Influence of rheopolyglucin and dextran dialdehyde on physicochemical properties and thermostability of human hemoglobin

A study was made of the influence of rheopolyglucin and dextran dialdehyde derived therefrom on the structural characteristics and thermostability of human hemoglobin. The effects of solution pH, incubation time and temperature, and the degree of dextran oxidation on the conjugation between human hemoglobin and dextran dialdehyde were assessed. Formation of the hemoglobin-dextran dialdehyde complex resulted in shielding of the protein chromophore groups by the polysaccharide and transition of a part of hemoprotein molecules from a low-spin (HbO₂) to a high-spin (Hb and MetHb) state. It was found that the temperature of denaturation transition for the native protein and hemoglobin in the presence of rheopolyglucin was 60°C versus 80°C for the hemoprotein-dextran dialdehyde conjugate. Presumably, the latter is determined by the enhancement of hydrophobic interactions within the protein globule caused by dextran dialdehyde and the ability of the surface-bound carbohydrate components to prevent the association of hemoglobin molecules.     

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.     

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.     

Physico-chemical properties of terrylytin modified by a copolymer based on vinylpyrrolidone

The proteolytic activity of terrylytin produced by the culture of Asp. terricola and modified by a water-soluble copolymer of vinylpyrrolidone and acrolein remained unchanged after enzyme modification. Using micro-thin layer chromatography, it was shown that the bulk of the epsilon-amino groups of lysine residues of the protein enter the reaction with the aldehyde groups of the polymeric matrix. The sedimentation and diffusion patterns of the polymerenzyme adduct demonstrated that the molecular weight of the modified enzyme is the total of molecular weights of its constituent components. Evidence from viscosimetry and gel chromatography allowed to develop a hydrodynamic model of the macromolecular product. It was shown that the rate of the enzyme inactivation in the solution calculated from the first order reaction equation depends on the nature of the enzyme electrochemical microenvironment. Under conditions close to physiological ones the rate inactivation constant for terrylytin modified by a neutral polymeric matrix is 10 times less than that for the native enzyme. At the isoelectric point (pH 4,6) a positively charged polymeric form of terrylytin is found to be the most stable one. The pH and temperature optima for casein hydrolysis remained unchanged throughout polymeric modification. The polymeric membrane did not hamper the diffusion during approximation of the substrates (casein and insulin) to the enzyme molecule during the catalytic act, which manifested itself in a constancy of Michaelis curves. Terrylytin modification by a copolymer causes an increase of stability with respect to trypsin proteolysis and a decrease of human blood plasma affinity for the inhibitors. The apparent inhibition constants for modified enzyme forms do not depend on the nature of electrochemical microenvironment and exceed that for native terrylytin 10-fold.     

Parameters of oxygen-binding function of human hemoglobin modified by carbon oxide and UV-light

The parameters of oxygen-binding function of human hemoglobin, modified by carbon oxide and UV-radiation: the pressure of half-saturation with the ligand (P50), Hill's constant (n), and arterial-venous difference of HbO2 concentration in the sample were studied. The presence of carboxyform in blood in boundaries of admissible values (lower than 10 per cents) did not noticeably influence to the oxygenation parameters. Functional properties of hemoproteid were substantially modified in case of HbCO concentration increasing from 30 up to 80 percent. It has been discovered, that the leading mechanism of protection from hemic hypoxia in case of poisoning with CO is decreasing of degree of cooperative interactions and relative affinity of hemoglobin for ligands. The stimulating influence of UV-light to the functional properties of modified with carbon oxide human hemoglobin observed in case carboxyform hemoprotein concentration in solution was lower than 10 percent. The disturbance of oxygen-binding ability of hemoglobin at the influence of higher concentrations of Hb-CO was inconvertible and was not correct with UV-radiation.     

Studies on camel hemoglobin. 1. Physico-chemical properties and some structural aspects of camel hemoglobin (Camelus dromedarius).

Hemoglobin from an adult camel (Camelus dromedarius) was prepared from the red cell lysate by CM- and DEAE-cellulose chromatography. The purified hemoglobin showed a lesser mobility on starch gel electrophoresis at pH 8.5 than that of human hemoglobin C. Native camel hemoglobin contains 95-99% alkali-resistant hemoglobin and in soluble in 2.94 M K2HPO4/KH2PO4 buffer. Different forms of camel hemoglobin show similar ammonium sulfate precipitation curves. Indirect evidence for the stability of camel hemoglobin solutions was obtained from several sources. Spontaneous met-hemoglobin formation is extremely slow and minimal quantities of degradation products appear on starch gel electrophoresis and on chromatographic separation. The alpha and beta chains of camel hemoglobin A were separated on a CM-23 column by the use of a pyridine formate gradient. Large peptide fragments were obtained by tryptic digestion of maleylated alpha and beta chains. The N-terminal structure of the alpha and beta chains and of tryptic maleylated peptides derived from alpha and beta chains are presented. Between adult camel hemoglobin and adult human hemoglobin six amino acid differences in the N-terminal 20 amino acid residues of the alpha chain, at residues: 4, 5, 12, 14, 17, and 19; eight amino acid substitutions were found in the beta chain at positions: 4, 5, 6, 9, 12, 13, 16, and 19. Substitutions at alpha5 Ala leads to Lys, and beta19 Asn leads to Lys, increase the net positive charge of camel hemoglobin by two, while other substitutions result in no charge differences. The molecular basis of the stability of camel adult hemoglobin is discussed.     

