Effect of polyanionic polymers on haemoglobin oxygen-binding properties: application to the synthesis of covalent conjugates with low oxygen affinity

Polyanionic water-soluble polymers containing sulphate, phosphate and polycarboxylate groups were synthesized. These compounds, when simply added to haemoglobin solutions, were shown to lower the affinity of the protein for oxygen. Their influence on oxygen affinity was regarded as the result of a specific interaction of the polymer anionic groups inside the 2,3-diphosphoglycerate-binding site of deoxyhaemoglobin. On the other hand, these polymers were linked to deoxyhaemoglobin to give covalent conjugates also exhibiting an oxygen affinity lower than that of free haemoglobin in the presence of 2,3-diphosphoglycerate, its natural effector, which means that after fixation, the polyanionic polymers are still acting as effectors.     

Temperature acclimation and oxygen-binding properties of blood and multiple haemoglobins of rainbow trout.

Acclimation of rainbow trout to 5, 15 and 22 degrees C for periods exceeding 4 months had no significant effect on the oxygen affinity of whole blood or on the concentration of ATP, which is the main organic phosphate in red cells. Slight differences were, however, found in the oxygenation properties of the haemolysates, which correlate with changes in the relative concentration of the multiple haemoglobins. The oxygen-binding properties of the main haemoglobin components account for the observed differences in the haemolysates. The possible thermoacclimatory significance of changes in haemoglobin multiplicity and co-factor concentrations is discussed.     

Theoretical analysis of effects of blood substitute affinity and cooperativity on organ oxygen transport.

Hemoglobin-based O(2) carriers (HBOCs), which are developed as an alternative to blood transfusion, provide O(2) delivery. At present, there is no model to predict the O(2) transport for a red blood cell-HBOC mixture on a whole organ basis. On the basis of the first principles of mass balance, a model of O(2) transport for an organ was derived to calculate venous Po(2) (Pv(O(2))) for a given inlet arterial Po(2) (Pa(O(2))), blood flow, and oxygen consumption. The model was validated by using several in vivo animal studies on HBOC administration for a wide range of HBOC oxygen-binding parameters and predicted Pv(O(2)) for various Pa(O(2)) in the same species. The model was also used to predict the effect of HBOC affinity and cooperativity on Pv(O(2)) for humans. The results indicate that Pv(O(2)) can be increased at a constant blood flow-to-oxygen consumption ratio by reducing the affinity of HBOC for normoxia and mild hypoxia; however, a high-affinity HBOC would be more efficient in maintaining higher Pv(O(2)) for severe hypoxia (Pa(O(2)) < 40 Torr).     

Crystalline ligand transitions in lamprey hemoglobin. Structural evidence for the regulation of oxygen affinity

The hemoglobins of the Sea Lamprey (Petromyzon marinus) exist in an equilibrium between low affinity oligomers, stabilized by proton binding, and higher affinity monomers, stabilized by oxygen binding. Recent crystallographic analysis revealed that dimerization is coupled with key changes at the ligand binding site with the distal histidine sterically restricting ligand binding in the deoxy dimer but with no significant structural rearrangements on the proximal side. These structural insights led to the hypothesis that oxygen affinity of lamprey hemoglobin is distally regulated. Here we present the 2.9-A crystal structure of deoxygenated lamprey hemoglobin in an orthorhombic crystal form along with the structure of these crystals exposed to carbon monoxide. The hexameric assemblage in this crystal form is very similar to those observed in the previous deoxy structure. Whereas the hydrogen bonding network and packing contacts formed in the dimeric interface of lamprey hemoglobin are largely unaffected by ligand binding, the binding of carbon monoxide induces the distal histidine to swing to positions that would preclude the formation of a stabilizing hydrogen bond with the bound ligand. These results suggest a dual role for the distal histidine and strongly support the hypothesis that ligand affinity in lamprey hemoglobin is distally regulated.     

