The use of 1H-NMR spectroscopy and refractometry for investigation of the distribution of nonelectrolytes of N-alcohol series between human red blood cells and extracellular medium

Comparative analysis of 1H NMR spectroscopy and refractometry with respect to their application for investigating the distribution of nonelectrolytes of n-alcohol series (ethanol, 1,2-propanediol, glycerol) and polyethylene glycols (PEGs) with molecular masses of 400, 600, 1500 between human erythrocytes and extracellular medium was performed. The distribution coefficients (Q) for solutions of ethanol, 1,2-propanediol, glycerol, PEG-400, PEG-600 and PEG-1500 were obtained. The Q values decreased with the increase in the nonelectrolyte molecular mass from 1.23+/-0.12 for ethanol to 0.40+/-0.08 for PEG-1500 (1H NMR spectroscopy) and from 2.6+/-0.12 for ethanol to 0.23+/-0.03 for PEG-1500 (refractometry). It was shown that 1H-NMR high-resolution spectroscopy ensures more precise determination of Q values for nonelectrolytes with low molecular masses; for PEGs with high molecular masses, the accuracy of Q value calculation by this method was about 20%. On the contrary, refractometry can be used for investigating substances with high molecular masses; the error of Q value determination for solution of low-refractive substances, such as ethanol, may be more than 50%.     

The study of Ca2+ influx in human erythrocytes in isotonic polyethylene (glycol) 1500 (PEG-1500) and sucrose media

Effects of isotonic solutions of polyethylene (glycol) 1500 (PEG-1500) and sucrose on Ca2+ influx into ATP-depleted red blood cells were studied using the Ca2+ -sensitive fluorescent dye fura-2AM. When incubated in isotonic low ionic strength media (containing 2 mM CaCl2 in addition to sucrose and PEG-1500), the initial rate of Ca2+ influx was higher than that for the cells in physiological (normal ionic strength) medium. After 20 minutes of incubation in the PEG-1500-containing solution, a 10-fold increase of Ca2+ influx was observed, whereas in the sucrose medium the rate of Ca2+ influx decreased compared to that in physiological medium. 1H-NMR data provided no evidence of direct interaction between PEG-1500 and the erythrocyte membrane. Moreover, PEG-1500 did not affect lipid peroxidation (LPO) induction in erythrocyte membranes. We propose that a change in the hydrogen environment of Ca2+ -ATPase of the erythrocytes suspended in the PEG-1500 solution is the primary cause of altered Ca2+ homeostasis in these cells. The activation of the Ca2+ -ATP-ase in sucrose medium may result in an incomplete suppression of the Ca2+-pump activity in ATP-depleted cells, which is accelerated when calmodulin binds with the Ca2+-ATP-ase under the conditions of rapid Ca2+ accumulation.     

The reaction between some dyes and synthetic hydroxyapatite 2. Nature of the binding reaction

Synopsis In order to study the reactions involved in some of the histochemical procedures used for demonstrating calcium in calcified tissues, it was considered appropriate to use well characterized synthetic hydroxyapatite in the first instance. In the first paper of this series (Speirs, 1970), it was found that many dyes not previously used in histochemistry were capable of staining hydroxyapatite; the purpose of the present paper is to describe the numerous experimental approaches that have been made in an attempt to elucidate the mechanisms involved in the adsorption of some of these dyes by hydroxyapatite. Dyes have been grouped according to their adsorption curves (in which dye uptake by solid was plotted against the concentration of dye in solution at equilibrium). From these graphs, predictions and calculations were made concerning the orientation of the dye molecules on the surface of hydroxyapatite, the type of bonding possibly involved and the area of surface covered by each molecule. These were then related to the dimensions and structure of the dye molecules. Saturation of surface sites was achieved in the adsorption of some dyes and the nature of these sites was investigated by studying (1) competition between several dyes for the surface, (2) the accessibility of surface calcium and phosphorus in stained and unstained hydroxyapatite, and (3) the release of³²P from surface labelled hydroxyapatite during dye adsorption. Most of the dyes adsorbed from 95% ethanol were displaced relatively easily by treatment with 0.5 mM phosphate in ethanol, but those adsorbed from tris buffer, pH 7.45, were more stable when exposed to phosphate in tris. Treatment of stained hydroxyapatite with solvents containing 0.5 mM calcium reduced the rate of elution of the dyes. Convincing evidence for chelation, hydrogen bonding, ion exchange and physical adsorption processes as the mechanisms of adsorption has not been obtained. Future studies to investigate these processes are discussed.     

