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The purpose of the present work was to investigate influence of different freeze-thawing protocols on structure and antioxidant properties of isolated proteins. In our experiments we have studied human serum albumin, human hemoglobin and cytochrome C derived from equine heart frozen down to 77.15K with 1-2 deg/min and 300 deg/min rate with following thawing on a water bath at 293.15 K. Native proteins were assumed as a control. Influence of freeze-thawing protocols on protein structure was investigated using spectrophotometric and fluorescent assays. Antioxidant activities of isolated proteins were estimated by their ability to reduce ABTS+ radical. It has been established that unfolding derived from freeze-thawing exposure leads to protein antioxidant activity increasing while decreasing of such an activity may be connected with macromolecule aggregation. Character of freeze-thawing influence on antioxidant activity of proteins depends on molecule structure peculiarities and freezing protocols. [ABSTRACT FROM AUTHOR]

Probable mechanisms of cytotoxicity of a total fraction of multi-walled carbon nanotubes using spin probe methods (in human erythrocytes and rat hepatocytes) and electrical breakdown (in mouse oocytes) have been studied. Erythrocyte membrane damage induced by carbon nanotubes is developed in time. After two-day exposure at 279 K the erythrocyte samples with carbon nanotubes concentrations of 10, 50, 100, and 200 μg / ml showed a part of damaged erythrocytes of 4, 10, 16, and 25%, respectively. A 4 h exposure of rat liver homogenate with carbon nanotubes at 273 K resulted in a significant decrease in liver homogenate mitochondrial activity. The study of the processes of oocyte electroporation has shown that probable mechanisms of nanotube cytotoxicity are associated with an increase in membrane conductivity, alteration of surface potential, formation of defects within the membrane, and as a consequence, with an increase in membrane permeability. In addition to their capability to affect membrane structural changes, the nanotubes have a capacity to inhibit delicate electrochemical processes in cells. [ABSTRACT FROM AUTHOR]

Background: Glucose oxidase enzyme may be a suitable model for studying the effect of low temperatures on structural and functional properties of biomacromolecules., Objective: The research aim was to study the freeze-thawing effect on glucose oxidase isolated and immobilized by cross-linking with glutaraldehyde, Methods: Effects of freezing rates on conformation and activity of glucose oxidase isolated and cross-linked in solution with gluteraldehyde was studied., Results: Freezing with slow rate (2 degree C per min) induces significant protein aggregation, activity reduction and conformational changes in polar and hydrophobic regions. Freezing at 100 degree C per min, however, causes conformational changes primarily in polar regions, insignificant aggregation, depending on the number of freeze-thawing cycles and increases enzyme activity., Conclusion: With the rise in glucose oxidase concentration in solution the low temperature-induced destabilization effect is reduced both during low and rapid freezing. At the slow cooling rate, cross-linking with glutaraldehyde results in more conformation alterations in polar regions of proteins, accompanied with an increase in enzyme activity.

Aqueous-saline human placenta extract (HPE) is known to possess antioxidant activity due to the high concentration of bioactive substances. This fact allows its application in clinical practice in order to treat oxidation-induced diseases. Extract antioxidant activity is mainly conditioned by proteins. Freezing of extracts has been shown to lead to their antioxidant activity increasing due to protein conformation changes.Different biological models are widely used in order to evaluate efficacy of novel antioxidants and mechanisms of their action. One such model appears to be erythrocytes under nitrite-induced oxidative stress. Nitrite is known to be able to penetrate erythrocyte membrane and to oxidize hemoglobin. In order to investigate whether HPE is able to decrease nitrite-induced oxidative injuries and to evaluate the protein contribution to this process, spectrophotometric and electron spin resonance (ESR) assays were used.Experimental data have revealed that antioxidant activity of extracts and of some of their fractions correlates with methemoglobin concentration lowering. Preliminary erythrocyte incubation with an extract fraction of 12 kDa allows preservation of the structural-dynamic cytosol state the closest to the control.

The influence of the freezing velocities and final temperatures of storage on the complex dielectric permittivity of cord blood serum have been studied. On the temperature dependences of the dielectric permittivity the non-monotonous changes at the characteristic temperatures accompanying with the change of the activation energy of the water molecules dielectric relaxation were found out. The drastic deflection of the temperature dependence of ?' from the monotone curve at 15 degrees C region correlates with the fracture of Arrhenius plots of the dielectric relaxation time and viscosity at the same temperature. Slow freezing (1-2 degrees C/min) of cord blood serum results in ?' diminution in comparison with control, that testifies an increase of bound water amount because of loosening in this case the surface of biomacromolecule polypeptide chains. Rapid freezing (300-400 degrees C/min) results in ?' increase of serum, that is caused, apparently, by the cryoaggregation of biomacro-molecules, which can thus happen.