Sickle cell anemia and the affinity of the blood for oxygen]

Blood affinity for oxygen is reduced in patients with homozygous HbS disease. The mechanisms were related to polymerisation and sickling process. In the HbS blood, P50 values were related to the percentage of sickling, the effect of prior deoxygenation on P50 measurement was established. This work underlines the influences of experimental conditions on the determination of sickle cell blood affinity and the difficulties inherent in the evaluation of in vivo oxygen transport in sickle cell disease.     

The role of oxygen-binding properties of hemoglobin in the development of oxygen deficiency in acute exogenous hyperthermia

Acid-base balance and oxygen-binding hemoglobin properties in mixed venous blood have been studied in 25 mongrel rabbits with acute environmental hyperthermia. As oxygen-hemoglobin affinity at standard pH, pCO2 and temperature increases, the effect of heat on oxygen-hemoglobin interaction is considerably attenuated. The Bohr effect increases. The mechanisms of changes in oxygen-binding properties of hemoglobin and their role in development of oxygen deficiency are discussed.     

The role of the oxygen-binding properties of the blood in maintaining pro-oxidant-antioxidant equilibrium in the body]

Tissue pro-oxidant generation under standard conditions is equilibrated with the activity of intra- and extracellular antioxidants; thus some optimal level of pro-oxidant-antioxidant balance is created. Oxygen-dependent nature of lipid peroxidation processes implicates its complex multilevel regulatory system, where systemic mechanisms may dominate upon the intracellular ones. This suggests a necessity in the investigation of body oxygen transport not only in terms of the requirements of energetic metabolism in electron acceptor but also as a physiological mechanism for antioxidant defense and, in general, as the mechanism involved in a maintenance of pro-oxidant-antioxidant balance. The hemoglobin-oxygen affinity has a special place in a complex antioxidant system hierarchy, because it determines the condition of oxygen diffusion to tissues and ultimately the value of tissue pO2. The blood oxygen-binding properties under different hyperthermic states with or without a correction of hemoglobin-oxygen affinity and L-arginine-NO pathway were shown to be involved into a complex integration with elements of different functional systems and to play an important role in complex physiologic mechanisms for the maintenance of pro-oxidant-antioxidant balance. The oxyhemoglobin dissociation curve shift leftwards may have an adaptive effect under conditions of low oxygen utilization because of limitation of oxygen fraction spent on a free radical generation and the following initiation of lipid peroxidation processes.     

Nervous mechanisms in the regulation of blood sugar

The aim of this paper is to precise the involvement of the nervous system in blood glucose regulation. The relevant mechanisms, triggered by blood glucose changes (increase or decrease of glycemia), intervene through the control of pancreatic and surrenal hormone release on the one hand, and hepatic glucose synthesis on the other hand. The part of various efferents and afferents, sensory endings and central "glucosensitive" neurons was analyzed in different situations. 1) Hyperglycemia increases the activation of the pancreatic parasympathetic fibres and decreases that of the surrenal sympathetic fibres. Hypoglycemia elicits reverse effects in the two types of efferents. 2) Hyperglycemia produces an activation in hepatic efferent vagal fibres and thus an acceleration of glycogen synthesis. Reversely, hypoglycemia stimulates both the hepatic sympathetic efferents and the glucose release by the liver. 3) The gustative receptors and the gastro-intestinal glucoreceptors are stimulated by glucose, which produces an insulin release. 4) The various kinds of afferents modify the efferent control of blood glucose level, through the "glucosensitive" central neurons located in hypothalamic and medullary regions.     