Parameters of interaction of sodium dodecyl sulfate with human hemoglobin

It was shown that sodium dodecyl sulfate at concentrations not exceeding the critical micelle concentrations (0-1.9 mM) induced the conversion of oxy- and methemoglobin but not deoxyhemoglobin to hemichrome. The concentration dependences of hemichrome formation were represented as Hill plots, and the parameters of detergent binding were estimated. OxyHb in 20 mM potassium-phosphate buffer, pH 6.8, has two groups of binding sites: the first group is characterized by the Hill constant n1 = 2 and the concentration of half saturation [SDS]50 = 0.8 mM, and the second group is characterized by the Hill constant n2 = 8 and [SDS]50 = 0.9 mM. In the case of metHb one group of binding sites with the Hill constant n = 2 and half saturation concentration [SDS]50 = 0.2 mM was observed. An increase in environmental pH to 7.9 decreased the affinity of Hb for SDS. It is suggested that primary binding sites for SDS in oxyHb coincide with the anion-binding center of the Hb molecule. The interaction of the detergent with these binding sites induced a structural transition of the hemoprotein molecule. As a result of this transition, secondary binding sites were exposed. In a model system (hemin--imidazole in ethanol solution), the enthalpy of the transition of hemin from a high-spin to a low-spin state was estimated to be 47 +/- 7 kJ/mol.     

Effect of substances with cryoprotective properties on surface marker CD44 in human erythrocytes

The changes in surface marker CD44 in human erythrocytes exposed to cryoprotective media, as well as the impact of oxidative modification of membrane-cytoskeleton proteins on the CD44 characteristics under the changed physicochemical parameters of the cellular environment, were investigated in this study. Prolonged exposure of glycerol, DMSO, sucrose, and PEG-1500 caused a decrease in CD44 expression level and in amount of CD44-positive cells. That may reflect subtle rearrangements in the system of protein–protein interactions in the erythrocyte membrane-cytoskeleton complex, which may affect the stability of cells during cryopreservation. Extracellular substances (sucrose and PEG-1500) exhibited a more pronounced effect on the CD44 in erythrocytes in comparison with the examined substances of an intacellular type. Modification of membrane-cytoskeleton proteins with oxidizing bifunctional reagent diamide enhanced the identified tendencies.     

Improvement of proteolytic resistance of immunoadsorbents by chemical modification with polyethylene glycol

Immunoadsorbents were modified with monomethoxy-polyethylene glycol (PEG; average molecular weights of 5000 (PEG-5000) and 1900 (PEG-1900)) activated with cyanuric acid (activated PEG) by four different methods. In the two methods, anti-BSA antibodies were modified with activated PEG with and without protection of antigen binding sites with BSA and then were coupled to CNBr-activated Sepharose 4B. In the other two methods, Immunoadsorbents, which were prepared by coupling anti-BSA antibodies to CNBr-activated Sepharose 4B, were modified with activated PEG with and without the protection. The effects of PEG modification by these four methods on the binding ratio (the ratio of the numbers of moles of antigen adsorbed to the numbers of moles of binding sites of antibody coupled), the antigen binding property and the resistance to proteolytic digestion of immunoadsorbents were studied. The decrease in the binding ratio by the modification with activated PEG was small enough to use modified immunoadsorbents for industrial purification processes. The resistance to proteolytic digestion of immunoadsorbents was improved by modification with activated PEG. The modification without protection of antigen binding sites gave higher resistance to proteolytic digestion than that with protection, while the former caused larger decrease in the binding ratio of modification. The immunoadsorbents modified with activated PEG-5000 showed higher resistance to proteolytic digestion than those modified with activated PEG-1900.     

