Thermal stability of beta-lactoglobulin in the presence of aqueous solution of alcohols and polyols

A systematic study concerning the effect of aqueous solution of alcohols and polyols with four carbon atoms on β-lactoglobulin stability is presented. The protein was chosen due to its functional properties and applications in food and pharmaceutical industries and because its structure and properties in aqueous solution have been widely described. The alcohols having a four carbon chain were selected to examine the effect of the gradual increase in the number of OH groups on protein stability.

Protein thermal stability in water, buffers and dilute aqueous solutions of 1-butanol, 1,2-butanediol, 1,2,4-butanetriol and 1,2,3,4-butanetetrol was evaluated by fluorescence spectroscopy. The results were used to determine the temperature range in which the unfolding process is reversible and the protein denaturation temperature in acetate buffer pH 5.5 and in the aqueous mixed solvents. Thermodynamic results show that alcohol denaturating effect diminishes gradually as the number of OH groups increase.     

Physico-chemical characteristics and functional properties of membrane-bound hemoglobin]

It was shown that fetal form of haemoglobin (Hb) is mainly connected with membrane of peripheral red blood corpuscles. Membrane-bound haemoglobin has relatively high affinity to O2, low Hill's coefficient, much higher peroxidase activity in comparison with cytosolic Hb.     

Thermal stability of human haemoglobin in the presence of sarcosine and sorbitol

Sarcosine and sorbitol at 10–30% (w/w) stabilized haemoglobin from human erythrocytes to thermal denaturation. At 65?°C, the protein's half life was increased from 0.85 to 50 min in 30% sarcosine and to 24 min in 30% sorbitol. A kinetic analysis based on the Lumry-Eyring mechanism of inactivation showed that the denaturation process can be described by a second-order rate expression with an apparent activation energy ranging from 56 to 87 kcal mol^?1.     

Interaction of hemoglobin with salts: Effects on the functional properties of human hemoglobin

Oxygen isotherms of human hemoglobin measured in distilled water and in solutions of different inorganic salts in the concentration range from below 10^?3 m to above 1·5 m at neutral pH indicate that the oxygen affinity decreases with increasing salt concentration in the lower range of ionic strength; above the physiological range, there is in most cases a further decrease in oxygen affinity, but this varies with the nature of the salt and, in some instances, the affinity goes through a maximum.The effect of cations, which is opposite to that of anions, operates primarily in the higher concentration range; i.e. above 0·1 m. This effect is especially large for Li⁺, Ca²⁺ and Mg²⁺.The alkaline Bohr effect depends strongly on anion concentration, being displaced towards higher pH values and being reduced in magnitude as chloride concentration is increased. On the other hand, the acid Bohr effect, observed below pH 6, appears to be independent of chloride concentration from 6 × 10^?2 m to 2 m.The overall heat of oxygenation has been determined for the isoionic protein as well as at different concentrations of chloride and phosphate. The average intrinsic heat of reaction of hemoglobin with oxygen in solution is found to be ?14·6 kcal/mol of O₂.     

Radiation-induced changes of structural and functional properties of human hemoglobin

Structural changes of human deoxyhemoglobin (deoxyHb) induced by water and ethanol radicals were investigated in this study using absorption spectroscopy. Spectra of deoxyHb samples irradiated under various conditions (the atmosphere of argon or N2O in the absence or presence of ethanol) indicate their conversion into methemoglobin (metHb), hemichrome- and cholehemichrome-like products. The absorbance at the characteristic maxima of these derivatives and also of the oxidized or reduced samples following irradiation depends on the dose of radiation and the conditions employed.     

Radiation-induced changes of structural and functional properties of human hemoglobin

Gel filtration and SDS-PAGE separation of hemoglobin (Hb) irradiated under argon or N2O show formation of covalent-aggregated Hb molecules. The production of covalent bonds is attributed mainly to the action of hydroxyl radicals, because addition of ethanol, a scavenger of these radicals, suppresses this reaction to a great extent. The oxidized heme iron forming metHb or hemichromes is found in all the separated fractions of irradiated Hb. It is also found that the radiation-modified Hb molecules exhibit a decrease of co-operative binding of oxygen.     

Effect of ethanol on functional properties of the human hemoglobin molecule

The effect of ethanol on the oxygenation of hemoglobin was studied by kinetic absorption spectroscopy. It was found that the efficiency of oxygen geminate rebinding decreased upon ethanol addition. At ethanol concentrations up to 4.5 M, its influence on the structure and functional properties of the hemoglobin molecule is determined by changes in the bulk dielectric constant of solution. The decrease in the rate constant of the bimolecular stage of rebinding k'4 was caused by an increase in the viscosity of solution, with k'4 being approximately 1/eta 0.5. Upon oxidation of hemoglobin to hemichrome initiated by ethanol, dramatic conformational changes in the region of the heme pocket took place. They lead to a more than twofold increase in the efficiency of exit of oxygen molecules from the protein matrix to the solution after photodissociation.     

