using a mercury lamp and compared with non-irradiated (control) hair. The cuticle damage with partial exfoliation is revealed with the aid of SEM and optical microscopy of semifine sections. Gel filtration chromatography shows that the molecular weight of soluble proteins ranges from 5 to 7kDa. Absorption spectroscopy proves an increase in amount of thiols in a heavier fraction of the soluble proteins of irradiated samples under study. Raman data indicate a decrease in the amount of SS and CS bonds in cystines and an increase in the amount of SH bonds due to irradiation. Such changes are more pronounced in peripheral regions of hair. Conformational changes of hair keratins presumably related to the cleavage of disulfide bonds, follow from variations in amide I and low-frequency Raman bands. An increase in the content of thiols in proteins revealed by both photometric data on soluble proteins and Raman microspectroscopy of hair cuts can be used to develop a protocol of the analysis of photoinduced hair modification.

[Disturbance of serum albumin conformation in patients with melancholic depression]. / Narushenie konformatsii al'bumina syvorotki u bol'nykh melankholicheskoi depressiei.

OBJECTIVE: Conformational protein changes may be an important component of the disturbance of molecular processes in the development of pathological process in the body. We studied conformations of albumin molecule in the blood of patients with depression using biophysical -nanotechnical approach. MATERIAL AND METHODS: We examined 19 patients with depression and 25 healthy controls. Properties of serum albumin were compared in patients with typical melancholic depression and controls using spectroscopy (subnanosecond range) with K-35 fluorescent probe. RESULTS AND CONCLUSION: The properties of albumin binding sites in patients before and after treatment differed from those in controls. The authors suggest that it points to the changes in albumin molecule conformation that may influence the functional state of the protein. It has been suggested that these changes may be considered as biomarkers of pharmacotherapeutic efficacy.

Effects of ultra violet radiation on the soluble proteins of human hair.

Exposure of hair fibers from healthy volunteers to Ultra Violet Radiation (UVR) under laboratory conditions enhanced protein elution from the hair tresses into a buffer solution (pH 10.5). At the same time the UVR decreased the intensity of tryptophan fluorescence in the eluted proteins. After mechanical homogenization of these hair samples, the increase of soluble protein was registered for UVR treated hair as well as the rise in sulfhydryl group content of these proteins. Analysis of soluble proteins from hair samples homogenized before and after protein elution has shown that mainly proteins rich in sulfhydryl groups were eluted and as a result sulfhydryl content of proteins in hair shaft decreased. The hypothesis concerning the effects of environmental factors on the properties of hair shaft proteins was examined, the proximal and distal parts of normal hair (0-5 cm and 15-20 cm from hair root) were compared. In the distal parts there was a higher quantity of soluble proteins registered after homogenization, with decreased sulfhydryl group content and tryptophan fluorescence. It could be supposed that this difference results from the steady rupture of cystine in sulfur bridges and tryptophan under exposure to environmental factors (mainly, UVR), followed by elution of the resulting peptides.

[The new diagnostic algorithms in early period of acute intoxications with psychotropic preparations].

The patients with acute intoxications with psychotropic preparations were analyzed for physical chemical characteristics of blood albumin at admission to hospital. The common level of reduced thiols in albumin fraction was analyzed too. The more frequent cases of unfavorable course of disease were noted at indicator rate less than 250 mkmol/l than at rates higher than 250 mkmol/l. This occurrence permits to consider this indicator as prognostically significant and to evaluate individual risk of unfavorable course of disease in the given category of patients. Under acute intoxications with psychotropic preparations appllying of fluorescent albumin test permits already at fist day of hospitalization to reveal a subgroup of patients with higher risk of subsequent development of pneumonia.

[On mechanisms of fluorescence quenching by water].

Mechanisms of fluorescence quenching of aromatic chromophores by water are reviewed. The mechanisms include polarity of chromophore environment, proton or electron transfer between the excited chromophore and water. A hypothesis is proposed that the quenching can be a result of chromophore-solvent hydrogen bond breaking in the excited state.

[Development of a technical arsenal of the method of fluorescent probes].

A brief history of the development of the method of fluorescent probes and examples of its application are presented. Works done at the 2nd Moscow medical institute and institute of physical chemical medicine in collabora'tion with other institutes on: (1) detection of T- and B-lymphocytes in immune pathology; (2) investigation of the structure and clinical estimation of lipoproteins from blood plasma or serum in relation to the assessment of risk factors for the development of cardiovascular diseases; (3) detection of changes in album molecule in a series of pathological processes improving the prognosis of the development of such diseases as peritonitis, pancreatitis, poisoning with psychotropic preparations etc.; (4) intravital measurement of the potentials in electric fields in leukocytes and changes of these fields in the-course of immunological diseases are described. With these approaches it is possible to study molecular events in the course of pathogenesis and also obtain diagnostically significant information on physical chemical aspect of these events. This information is not a conventional method used in the clinical laboratory.