Intracellular free calcium content increase in the erythrocytes treated with the cryoprotective medium based on polyethylene glycol 1500 (PEG-1500)

Changes in intracellular free calcium content ([Ca2+]i) in human erythrocytes treated with the cryoprotective medium based on low toxic polymer--polyethylene glycol 1500 (PEG-1500) and then transferred to physiologic salt solution containing 2 mM CaCl2 were studied using fluorescent calcium probe--fura-2. A method of [Ca2+]i calculation with allowance for haemolysis of the cells during the experiment was proposed. It was shown that ignorance of the cell haemolysis resulted in significantly higher [Ca2+]i values obtained. Significant time-dependent increase of [Ca2+]i in the cells treated with PEG-1500 cryoprotective medium at +4 degrees C as well as at +22 degrees C (without freezing) and then transferred in the 2 mM CaCl2 containing physiological salt solution at +37 degrees C was observed. Freezing-thawing of the cells treated with the PEG-1500 cryoprotective medium enhanced haemolysis and further accumulation of calcium in the cells. The results of the study prove that the use of PEG-1500-based cryoprotective medium which does not require washing for human erythrocytes will be accompanied by progressive destruction (haemolysis) of the cells in the blood vessels and may have some negative consequences connected with [Ca2+]i increase in the cryopreserved erythrocytes.     

Changes in the erythrocyte membrane-cytoskeleton complex induced by dimethyl sulfoxide, polyethylene glycol, and low temperature

The effect of the cryoprotectants DMSO and PEG-1500 as well as freezing-thawing on the proteins of the canine erythrocyte membrane-cytoskeleton complex was studied using the cross-linking agent diamide. It was shown that the intensity of disturbances in the protein network structure correlated with the increased SH-group accessibility for oxidative bridging by this compound and accordingly, enhanced formation of high-molecular-weight protein aggregates. The maximum level of diamide-induced aggregability was revealed upon freezing of erythrocytes in liquid nitrogen without cryoprotectant. Electrophoretic analysis of the ghosts of erythrocytes incubated with cryoprotectants showed a significant increase in the aggregation level only for the cells in the polymer solution. After the freezing-thawing cycle, the diamide-induced protein aggregability in erythrocytes cryopreserved with PEG-1500 strongly increased; when DMSO was used for cell protection, the aggregation was much less pronounced than in the unprotected cells. One can suppose that the exocellular cryoprotectant PEG-1500, as distinct from the endocellular cryoprotectant DMSO, is unable to provide for preservation of the structure of the membrane-cytoskeleton protein complex at a level necessary for the maintenance of cell integrity after the return to physiological conditions.     

Fermentation of glycerol by Anaerobium acetethylicum and its potential use in biofuel production

Growth of biodiesel industries resulted in increased coproduction of crude glycerol which is therefore becoming a waste product instead of a valuable ‘coproduct’. Glycerol can be used for the production of valuable chemicals, e.g. biofuels, to reduce glycerol waste disposal. In this study, a novel bacterial strain is described which converts glycerol mainly to ethanol and hydrogen with very little amounts of acetate, formate and 1,2‐propanediol as coproducts. The bacterium offers certain advantages over previously studied glycerol‐fermenting microorganisms. Anaerobium acetethylicum during growth with glycerol produces very little side products and grows in the presence of maximum glycerol concentrations up to 1500 mM and in the complete absence of complex organic supplements such as yeast extract or tryptone. The highest observed growth rate of 0.116 h^?1 is similar to that of other glycerol degraders, and the maximum concentration of ethanol that can be tolerated was found to be about 60 mM (2.8 g l^?1) and further growth was likely inhibited due to ethanol toxicity. Proteome analysis as well as enzyme assays performed in cell‐free extracts demonstrated that glycerol is degraded via glyceraldehyde‐3‐phosphate, which is further metabolized through the lower part of glycolysis leading to formation of mainly ethanol and hydrogen. In conclusion, fermentation of glycerol to ethanol and hydrogen by this bacterium represents a remarkable option to add value to the biodiesel industries by utilization of surplus glycerol.     