Solvent regulation of oxygen affinity in hemoglobin. Sensitivity of bovine hemoglobin to chloride ions

Under physiological conditions of pH (7.4) and chloride concentration (0.15 M), the oxygen affinity of bovine hemoglobin is substantially lower than that of human hemoglobin. Also, the Bohr effect is much more pronounced in bovine hemoglobin. Numerical simulations indicate that both phenomena can be explained by a larger preferential binding of chloride ions to deoxyhemoglobin in the bovine system. Also, they show that the larger preferential binding may be produced by a decreased affinity of the anions for oxyhemoglobin, thereby stressing the potential relevance of the oxy conformation in regulating the functional properties of the protein. The conformation of the amino-terminal end of the beta subunits appears to regulate the interaction of hemoglobin with solvent components. The pronounced sensitivity of the oxygen affinity of bovine hemoglobin to chloride concentration and to pH suggests that in bovine species these are the modulators of oxygen transport in vivo.     

A comparative study of the temperature dependence of the oxygen-binding properties of mammalian hemoglobins.

The effect of temperature on the oxygen-binding properties of hemoglobin (Hb) from ruminants, such as ox, reindeer, musk ox, mouflon and egyptian water buffalo is compared to that of human adult Hb (HbA). A striking difference emerges where in the presence of chloride ions and in the absence of 2,3-diphosphoglycerate [Gri(2,3)P2] a strongly reduced exothermic oxygenation process is observed for all ruminant Hb investigated with respect to HbA. Next, in the presence of physiological concentrations of Gri(2,3)P2, HbA displays a less exothermic oxygenation process, with values tending toward those observed in ruminant Hb [where Gri(2,3)P2 is not a physiological effector and for which the addition of Gri(2,3)P2 has essentially no effect on the oxygenation enthalpy]. Different from HbA, the intrinsically less exothermic oxygen binding seems to be independent of the experimental conditions for ruminant Hb, underlying specific structural characteristics which might be responsible for this feature.     

Genetic regulation of plasma and red blood cell magnesium concentration in man. II. Segregation analysis.

A previous paper in this series reported that genetic factors play a major role in the familial transmission of plasma (P) and red blood cell (RBC) magnesium (Mg) concentrations. We report here the results of commingling analysis based on a random sample of unrelated individuals, and complex segregation analysis of a random sample of nuclear families. For RBC Mg, there is evidence for a mixture of two distributions, but not for three. For P Mg, there is no evidence for commingling. Complex segregation analysis under a mixed model yielded significant support for a major gene effect on RBC Mg, but not on P Mg. Parameter estimates indicated that the data are compatible with a rather common major gene (q = .23) for elevated RBC Mg, roughly 5% of the population being homozygotes for this gene, that the nonfamilial factors account for a small fraction of the total variance, and that the overlap of distributions of homozygotes is not large.     

Genetic mechanisms of age regulation of protein C and blood coagulation.

Blood coagulation activity in humans increases with age. We previously identified two genetic elements, age-related stability element (ASE; GAGGAAG) and age-related increase element (AIE; unique stretch of dinucleotide repeats), which were responsible for age-related stable and increasing expression patterns, respectively, and together recapitulated normal age regulation of the human factor IX (hFIX) gene. Here we report the age-regulatory mechanisms of human anticoagulant protein C (hPC), which shows an age-stable pattern of circulatory levels. The murine protein C gene showed an age-related stable expression pattern in general agreement with that of the hPC. Through longitudinal analyses of transgenic mice carrying hPC minigenes, the hPC gene was found to have a functional age-related stability element (hPC ASE; CAGGAAG) in the 5'-upstream proximal region but was found to lack any age-related increase element. Three other ASE-like sequences present in the hPC gene, GAGGAAA and (G/C)AGGATG, also bound nuclear proteins but were not active in the age regulation of the hPC gene. Functional hPC ASE and hFIX ASE were apparently generated through convergent evolution, and hFIX ASE can fully substitute for the hPC ASE in conferring age-related stable expression pattern of the hPC gene. In the presence of the hPC ASE, hFIX AIE can convert the age-stable expression pattern of the hPC gene to a hFIX-like age-related increase pattern. These results support the universality of ASE and AIE functions across different genes. Clearance of hPC protein from the circulation was not significantly affected by age. We now have established the basic mechanisms responsible for the age-related increase of blood coagulation activity.