[Stress effect on the development of hemorrhagic stroke].

The objective of this work was to study how stress, activity in the open field test, and conformational properties of albumin-binding sites are associated with experimental hemorrhagic stroke in rats. The open-field behavioral pattern in rats was characterized by the previously developed by us activity index. In accordance with this activity index, rats were divided into two groups, i.e., active and passive animals. The animals were subjected to experimental hemorrhagic stroke with or without previous emotional stress. It was shown that the previous stress affected the stroke development. Stress loading before experimental stroke changed albumin conformational properties in rats with active and passive behavioral patterns in different ways. It was associated with different ability of the albumin globule to undergo pH-induced transition N-F and in different accessibility of albumin-bound fluorescent probe CAPIDAN to nitrate-induced fluorescence quenching.

Studies of oxidant-induced changes in albumin transport function with a fluorescent probe K-35. Metal-catalyzed oxidation.

The dynamics of albumin transport function was studied during metal-catalyzed oxidation of albumin in diluted blood plasma from healthy donors and in the solution of purified albumin using fluorescent probe K-35. The changes were compared with the dynamics of free radical oxidation markers. For oxidation, different concentrations of Cu(2+), Fe(2+), Fe(3+) ions as well as EDTA and H(2)O(2) were used. Oxidative modification of proteins was assessed by carbonyl and bityrosine fluorescent products. Oxidation of plasma lipids was assessed by the levels of TBA-reactive products. It was found that oxidation markedly decreased effective concentration of albumin characterizing albumin binding capacity, and leads to accumulation of carbonyl products of protein oxidation, bityrosine fluorescent products in proteins, and TBA-active products of lipid oxidation. It was hypothesized that reduced effective concentration of albumin is related to impairment of its binding sites and/or accumulation of free-radical oxidation products filling the binding sites of albumin.

Effects of fatty acids on human serum albumin binding centers.

Albumin is a carrier of nonesterified long-chain fatty acids and many other ligands. The status of its binding centers was studied for various proportions of nonesterified long-chain fatty acids and albumin as exemplified by palmitic acid. The status of the binding center was tested by recording K-35 probe fluorescence decay in the subnanosecond band. This method showed the work of three types of centers. Palmitic acid enhanced binding activity of all centers, though to a different degree: if the palmitic acid/albumin proportion increased to 2-3, the probe binding to type 1 centers (located in the drug center I region) increased 1.5 times, while binding to type 3 centers increased more than 3-fold. Modification of these centers by nonesterified long-chain fatty acids was similar in the isolated human albumin preparation and in diluted blood serum. Hence, K-35 probe showed the actual status of various albumin centers, their binding capacity depending to a different measure on the fatty acid charge of albumin.

[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.

Studies of oxidant-induced changes in albumin transport function with a fluorescent probe k-35. Effect of hypochlorite.

The dynamics of changes in albumin transport function during hypochlorite-induced oxidation of isolated albumin in blood plasma and serum was studied with a fluorescent probe K-35. Binding of the probe K-35 to albumin was characterized by effective concentration of albumin. Oxidative modification of proteins was evaluated by the content of carbonyl products of protein oxidation and bityrosine fluorescent products. Oxidation with hypochlorite was accompanied by a decrease in the effective concentration of albumin in albumin, diluted plasma, and serum and accumulation of carbonyl products of protein oxidation and bityrosine fluorescent products. The decrease in the effective concentration of albumin during oxidation with hypochlorite can be explained by oxidative damage to albumin binding sites. Oxidative modification of probe K-35 binding sites with hypochlorite contributes to a decrease in effective concentration of albumin under pathological conditions.

Study of high-resolution H1 nuclear magnetic resonance spectra of the serum and its albumin faction in patients with the first schizophrenia episode.

We studied high-resolution (1)H nuclear magnetic resonance spectra of the serum and serum albumin from patients with the first episode of schizophrenia and healthy individuals. A relative increase in signal intensities of CH(2) protons in serum LDL and VLDL in schizophrenia was demonstrated. Higher intensities of CH(2) and CH(3) protons of non-esterified fatty acids were found in (1)H nuclear magnetic resonance spectra of serum albumin. These data attest to an essential role of changes in lipid metabolism and changed ligand load of albumin in schizophrenia.

[Molecular motility of a fluorescent probe in binding sites of albumin molecules].

The molecular mobility of the fluorescent probe, N-(carboxymethyl)imide of 4-(dimethylamino)naphthalic acid (K-35) in three types of binding sites on a human serum albumin (HSA) molecule has been studied. The time-resolved decay of K-35 polarized fluorescence in HSA has been studied and it has been shown that probe molecules bound to different sites have different fluorescence decay time, which poses problems in the interpretation of polarization decay. However, it has been found that, in the case of rather slow thermal rotation of the probe, the decay of each of vertical and horizontal polarized fluorescence components can be approximated by three exponentials corresponding to three types of binding sites. The mobility of the probe in different sites was estimated. The mobility was different but hindered by tens of times in all sites as compared with the rotation of K-35 in water. The slowest motion occurred in the sites of the first type localized in the region of the well known first drug-binding site: here the rotational correlation was close to 72 ns or more. In the sites of the second type, the time was about 40 ns, and in the sites of the third type, the time was about 10 ns. It was found that the higher the rotation rate, the higher the fluorescence quenching rate. Probably, it is this motion that is responsible for different fluorescence decay times in different HSA sites.