The changes in erythrocyte Ca<Superscript>2+</Superscript>-ATPase activity induced by PEG-1500 and low temperatures

Various organic compounds are applied upon cryopreservation and their adding into cell suspension causes modification of subcellular systems, providing cell survival during freeze–thawing. The aim of the study was to assess the modifying effect of cryoprotectant PEG-1500 and low temperatures on Ca²⁺-ATPase activity in saponin-permeabilized erythrocytes. PEG-1500 was revealed to inhibit erythrocyte Ca²⁺-ATPase activity despite the presence of endogenous effectors able to stimulate the enzyme function. Presumably, the Ca²⁺-ATPase modification was determined by the physicochemical properties of the polymer solution, since the removal of PEG-1500 out of the medium recovered the enzyme activity. Reversibility of Ca²⁺-ATPase inhibition was characteristic of erythrocytes both exposed to cryoprotectant without freezing and frozen–thawed in the PEG-1500 presence. The cell freeze–thawing without cryoprotectant had no effect on Ca²⁺-ATPase, suggesting that membrane form of enzyme is cryoresistent. Although the efficiency of erythrocyte cryopreservation with PEG-1500 depends on the incubation temperature before freezing stage, the functional indices of Ca²⁺-ATPase in erythrocytes exposed to PEG-1500 at 37 and 5–7°C had no significant distinctions if the subsequent ATP hydrolysis was conducted at 37°C. However, the enzyme activity was additionally slowed down when the temperature of enzymatic reaction was decreased to 5–7°C after erythrocyte preincubation with PEG-1500 under the same conditions. The identified changes in Ca²⁺-ATPase activity in erythrocytes in the PEG-1500 presence were most likely determined by a modifying effect of the cryoprotectant on the membrane structure; as a result, the Ca²⁺-ATPase endogenous effectors present in the medium could not overcome the restrictions imposed on the enzyme function by a modified membrane macroenvironment.     

Reduction of ferricytochrome c, methemoglobin and metmyoglobin by hydroxyl and alcohol radicals.

We have studied the reaction of ferricytochrome c, methemoglobin and metmyoglobin with OH and alcohol radicals (methanol, ethanol, ethylene glycol and glycerol). These radicals can be divided into three groups: 1. The OH radicals which reduce the ferricytochrome c with a yield of (30 +/- 10)% and methemoglobin with a yield of (40 +/- 10)%. They do not reduce metmyoglobin. The reduction is not a normal bimolecular reaction but is most probably an intramolecular electron transfer of a protein radical. 2. Methanol and ethanol radicals which reduce all three hemoproteins with a yield of (100 +/- 5)%. This reduction is a normal bimolecular reaction. 3. Glycerol radicals which do not reduce the ferrihemoproteins under our experimental conditions. Ethylene glycol radicals do not reduce ferricytochrome c and metmyoglobin but they do reduce methemoglobin with a yield of (30 +/- 10)%.     

Identification, effective purification and functional characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-640

The extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-640 was purified using polyethylene glycol fractionation (PEG) and gel-filtration. The cell free extract was subjected to fractionation by PEG-200, 400 and 1500. The 10% (w/v) PEG-1500 gave dextransucrase with maximum specific activity of 23 with 40 fold purification in a single step. The purified enzyme showed multiple molecular forms on SDS-PAGE, however the same sample showed a single band on non-denaturing native-PAGE. The purified dextransucrase fractions obtained from PEG-1500, confirmed the presence of dextran, when run on SDS-PAGE under non-denaturing gels for in situ activity detection by Periodic Acid Schiff's staining. The activity bands corresponded to the native and active form of the purified dextransucrase of approximately, 180kDa molecular size, that appeared on the denaturing gels stained with Coomassie Brilliant Blue. No bands appeared after staining the activity of dextransucrase on non denaturing SDS-PAGE gels with raffinose, which excluded the presence of fructosyltransferases. Further purification of 10% PEG-1500 purified dextransucrase by gel-filtration gave dextransucrase with specific activity of 35 with 61 fold purification.     

