Photochemical transformation of lactate dehydrogenase isoforms: UV-sensitivity in the presence of biogenous amines

It has been all round investigation of UV-irradiation influence in a wide dose range on the structural and functional properties of human blood lactate dehydrogenase (LDH) isoenzymes. The photoprotective action of biogenous amines on the functional activity of different enzyme isoforms was found. It has been established that the protective action of biogenous amines is caused by the formation of complex LDH--biogenous amine and by the acception of the active oxygen forms by the molecules of lactate dehydrogenase. Under the conditions of exogenous singlet oxygen generation in the presence of methylene blue, the inactivation of immobilized LDH tetramers and subunits was observed, that shows participation of this active intermediate in the processes of UV-modification of the enzyme in soluble and immobilized states. The scheme of processes of LDH molecules phototransformations in the presence of biogenous amines has been suggested.     

Effects of ultraviolet irradiation on structural and functional condition of T- and B-lymphocytes of human blood

The influence of ultraviolet (UV) radiation on structural and functional condition of membranes T- and B-lymphocytes of human blood is investigated. It is shown, that intergral flow of UV-light (240-390 nm) in doses 151-1359 J/m2 leads to the increase of the expression of some HLA-antigen (DR1, DR2, DR5, DR7) and Fc-receptors on membranes of B-lymphocytes. The level of HLA-antigen was increased by 20-500% from the initial state after exposure to different doses. The growth of the expression of Fc-receprots was registered in 80% of the donors in the range from 20 up to 540%. The minimum dose of UV-light (151 J/m2) caused the maximum effect. The reduction of the level of Fc-receptors by 10-90% relatively to the native condition was registered for T-cells in 80% of the donors. Groups of the donors were revealed, T-lymphocytes of which differed by the sensitivity to UV-radiation. The effect of "strengthening--weakening", resulting to the reduction of the initially high parameters and to increase in the initially low ones was found.     

Structural and functional characteristics of hemoglobin molecules from the preserved donor blood during long-term storage

The human blood haemoglobin molecules keep their quaternary structure for 25 days at a long-term storage of the blood stabilised with glugicyr. The molecules' electronic characteristics did not change during 5 days, and their oxygen-binding ability remained unaltered just during 2 days after taking the blood.     

Effect of rheopolyglucin and dialdehyde dextran on thermostability of human haemoglobin molecules

Structural characteristics and thermostability of human haemoglobin molecules, modified by reopolyglukine and dialdehyddextrane (DAD) have been studied. Haemoglobin mainly preserves its oxyform when including reopolyglukine in mixture (the mol. ratio of protein to dextrane was 1:4), and disordering of polypeptide chains and increasing the volume of haemoproteids molecules are noticed. Assessment of the influence of mixture pH, exposure and temperature of incubation and the rate of dextrane oxidation on the intensity of the process of human haemoglobin conjugation with DAD have been chosen. Formation of the haemoglobin-DAD complex leads to shielding protein chromophoral groups by polysugar matrix and to transforming the part of haemoproteid molecules from lowspin form (HbO2) to highspin forms (Hb, MetHb). It has been detected that temperature of denaturational points for native protein and haemoglobin in the presence of reopoliglukine is 60 degrees C, but it is 80 degrees C for the haemoglobin-DAD conjugate. The latter is probably determined by increasing hydrophobic interactions inside the protein globule under the effect of DAD and by ability of the surface-bound carbohydrate components prevent association of haemoglobin molecules.     

Thermal denaturation of oligomeric proteins: structural and functional modifications, the sequence of steps

On the basis of literature and own experimental data changes in the structural and functional properties of some oligomeric proteins of the blood system (hemoglobin, lactate dehydrogenase, superoxide dismutase, catalase) exposed to the influence of temperature in a broad range were analyzed. The many-phase character of the temperature modification of protein molecules with different values of functional and kinetic parameters for each of revealed stages was discovered. At a critical temperature and at higher values, the dissociation of oligomeric proteins into separate subunits was shown to occur along with the typical "loosening" of the protein globule. It was shown that low-molecular components (subunits) can subsequently associate with one another and with oligometic proteins, which leads to irreversible denaturation and to the unusual physicochemical behavior of protein molecules. Schemes of processes underlying the temperature modifications of the proteins studied were elaborated.     