Physicochemical and functional properties of hemoglobin of mice infected with Ehrlich's carcinoma]

Injection of white mice with Ehrlich's carcinoma triggers an increase in the mice blood erythrocyte fraction suspended in the 14% sucrose concentration zone. As established by acid erythrograms, a quantitative increase of this red blood cell population is due to an increased rate of erythroblast maturation and occurrence of immature cell forms in the blood stream. As shown by alkali denaturation of hemoglobin, tumour development causes an increase in alkali-resistant hemoglobin fraction in the erythrocytes. On the basis of the data on alkali denaturation of hemoglobin, it is suggested that in the infected mice increased rate of erythrocyte maturation go in line with selective binding of alkali resistant hemoglobin fraction to red cell membrane.     

Mechanisms of aliphatic alcohols oxidation by enzymatic systems of the liver]

The main pathways of aliphatic alcohols oxidation in human and mammalian liver, i.e. dehydration of alcohols by cytosolic alcohol dehydrogenases and oxidation in the presence of microsomal enzymatic system, catalase and hydrogen peroxide are described. A special emphasis is laid upon the interaction of alcohols with terminal oxidase of the microsomal hydroxylating system, i.e. cytochrome P-450. The relative role of these three oxidative pathways in alcohol conversions is evaluated.     

The effect of hypoxic hypoxia on the physicochemical and functional properties of hemoglobin in rats]

The effect of intermittent altitude chamber hypoxia is established to cause an increase in solvability of laboratory rat hemoglobin. Results of immunochemical and fluorescent analysis of the samples of hemoglobin and its component are presented and discussed. They prove that changes in solvability of hemoglobin are determined by the conformational reconstructions of the respiration protein as a result of formation of the complexes with internally erythrocytic metabolites.     

The effect of macromolecular polyanions on the functional properties of human hemoglobin.

The binding of dextran sulphate and heparin to human hemoglobin and their effect on the properties of gas transport have been investigated. Both dextran sulphate and heparin are strongly bound by oxy-hemoglobin as well as deoxyhemoglobin and the stoichiometry of the binding (polyanion/tetrameric hemoglobin) is less than unity; sedimentation analysis gives indication for the existence of octomers. The oxygen affinity of hemoglobin is decreased, to the same extent, by both dextran sulphate and heparin. This effect is pH-dependent. In addition the polyanions affect the position and the magnitude of the Bohr effect. In the presence of dextran sulphate the recombination of hemoglobin with carbon monoxide after flash photolysis is biphasic and the fraction of quickly reacting material increases with dilution of the protein.     

Autooxidation and oxygenation of human hemoglobin]

The processes of reversible oxygen binding and nonreversible autoxidation of human hemoglobin were studied. The activation energy of the oxygen binding, as determined by the temperature dependence of the P50 parameter, was 26 +/- 4 kJ/mol, the activation energy of the autoxidation, as determined by the temperature dependence of the apparent rate constant of autoxidation, was 120 +/- 15 kJ/mol. Pyridoxal phosphate decreased the oxygen affinity of hemoglobin, slightly diminished the cooperativity of the oxygenation process and unaffected the activation energy of the oxygen binding. Pyridoxal phosphate slightly reduced the Bohr coefficient value from 0.70 to 0.65. Pyridoxal phosphate, but not pyridoxal, raised the apparent rate constant of autoxidation reaction. The rate of autoxidation significantly increased as the pH value of the medium decreased, reflecting, probably, protonation of the distal histidine of the hemoglobin. The activation energy of autoxidation was independent of pH. Aliphatic alcohols also increased the rate of the autoxidation process, probably, either by stabilization of the hemoglobin T-state, or by direct nucleophilic displacement of the oxygen molecule.     

Effect of temperature on functional properties of carp hemoglobin

Precise oxygen equilibrium curves of carp hemoglobin have been obtained in 0·1 m-phosphate from 10 to 25 °C. The equilibrium data were analyzed according to the stepwise oxygenation model of Adair (1925) to obtain the enthalpy change (ΔH_i), entropy change (ΔS_i) and free energy change (ΔG_i) for the i (= 1, 2, 3, 4) individual oxygenation steps. The values of ΔH_i are definitely non-uniform with dependencies on i and the pH of the medium. The co-operative effects in carp hemoglobin are due mainly to enthalpic contributions under the conditions studied here. The thermodynamic properties suggest a structural transition with pK ~8·5 as was also seen in other functional and spectroscopic measurements.