Effect of the addition mode of carbon nanotubes for the production of chitosan hydrogel core-shell beads on adsorption of Congo red from aqueous solution

The adsorption performance of chitosan (CS) hydrogel beads (CSBs) generated by sodium dodecyl sulfate (SDS) gelation with multi-walled carbon nanotube (CNT) impregnation was investigated for Congo red removal as a model anionic dye. CNT-impregnated CSBs were prepared by four different strategies for dispersing CNTs: (a) in CS solution (CSBN1), (b) in SDS solution (CSBN2), (c) in CS solution containing cetyltrimethylammonium bromide (CTAB) (CSBN3), and (d) in SDS solution for gelation with CTAB-containing CS solution (CSBN4). It was observed from FE-SEM study that depending on nature of CNT dispersion, CNTs were found on the outer surface of CSBN2 and CSBN4 only. The adsorption capacity of the CSBs varied with the strategy used for CNT impregnation, and CSBN4 exhibited the highest maximum adsorption capacity (375.94 mg/g) from the Sips model. The lowest Sips maximum adsorption capacity by CSBN3 (121.07 mg/g) suggested significant blocking of binding sites of CS by CNT impregnation.     

Interaction of phosphorylase b with eosin. Influence of substrate and effectors on eosin-enzyme complexes.

The interactions of rabbit muscle glycogen phosphorylase b with Eosin (2',4',5',7'-tetrabromofluorescein) was studied. Eosin was found to be an effective inhibitor of the enzyme. The inhibition constants for the dye were estimated to be approx. 36 and 60 microM with respect to AMP and glucose 1-phosphate respectively. The binding of Eosin to phosphorylase b is accompanied by a red-shift of about 12 nm in the dye absorption-spectrum maximum, indicating low-polarity binding sites on the enzyme molecule for the dye. The absorbance in the difference absorption maximum at 537 nm was utilized to follow the conjugation of phosphorylase b with Eosin. Scatchard plots of the titration data revealed the existence of at least two classes of binding sites on the protein molecule for Eosin, and the dissociation constants measured in Tris/HCl buffer, pH 7.0 (IO.091), were 7.7 and 41.7 microM respectively. The influence of the substrates and effectors on Eosin-enzymes complexes was used to study the ligand-phosphorylase b interactions. IMP displaced the dye completely from the enzyme, indicating that there are two IMP-binding sites per phosphorylase b monomer. AMP binding to the enzyme with respect to Eosin concentration is of two types: a non-competitive one for the high-affinity site for AMP and a competitive one for the low-affinity site for the activator. The effects of glucose 6-phosphate, ATP, Pi and glycerol 2-phosphate in the system are in according dance with a partially competitive model. Glucoes 1-phosphate and UDP-glucose appear to affect only the high-affinity site for Eosin, whereas glucose and glycogen have no effect on Eosin-phosphorylase b complexes. Our results suggest that Eosin can be used as an efficient optical probe for studying the phosphorylase b system.     

Multiple binding modes for Hoechst 33258 to DNA

Two binding modes for the bisbenzimidazole Hoechst 33258 to native DNA at physiological conditions have been distinguished. Type 1 binding, which dominated at low dye/phosphate ratios (D/P less than 0.05) or low dye concentrations, had a high quantum yield of fluorescence with maximum emission at 460 nm. Binding of the dye at type 2 sites (0.05 less than D/P less than 0.4) lead to quenching of fluorescence from type 1 bound dye, presumably by nonradiative energy transfer. Fluorescence quantum yield of type 2 bound dye was low (phi = 0.05-0.1) and it peaked around 490 nm. At D/P greater than 0.4, the dye/DNA complex precipitated. This was caused by an additional dye-DNA interaction that was strongly cooperative. The anomalous dispersion of the refractive index of the complex changed abruptly around D/P = 0.4, indicating that the precipitating dye-DNA interaction involved strong electronic interaction between dye molecules. Hoechst 33258 precipitated polynucleotides irrespective of strandedness and base composition when dye concentration was raised above 1 X 10(-5) M. In the presence of 25% ethanol, type 2 binding to DNA did not occur, whereas the binding constant for type 1 binding (kappa = 2 X 10(3) M-1) was about two orders of magnitude smaller than in physiological buffer. DNA was not precipitated by high concentrations of Hoechst 33258 in 25% ethanol.     