Physico-chemical properties of ribonuclease A modified with pyridoxal-5'-phosphate]

The physico-chemical properties have been studied of RNase A selectively modified at the E-NH2-group of Lys-7 and Lys-41 with pyridoxal-P. Modification did not affect conformational stability of the protein globule, thus all changes in the molecule of the modified RNase A were localised around the alkylated Lys residue. In the both cases pyridoxyl-P. The residue was shown to be localized in the active site region of the (P-Pxy)-Lys-7-RNase A and its chromophore parts was highly exposed to the solvent. (P-Pxy) E-Lys-7-RNase A and its chromophore parts was highly exposed to the solvent. In the Lys-41 derivative, pyridoxamine-P was situated exactly in the active site and is partially hidden in the protein grobule. The pH-dependence of absorption spectra indicates that the chromophore of pyridoxyl-P in modified proteins is quite sensible to the ionic state of its surrounding. The usefulness of pyridoxyl-P as a reporter group was proved in the study with (P-Pxy)-Lys-7-RNase A. Some conformational changes involving His-119 were shown to take place in the course of the enzyme-nucleotide complex formation.     

Increased thermostability and phenol removal efficiency by chemical modified horseradish peroxidase

Horseradish peroxidase was modified by phthalic anhydride and glucosamine hydrochloride. The thermostabilities and removal efficiencies of phenolics by native and modified HRP were assayed. The chemical modification of horseradish peroxidase increased their thermostability (about 10- and 9-fold, respectively) and in turn also increased the removal efficiency of phenolics. The quantitative relationships between removal efficiency of phenol and reaction conditions were also investigated using modified enzyme. The optimum pH for phenol removal is 9.0 for both native and modified forms of the enzyme. Both modified enzyme could suffer from higher temperature than native enzyme in phenol removal reaction. The optimum molar ratio of hydrogen peroxide to phenol was 2.0. The phthalic anhydride modified enzyme required lower dose of enzyme than native horseradish peroxidase to obtain the same removal efficiency. Both modified horseradish peroxidase show greater affinity and specificity of phenol.     

Association-dissociation of molecules of hemoglobin and polymeric hemoglobin in solutions

The process of association-dissociation of hemoglobin molecules into dimers of its subunits in water and water-saline solutions is studied by the method of gel-penetrating chromatography and ultrafiltration. The quantitative assessment of stabilization of quaternary structure of hemoglobin in chemically bound polymer derivative in comparison with native peptide on the basis of building differential concentration curves is conducted for the first time. By the method of atomic-force spectroscopy, the morphology of nanoparticles of hemoglobin and its modified polymeric derivative is studied.     

Mathematical models of the oxygen-binding function of intact human hemoglobin and hemoglobin modified by UV-radiation in the presence of carbon oxide

The influence of carbon oxide and UV-radiation in doses of 151-453 J/m2 on the physiological properties of human oxyhemoglobin has been studied. Mathematical models of the oxygen-binding function of intact and modified hemoprotein have been developed. It has been found that saturation of human hemoglobin with oxygen obeys the logistic dependence. In the presence of carboxyhemoglobin, the oxygenation parameters change and saturation curves are described by the equations of degree dependence. It has been shown that UV light had the stimulating influence on the functional properties of human hemoglobin modified by carbon oxide if the concentration of carboxyform of the hemoprotein in solution was no higher than 10 per cent. The disturbance of the oxygen-binding ability of hemoglobin by the action of higher concentrations of carboxyhemoglobin was irreversible and was not corrected by UV-radiation.     

