ABSTRACT
The process of association-dissociation of hemoglobin molecules into dimers of its subunits in 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.
Subject(s)
Hemoglobins/chemistry , Polymers/chemistry , Chromatography, Gel , Microscopy, Atomic Force , Protein Structure, Tertiary , Sodium Chloride , Solutions , UltrafiltrationABSTRACT
Polycondensation of a catalase (EC 1.11.1.6) with glutaraldehyde in order to stabilize the quaternary structure of an enzyme, maintain its activity, and protect it from thermal denaturation was studied. Synthesis showed a superequivalent utilization of the aldehyde groups relative to the catalase amine groups, as a result of the formation of glutaraldehyde oligomers linked to the enzyme.
Subject(s)
Catalase/chemistry , Cross-Linking Reagents/chemistry , Glutaral/chemistry , Animals , Cattle , Protein Denaturation , Protein Structure, QuaternaryABSTRACT
Polymer aspects of polycondensation of pyridoxylated hemoglobin with glutaraldehyde have been considered. On the basis of the investigation of reaction kinetics, the mechanism of chemical crosslinking of hemoglobin molecules into oligohemoglobin is proposed. Owing to the statistical character of the reaction, the resulting macromolecules are polydisperse with respect to the degree of modification of hemoglobin amino groups, and size of oligohemoglobin molecules. The formation of hemoglobin oligomers was studied by varying the following reaction conditions: pH, the components ratio, and their concentrations. It is shown that the net electric charge of the oligohemoglobin molecule depends on the terminating agents. However, these agents have no effect on the electrophoretic mobility of erythrocytes in oligohemoglobin solutions. The efficiency of oxygen transport of these solutions is close to that of human blood erythrocytes. Oligohemoglobin circulation in the blood of animals after intravenous infusion leads to rapid removal of low molecular weight fractions from blood and to the accumulation of high molecular weight fractions in plasma. The period of half-release of oligohemoglobin from the organism is 14-16 h.
Subject(s)
Blood Substitutes/chemistry , Blood Substitutes/chemical synthesis , Hemoglobins/chemistry , Hemoglobins/chemical synthesis , Pyridoxal Phosphate/analogs & derivatives , Animals , Blood Substitutes/pharmacology , Dogs , Electrochemistry , Erythrocytes/chemistry , Erythrocytes/drug effects , Hemoglobins/pharmacology , Humans , Molecular Weight , Oxygen/blood , Oxygen/chemistry , Oxyhemoglobins/chemistry , Pyridoxal Phosphate/chemical synthesis , Pyridoxal Phosphate/chemistry , Pyridoxal Phosphate/pharmacology , RatsABSTRACT
Disperse systems based on hemoglobin sorption immobilization in reticular carboxylic polyelectrolytes are proposed as model of erythrocytes. The efficiency of oxygen transport of these systems is much higher than that of native hemoglobin and is comparable with the gas transport of erythrocytes. This is believed to be due to highly selective sorption of hemoglobin in microdisperse forms of permeable polyelectrolytes. Microparticles of immobilized hemoglobin exhibit high local concentration of hemoglobin: the protein mass being one order of magnitude than that of polymer-carrier. Besides, it is presumed that structure of the carboxylic polyelectrolyte matrix is a polymer analog of 2,3-diphosphoglycerate. Microdispersion of immobilized hemoglobin exhibits a aggregative resistance and have identity of surface charge with erythrocytes. Studies in vivo have revealed biocompatibility of immobilized hemoglobin.
Subject(s)
Erythrocytes/chemistry , Hemoglobins/analysis , Adsorption , Biocompatible Materials , Biopolymers , Electrolytes , Humans , Models, Biological , Particle Size , Plasma SubstitutesABSTRACT
Colloid-disperse systems based on haemoglobin sorption immobilization in reticular polyelectrolytes are proposed and investigated. The efficiency of oxygen transport of these systems is much higher than that of native haemoglobin and is comparable with the efficacy of gas transport of erythrocytes. This is believed to be due to highly selective sorption immobilization of haemoglobin in microdisperse forms of permeable carboxylic reticular polyelectrolytes. Microparticles exhibit high local concentration of haemoglobin, the protein mass being by one order of magnitude higher than that of the polymer carrier.
Subject(s)
Erythrocytes/metabolism , Hemoglobins/metabolism , Models, Biological , Oxygen/blood , Biological Transport, Active , Humans , In Vitro Techniques , Kinetics , Microspheres , Oxyhemoglobins/metabolism , PolymersABSTRACT
Polycondensation of human hemoglobin with glutaraldehyde premodified with reagents bearing acid groups (glutamic acid or sodium bisulfite), has been carried out. The resulting modified hemoglobin displays better oxygen transport characteristics in comparison with native hemoglobin.
Subject(s)
Hemoglobins/metabolism , Oxygen/metabolism , Allosteric Regulation , Biological Transport , Glutaral/chemistry , Hemoglobins/chemistry , Humans , Protein Binding , Pyridoxal Phosphate/metabolismABSTRACT
Solutions of oligohemoglobins (OHb) with middle molecular mass 100 x 10(3) = 400 x 10(3), obtained after polycondensation of hemoglobin with glutaric aldehyde as well as oligohemoglobin modified with pyridoxal-5-phosphate, were studied in vivo and in vitro (plethoric administration, isovolemic metabolic substitution, hemorrhagic shock; at a dose of 0.5-1.8 g/kg of body mass; dogs, rabbits, rats). With increase of the OHb molecular mass period of its circulation in blood was elevated; blood plasma protected OHb from autooxidation; half-life of OHb was about 12-18 hrs in isovolemic metabolic substitution and in hemorrhagic shock and about 5 hrs--in plethoric administration. During circulation molecular mass selection of OHb occurred, where low molecular fraction decreased and high molecular fraction--accumulated; sign and value of human erythrocyte charge were similar both in OHb solutions with concentration up to 5% and in physiological solution.
Subject(s)
Hemoglobins/chemistry , Animals , Dogs , Erythrocytes/chemistry , Glutaral/chemistry , Humans , In Vitro Techniques , Molecular Weight , Oxidation-Reduction , Pyridoxal Phosphate/chemistry , Shock, Hemorrhagic/blood , SolutionsABSTRACT
Electrochemical properties of macromolecules of modified haemoglobin obtained by polycondensation with glutaric aldehyde have been investigated by means of potentiometric titration, PAG-electrophoresis, ion-exchange chromatography, and (for evaluation of isoelectric point) distribution between two aqueous polymeric phases. Introduction of additional functional groups into the macromolecule is possible by using various agents blocking polycondensation, which makes it possible to change the resulting charge and the isoelectric point.
Subject(s)
Aldehydes , Glutaral , Hemoglobins , Chromatography, Ion Exchange , Electrochemistry , Electrophoresis, Polyacrylamide Gel , Hemoglobins/analysis , Humans , Indicators and Reagents , Isoelectric FocusingABSTRACT
Oligomeric hemoglobins were produced after polycondensation with glutaric aldehyde (OHb) as well as after modification with pyridoxal-5-phosphate (OHb-PLP). OHb exhibited high affinity to oxygen (P50 = 17 torr) as well as a decreased rate of the subunits cooperative interaction (n = 1.5-1.6); OHb-PLP possessed P50 = 27 torr, n = 2.2 (for pO2 greater than P50), Bore effect -0.4. The P50 pattern was not distinctly altered in presence of approximately 15% Met hemoglobin, while already 8% of the Met form affected the "n" parameter. Biological activity of OHb and OHb-PLP was studied.