At present, the question of how the structural state of the erythrocyte cytosol is arranged to maintain essential permeabilities successfully both at normal temperature and during periods with a significant body temperature reduction during hypobiosis remains unanswered. In the present work, we performed comparative investigations of temperature-dependent changes in the cytosol state of erythrocytes from animals subjected to natural (winter hibernating ground squirrels) or artificial hypobiosis. The cytosol state was evaluated by the ESR method of spin probes (TEMPON) within the temperature range of 0-50 degrees C. Erythrocyte resistance to acid hemolysis, which is limited by the permeability of membranes for protons and the state of the anion channel, were determined using the method described by Terskov and Getelson [Biofizika 2 (1957) 259]. A change in cytosol microviscosity of erythrocytes was found as well as a temperature-dependent increase in acid resistance of erythrocytes. Our investigations allow us to conclude that physiological changes occurring in a mammalian organism during natural and artificial hypobiosis are accompanied by structural modifications of the erythrocyte cytosol. The temperature range where these modifications are observed (8, 15, 40 degrees C) suggests that the most probable modifying link is spectrin and/or the sites of its interaction with membrane. The interaction of cytoskeletal components with the cell membrane plays a key role in regulation of membrane permeability, suggesting an important role of this interaction in the adaptive reactions of erythrocytes.

The effect of rapid freeze-thawing on the structural rearrangement of integral and peripheral membrane proteins was studied using fluorescent spectroscopy. It was discovered that a long wave shift occurred in the fluorescence spectra of cytochrome P450, both in solution and within phospholipid vesicles. The efficiency of quenching of membrane-bound cytochrome P450 with acrylamide also increased after freezing. Similarly, a long wave shift in the fluorescence, spectra occurred after freezing spectrin solution and erythrocyte ghosts. These data are interpreted in terms of physical and chemical factors in the medium affecting the spectral properties of protein chromophores, and suggest that an increase occurs in the polarity of the microenvironment of the protein molecules, resulting in conformational modification. Study of the temperature dependence of spectrin fluorescence in solution, and within erythrocyte ghosts, suggests that modification of protein dynamics occurs both during freezing and during thawing. Despite various differences in the structure of the protein molecules, and in the phospholipid environment and solvent composition, freeze-thawing results in unilateral, irreversible conformation changes, more prominent in peripheral than integral proteins. The possibility that these changes contribute to cryoinjury to cell membranes is discussed.

EPR spin-probe method was used to investigate the dependence of erythrocyte cytosol microviscosity (ECM) on the concentration of different substances and temperature. The critical ECM value has been determined after reaching of which erythrocytes become destroyed. It is shown that this ECM value is reached at different NaCl concentrations depending on the temperature.

By means of the fluorescence probes pyrene, DSP-12 and n-terphenyl the effect of low temperature, glycerol and 1,2-propylene glycol on the structure of lecithin liposomes and proteoliposomes formed by lecithin and cytochrome P-450 has been studied. Freezing-thawing and cryoprotectants were shown to modify surface area and internal volume of the lipid bilayer.

We have studied the influence of the erythrocyte hypothermic storage of long-tailed ground squirrels at various physiological states and white mongrel rats on the impairment of the barrier function of their membranes for ferricyanide. The membranes of sleeping and awakening ground squirrels demonstrate the highest stability (the structural integrity is sustained for 3-3,5 months), meanwhile rat blood erythrocytes maintain the barrier function only for 2 weeks. Beside structural and viscous changes in the erythrocyte membrane and the cytosol of hibernating ground squirrel an important role in maintaining barrier function under conditions of hypothermic storage is played by the content of animal blood plasma. Appearance of a non-typical EPR spin probe spectrum in some ground squirrel blood samples in non-equilibrium state of entering or arousing from hibernation has been observed.

The results of spectroscopic examination of mitochondria and lysosomes indicate that freeze-thawing leads to alterations of different character and extent in membrane structural organization which manifest as changes in the molecular packing of the organelle membrane lipid bilayer, lateral separation of lipids into individual domains, and impairment of membrane permeability. Supercooling of organelle suspensions without crystallization of external water has been found not to affect membrane barrier function markedly; however, such a decrease in the temperature results in a slight loosening of the membrane with an increase in the volume of subcellular structures. The crystallization of external water causes dehydration of organelles, which favors a decrease in their volume, increasing the viscosity of the liquid phase inside subcellular structures and packing the lipid bilayer. Changes in the permeability of mitochondrial and lysosomal membranes manifest during thawing after the formation of an external liquid phase and might be due to the sharp rehydration of these membranes through latent membrane defects formed upon freezing.

Changes in permeability of lysosomal and mitochondrial membranes were studied under different temperatures at which cryoextracts from organelles inhibit most distinctly the protein synthesizing activity of the cell-free system from the rat liver. It is found that mitochondria are more sensitive to the effect of low temperature effect than lysosomes. Overcooling of the mitochondria suspension to the temperature of the free water crystallization is shown to cause no release of the protein synthesis inhibitor. The inhibitor release from organelles occurs from the moment of crystallization and reaches its maximum at the eutectic temperatures of the freezing medium which is due to the injury effect of the complex of physicochemical factors on the membrane structures, occurring during the phase transition of the solvent.

Possibility of studying erythrocyte dehydration by ESR-spin probe is substantiated. Dehydration of erythrocytes in relation to osmolarity of sodium chloride solutions is investigated. The results are shown to agree with the data obtained by radioisotope method.

Possible usage of ESR probe method for studying low temperature effect on structural and functional state of mitochondria is under study. It is shown that during freezing out of mitochondrial water there is sharp dehydration of intermitochondrial matrix and membrane thickening. The facts are given about damage of barrier function of the membrane at the moment of appearance of extramitochondrial liquid phase during thawing.

It was shown by means of ESR-spin probe method that microviscosity of erythrocyte cytosol had a limiting value which could be reached by various concentrations of sodium chloride depending on temperature. With a temperature decrease when reaching this value haemolysis occurs.