Effect of alcohols on photochemical transformations of mouse oxyhemoglobin

The spectral method was used to study the influence of such alcohols as xylene, mannitol, sorbitol and dulcitol on photochemical oxidation of mouse oxyhemoglobin molecules (6.68 X 10(-5) M). Mannitol (5.35 X 10(-4) M) was shown to exert a protective action with regard to UV-irradiated hemoproteid: the extent to which oxyhemoglobin molecules were oxidized in the presence of mannotol was lesser than that in its absence. Sorbiol or xylene (5.35 X 10(-4) M) added to an aqueous solution of hemoproteid failed to exert a photoprotective effect with respect to protein molecules.     

The role of the complex formation between cytochrome c and NAD in the manifestation of the photoprotective effect of the dinucleotide in relation to hemeprotein

UV irradiation of free ferricytochrome solutions, pH 8, induces photorecovery of protein molecules. Hemoproteide photorecovery does not occur after irradiation of the ferricytochrome c/NAD mixture, pH 6 and 8: dinucleotide exerts a photoprotective effect with respect to ferricytochrome. This NAD effect is not observed after exposure of the ferricytochrome c/NAD system, pH 4. With this pH value, each component of the above mixture is eluted from a gel chromatographic column by its peak, whereas with pH 6 and 8, NAD and ferricytochrome c leave the column as one fraction. This indicates that the photoprotective effect of the coenzyme manifests itself upon formation of complex with hemoprotein.     

Physico-chemical properties and thermostability of human hemoglobin molecules modified by rheopolyglucin and dialdehydedextrane

The structural characteristics and thermostability of human hemoglobin molecules modified by rheopolyglucin and dialdehydedextrane have been studied. The influence of mixture pH, the exposure time, and temperature of incubation and the rate of dextrane oxidation on the intensity of conjugation of human hemoglobin with dialdehydedextrane has been estimated. Optimal conditions for the binding of hemoglobin to dialdehydedextrane have been determined. The formation of the hemoglobin- dialdehydedextrane complex leads to the shielding of protein chromophore groups by the polysaccharide matrix and the transformation of a part of hemoproteid molecules from the low-spin form (HbO2) to high-spin forms (Hb, MetHb). It has been found that the temperature of denaturation transition for the native protein and hemoglobin in the presence of rheopolyglucin is 60 degrees C, and that for the hemoglobin-dialdehydedextrane conjugate is 80 degrees C. The latter is probably determined by the enhancement of hydrophobic interactions inside the protein globule under the effect of dialdehydedextrane and by the ability of the externally-bound carbohydrate components to prevent the association of hemoglobin molecules.     

Influence of UV-light and dexamethasone on functional properties of lymphocytes and neutrophils

UV-light and dexamethasone influence on functional properties of lymphocytes and neutrophils of peripherical donors' blood was studied. An increase of phagocytic activity of neutrophils was observed after their incubation with photomodified lymphocytes. It was found that UV-irradiation of lymphocytes activated synthesis of interleukines 1beta and 2. Dexamethasone presence in lymphocyte suspension inhibited the synthesis of the studied cytokines, especially by the incubation with photomodified cells. It was shown by the method of fluorescent labels that UV-irradiation improved interaction between dexamethasone and cell membrane.     

The use of commercial immunoenzyme and latex preparations for the demonstration of human rotavirus antigens

The results of using enzyme immunoassay and latex preparations for the diagnosis of rotavirus gastroenteritis are presented. High effectiveness of the enzyme immunoassay system developed in the USSR with latex diagnostic agents, such as Rotalex (Orion Diagnostica, Finland), Slidex Rota Kit (BioMérieux, France), The Wellcome Rotavirus Latex Kit (Wellcome Foundation Ltd., Great Britain), 48-63% and 21-41% respectively, has been noted. The results of the comparison of the system developed in the USSR with Wellcozyme Rotavirus, an enzyme immunoassay system manufactured by Wellcome Foundation Ltd. (Great Britain), are practically comparable. The results of the block test and the confirmation test used for control indicate that the Soviet preparation is specific. Materials on the practical evaluation of the assay system at health institutions are presented. Good prospects for the use of this system in the diagnosis of rotavirus gastroenteritis, as well as in the realization of epidemiological surveillance on this infection, are substantiated.