Improved glycerol to ethanol conversion by <Emphasis Type="Italic">E. coli</Emphasis> using a metagenomic fragment isolated from an anaerobic reactor

Crude glycerol obtained as a by-product of biodiesel production is a reliable feedstock with the potential to be converted into reduced chemicals with high yields. It has been previously shown that ethanol is the primary product of glycerol fermentation by Escherichia coli. However, few efforts were made to enhance this conversion by means of the expression of heterologous genes with the potential to improve glycerol transport or metabolism. In this study, a fosmid-based metagenomic library constructed from an anaerobic reactor purge sludge was screened for genetic elements that promote the use and fermentation of crude glycerol by E. coli. One clone was selected based on its improved growth rate on this feedstock. The corresponding fosmid, named G1, was fully sequenced (41 kbp long) and the gene responsible for the observed phenotype was pinpointed by in vitro insertion mutagenesis. Ethanol production from both pure and crude glycerol was evaluated using the parental G1 clone harboring the ethanologenic plasmid pLOI297 or the industrial strain LY180 complemented with G1. In mineral salts media containing 50 % (v/v) pure glycerol, ethanol concentrations increased two-fold on average when G1 was present in the cells reaching up to 20 g/L after 24 h fermentation. Similar fermentation experiments were done using crude instead of pure glycerol. With an initial OD₆₂₀ of 8.0, final ethanol concentrations after 24 h were much higher reaching 67 and 75 g/L with LY180 cells carrying the control fosmid or the G1 fosmid, respectively. This translates into a specific ethanol production rate of 0.39 g h^?1 OD^?1 L^?1.     

A comparison of the binding of imidazole to valence hybrid and methemoglobin: an assignment of individual heme reactivity

The ferric hemes of valence hybrid hemoglobins combine with imidazole in a manner analogous with the hemes of methemoglobin. Equilibrium studies show that imidazole binding to methemoglobin is minimally described by the sum of two independent processes (K₁ = 200 M^?1 and K₂ = 37 M^?1), both of which contribute equally to the observed difference spectrum. Using valance hybrid hemoglobins, which show single binding processes under similar conditions, it is possible to identify the high affinity sites in methemoglobin with the α chains and the low affinity sites with the β chains.Kinetic studies show that the valance hybrid hemoglobins react in a single exponential fashion with imidazole in contrast with methemoglobin which shows a biphasic reaction (k₁ = 85 M^?1 sec^?1k₂ = 25 M^?1 sec^?1). A comparison of the rates of reaction of the hybrids allows the assignment of the fast phase in methemoglobin to the β chains and the slow phase to the α chains.The heterogeneity of the imidazole reaction with methemoglobin occurs over the pH range 5.5–9.5 within which two ionization processes are discernable at pH 6.9 and 7.5.     

Adsorption of methylene blue dye from aqueous solution by sugar extracted spent rice biomass

This study was aimed at using sugar extracted spent rice biomass (SRB) as a potential adsorbent to remove methylene blue (MB) dye from aqueous solution. The SRB was used without any modification. A three factor full factorial experimental design (2(3)) was employed to investigate the effect of factors (adsorbent dose, dye concentration, temperature) and their interaction on the adsorption capacity and color removal. Two levels for each factor were used; adsorbent dose (0.25-0.5g/100mL), dye concentration (25-50mg/L), and temperature (25-45°C). Initial dye concentration and adsorbent dosage were found as significant factors for the adsorption of MB dye. Langmuir isotherm (R(2)>0.998) best explained the equilibrium of MB adsorption on SRB with monolayer adsorption capacity of 8.13mg/g. The pseudo-second order model (R(2)>0.999) was best fitted to explain the adsorption kinetics. Thermodynamic investigation revealed that the adsorption process was spontaneous, endothermic, and was feasible to treat dyeing wastewater.