修饰血红素提高血红蛋白类过氧化物酶活性

利用苯酚或对羟基联苯对血红蛋白的血红素辅基进行化学修饰,将修饰后的血红素与脱辅基血红蛋白进行重组得到新的血红蛋白。以光吸收扫描分析修饰血红素和重组血红蛋白,证明新的重组血红蛋白构建成功。酶活力测定表明,修饰血红素得到的重组血红蛋白的类过氧化物酶活性都比天然血红蛋白的酶活力高,用对羟基联苯修饰血红素得到的重组血红蛋白的酶活提高明显,约是天然血红蛋白的1.6倍。     

A recombinant human hemoglobin with anti-sickling properties greater than fetal hemoglobin

A new recombinant, human anti-sickling beta-globin polypeptide designated beta(AS3) (betaGly(16) --> Asp/betaGlu(22) --> Ala/betaThr(87) --> Gln) was designed to increase affinity for alpha-globin. The amino acid substitutions at beta22 and beta87 are located at axial and lateral contacts of the sickle hemoglobin (HbS) polymers and strongly inhibit deoxy-HbS polymerization. The beta16 substitution confers the recombinant beta-globin subunit (beta(AS3)) with a competitive advantage over beta(S) for interaction with the alpha-globin polypeptide. Transgenic mouse lines that synthesize high levels of HbAS3 (alpha(2)beta(AS3)(2)) were established, and recombinant HbAS3 was purified from hemolysates and then characterized. HbAS3 binds oxygen cooperatively and has an oxygen affinity that is comparable with fetal hemoglobin. Delay time experiments demonstrate that HbAS3 is a potent inhibitor of HbS polymerization. Subunit competition studies confirm that beta(AS3) has a distinct advantage over beta(S) for dimerization with alpha-globin. When equal amounts of beta(S)- and beta(AS3)-globin monomers compete for limiting alpha-globin chains up to 82% of the tetramers formed is HbAS3. Knock-out transgenic mice that express exclusively human HbAS3 were produced. When these mice were bred with knock-out transgenic sickle mice the beta(AS3) polypeptides corrected all hematological parameters and organ pathology associated with the disease. Expression of beta(AS3)-globin should effectively lower the concentration of HbS in erythrocytes of patients with sickle cell disease, especially in the 30% percent of these individuals who coinherit alpha-thalassemia. Therefore, constructs expressing the beta(AS3)-globin gene may be suitable for future clinical trials for sickle cell disease.     

Aggregation of hemoglobin S modified by bifunctional imidoesters

Sickle hemoglobin (Hb S) was cross-linked by two types of bifunctional imidoesters, dimethyladipimidate (DMA) and dimethyl-3,3'-dithiobispropionimidate (DTBP). These modified hemoglobins were separated into monomer, dimer and polymer fractions by gel filtration. All of these modified hemoglobins showed extremely left-shifted oxygen equilibrium curves with no cooperativity. The stabilities of these hemoglobins were also decreased. The solubilities of these modified hemoglobins in high-phosphate buffers were lower than those of native Hb S. Studies on the kinetics of the aggregation of these modified hemoglobins showed that intracross-linked Hb S with DMA and DTBP (DMA- and DTBP-modified monomeric Hb S) still retained the capability of aggregation with a delay time, while intercross-linked Hb S with DMA and DTBP (DMA- and DTBP-modified oligomeric Hb S) aggregated without a delay time. When the kinetics of aggregation was measured for mixtures of modified and native deoxy-Hb S, DMA-modified monomeric deoxy-Hb S shortened the delay time prior to aggregation of native deoxy-Hb S. The other modified deoxy-Hb S did not affect the delay time, suggesting that these modified oligomeric hemoglobins neither participate in the formation of nuclei nor copolymerize with native deoxy-Hb S.     

Studies of physical and chemical properties of the vacuum UV-irradiated molecules of human hemoglobin and its components (heme and globin)

The purpose of the present work is to study the vacuum ultraviolet radiation action on the optical and chromatographic characteristics of human hemoglobin molecules and their components - haem and globin. Using the methods of spectrophotometry and thin layer chromatography (TLC), we have investigated into the structural changes of molecules of human hemoglobin, haem and globin, induced by the influence of vacuum UV light (gamma = 118-134 nm, dose - 1.2 kJ/m2). It has been shown that vacuum ultraviolet radiation induces an infringement of the higher types of the spatial organization ofglobin molecules, thus leading to the changes in the structural state of the albuminous globule.     

Physico-chemical and immunological properties of allophycocyanins

Allophycocyanins were purified from diverse cyanobacteria and one rhodophytan alga (Cyanidium caldarium). The native proteins are trimeric molecules with the structure ()₃. Representative native allophycocyanins and their and subunits were characterized with respect to molecular weight, amino acid composition, isoelectric point, absorption and fluorescence spectra and immunological properties. All of the allophycocyanins studied were strikingly similar with respect to each of these properties.Renatured and subunits of allophycocyanin were distinct immunologically from each other, and both cross-reacted with the antiserum to the native protein.Trimeric allophycocyanin was readily reconstituted from the purified and subunits. Formation of hybrid allophycocyanins was demonstrated by direct isolation and characterization of the hybrid proteins and by immunological techniques.The results support the view that allophycocyanins are a highly conserved group of proteins.Abbreviation Used SDS sodium dodecyl sulfate     

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.