[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.

[Physico-chemical investigation of blood components using special fluorescent probes].

The objective of this paper was to overview results of the works aimed at the development and practical use of fluorescent dies (molecular flourescent probes) for the study of blood components carried out at the Institute of Physico-Chemical Medicine under the guidance of prof. Yu.M. Lopukhin. The new probes were used to directly detect and measure lipoproteins in unfractionated plasma and serum for the identification of risk factors of cardiovascular diseases. The study revealed changes in physico-chemical characteristics of albumin molecules in many pathological processes closely related to clinical features and severity of the disease. These data were used to develop a simple albumin test for clinical application. Probes for measuring electric field potential in leukocytes make it possible to diagnose changes of this parameter in patients with immune disorders. It is concluded that they carry diagnostically valuable information and may be helpful for recording pathological changes at the cellular and molecular levels that can not be observed by traditional laboratory methods.

[Conformational disturbances of serum albumin binding sites in schizophrenia].

The properties of serum albumin binding sites were studied using quenching of fluorescence of the molecular probe CAPIDAN (N-carboxyphenylimide of dimethylaminonaphthalic acid) with the nitrate anion. The samples of serum were obtained from 24 patients with paranoid schizophrenia and 24 healthy volunteers. In the absence of quencher the specific probe fluorescence was 1,4 times higher in patients than in volunteers. Fluorescence quenching constant for the probe bound to albumin was (M+/-m) 2,48+/-0,17 l/mol in patients versus 4,65+/-0,37 l/mol (p<0,01) in volunteers (p<0,01). The fluorescence fraction assessable to quenching was significantly (p<0,01) lower in schizophrenic patients as compared to controls (0,60+/-0,03 and 0,76+/-0,03) respectively). Thus, it is shown that in patients with schizophrenia the conformational state of albumin binding sites is significantly changed as compared to controls that can lead to the changes in the protein-ligand interaction and, thus, contribute to the pathogenesis of the disease and patient's response to treatment.

[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.

Creation of new physicochemical methods for blood analysis on the base of fluorescent probes.

This review deals with the development of new methods for studies of blood cells and plasma, based on the use of special dye molecules, the so-called fluorescent probes. These probes can also be used for clinical diagnosis. Probes and new methods on the basis of these probes were created for measurements of plasma and serum lipoproteins, serum albumin binding centers, blood leukocyte intracellular lipoproteins, allergens.

Characteristics of albumin molecule binding centers in patients with anxious depression. Study by the fluorescence quenching method.

The state of binding centers in albumin molecule in patients with anxious depression was studied by the method of quenching of fluorescence of molecular probe (dimethyl-aminonaphthaleic acid carboxyphenylimide) with nitrate ions. Serum samples from 24 donors without somatic and mental diseases and 26 patients were analyzed. In the absence of the quenching agent, specific fluorescence of the probe (standardized by albumin concentration) was lower in patients with depression. The fluorescence quenching constant and the percentage of fluorescence available for quenching were also lower in serum samples from patients. These data indicate that the parameters of binding centers in albumin molecule in patients with anxious depression are significantly modified in comparison with normal subjects. The detected changes can play a role in the pathogenesis of depressive disorders.

Dipolar relaxation in a lipid bilayer detected by a fluorescent probe, 4''-dimethylaminochalcone.

The dynamic behavior of polar molecules in egg phosphatidylcholine (PC) bilayers has been studied using a membrane fluorescent probe, 4''-dimethylaminochalcone (DMAC). Time and spectrally resolved fluorescence spectroscopy of DMAC incorporated in PC liposomes, as compared to studies of the probe in organic solvents, shows the existence of two independent populations, associated with different extent and speed of dipolar solvent relaxation. The first DMAC population represents approximately 69% of the fluorescence-emitting molecules, has a short fluorescence decay time (0.32 ns) and undergoes Stokes shift of 80 nm. The remaining 31% fraction of DMAC molecules has a decay time of 0.74 ns and undergoes a high (106 nm) Stokes shift. A fraction of the shift, ca. 24 nm for the first and 46 nm for the second population, is attributed to the fast (<0.1 ns) rotational relaxation of nearby dipolar molecules, which might be water. This two-state model accounts well for the detailed fluorescence properties of DMAC in egg PC, i.e. its broadened steady-state spectrum, its average fluorescence quantum yield and its complex wavelength-dependent fluorescence decays.