Electrochemical investigation of the effect of some organic phosphates on haemoglobin

The effects of DPG, IHP, GTP, GDP and GMP on the structure and stability of haemoglobin were electrochemically investigated with an iodide-modified silver electrode in 0.01 M KNO₃ at pH 7.0. Anodic and cathodic peaks of haemoglobin were observed at 250 mV and 12 mV with a formal potential value of 133 mV vs. Ag/AgCl. The effects of different concentrations of DPG, IHP, GTP, GDP and GMP on the anaerobic redox reaction were determined. The results showed that DPG and IHP can lead to a positive shift in the reduction peak of haemoglobin, indicating that the oxidation peak shift of haemoglobin was small as a result of stabilization of the reduced state and destabilization of the R-like state of haemoglobin. GTP elicited a more positive shift in the cathodic and anodic peaks of haemoglobin at a higher concentration, signifying that it has a low-affinity binding site on haemoglobin. The positive shift of the cathodic and anodic peaks revealed a slight variation in the structure and indicated the unfolding of haemoglobin in the presence of high concentrations of GTP. Our study also showed that GDP and GMP did not cause significant shift the cathodic and anodic peaks of haemoglobin even at high concentrations, refuting the existence of specific GDP-and GMP-binding sites on the protein. Moreover, the iodide-modified silver electrode method proved to be easy and useful in investigating the effects of ligands or other effectors on haemoglobin in solution.     

Autologous transfusion of erythrocytes with high or low concentration of 2,3-diphosphoglycerate. Survival rate and time and hemoglobin-oxygen affinity]

1. In human erythrocytes the 2.3 DPG concentration was increased three to fourfold of the norm as IPP re-suspension by an incubation time of four hours at 37 degrees C or as ACD-AG blood was lowered below 20% of the norm respectively. After an autologous transfusion the 24 hours' surviving rate and the apparent half survival time of cells as well as the affinity of haemoglobin to oxygen in the total blood were measured. 2. The 24 hours' surviving rate for fresh erythrocytes with increased 2.3 DPG and ATP concentration amounts to 73% and the apparent half survival time amounts to 6 days. If erythrocytes are stored for four weeks as IPP resuspension at 4 degrees C, the 24 hours' surviving rate is 59%. Erythrocytes from fresh ACD-AG blood with lowered 2.3 DPG and a normal ATP concentration have a 24 hours' surviving time of 85% and an apparent half survival time of 24 days. 3. After autologous transfusion of 400 ml of erythrocytes with increased 2.3 DPG concentration the P50 value of the total blood will increase by 3 mm of Hg, after administering 400 ml of erythrocytes with lowered 2.3 DPG concentration it will fall by 1.8 mm of Hg. 4. The findings are discussed in connection with the significance of the changes of affinity of haemoglobin to oxygen produced by 2.3 DPG for the oxygen supply of tissues and under the aspect of using stored blood with increased 2.3 DPG concentration for practical purposes.     

Influence of cerebral spinal fluid on the affinity of human red cells for oxygen

The oxygen affinity of washed red cells suspended in their own plasma or in CSF has been studied to demonstrate a possible effect of CSF on the oxygen affinity of human haemoglobin. The CSF was found to reduce the oxygen affinity of haemoglobin significatively, and this was not imputable to the action of pH, PCO2, temperature, 2,3DPG A hypothesis to explain the data found and their clinical interest towards the regulation of brain oxygenation was formulated considering the Monod-Wyman-Changeux model and the effect of solutions on proteins.     

The function of hemoglobins

Haemoglobin is primarily responsible for the oxygen supply and a normal acid-base economy in mammals. An optimal loading of haemoglobin with oxygen in the lungs and a high desaturation in the periphery are ensured by a highly cooperative oxygen binding of 4 subunits as well as by different effectors--2,3 DPG, chloride, pH, temperature...-.A number of differently built-up haemoglobins will ensure a sufficient oxygen supply of individual ontogenetic stages of development in the human organism.     

Oxygen transport to tissue modified by entrapment of an allosteric effector of haemoglobin in erythrocytes

Oxygen affinity of haemoglobin is modulated by several parameters such as the allosteric effector 2-3 DPG for most mammalians. Inositol hexaphosphate (I.H.P.) exerts the same effect on haemoglobin. A previously developed new methodology for the entrapment of drugs into erythrocytes has been adapted to I.H.P.; it is based on a reversible osmotic shock. I.H.P. loaded red blood cells have characteristics very similar to those of native cells. The decrease in oxygen affinity is related to the dose of encapsulated I.H.P. In piglets, transfusion of such cells has led to an increase of oxygen extraction from haemoglobin. Normal anesthetized animals regulate their oxygen consumption by reduction of cardiac output.     

Diphosphatidylglycerol reactivation of lipid-depleted beef heart cytochrome c oxidase: effect of subunit III removal

Beef heart cytochrome c oxidase can be fully delipidated by Triton X-100 using an alkaline pH, high ionic strength incubation followed by glycerol gradient centrifugation at pH 8 in 1% Triton X-100. Unfortunately, this procedure removes not only the 2-3 diphosphatidylglycerol (DPG) molecules that are tightly bound to each heme aa3 complex, but also removes subunit III. A one-to-one correlation exists between the percent of subunit III removed and the percent of tightly bound DPG extracted, suggesting a possible tight binding of these two molecules. However, based upon regeneration of most of the electron transport activity of the subunit III deficient complex by exogenous DPG, it appears that the functional DPG binding site must be located on a subunit other than subunit III.     

Spectrin interactions with globin chains in the presence of phosphate metabolites and hydrogen peroxide: implications for thalassaemia

We have shown the differential interactions of the erythroid skeletal protein spectrin with the globin subunits of adult haemoglobin (HbA); these indicate a preference for alpha-globin over that for beta-globin and intact HbA in an adenosine 5'-triphosphate (ATP)-dependent manner. The presence of Mg/ATP led to an appreciable decrease in the binding affinity of the alpha-globin chain to spectrin and the overall yield of globin-spectrin cross-linked complexes formed in the presence of hydrogen peroxide. Similar effects were also seen in the presence of 2-,3-diphosphoglycerate (2,3 DPG), the other important phosphate metabolite of erythrocytes. The binding affinity and yield of cross-linked high molecular weight complexes (HMWCs) formed under oxidative conditions were significantly higher in alpha-globin compared with intact haemoglobin, HbA and the beta-globin chain. The results of this study indicate a possible correlation of the preferential spectrin binding of the alpha-globin chain over that of the beta-globin in the haemoglobin disorder beta-thalassaemia.     

Further improvement of the ACD-AG protective solution for blood. III. Improvement of the oxygen transport function of erythrocytes in sorbitol xylitol pyruvate solutions with elevated Ph]

If the pH value in the ACD or in the ACD-AG storage solution is enhanced, the glucose in the autoclaving with undergo a caramelizing process. For this reason glucose was replaced by sorbite in the storage solutions with a pH value of 6.0 and additions of xylitol and pyruvate. The initial pH value in the blood amounted to 7.3. The content of 2.3 DPG of the erythrocytes remained fully preserved in the blood with sorbitol and additions of xylitol and pyruvate during the first 2 weeks of storage and decreased to 30% only in the third week. There were only slight amounts of 2.3 DPG in the ACD-AG blood at that time of storage. Up to the third week of storage the ATP content of erythrocytes as well as the haemoglobin level in the plasma revealed no essential differences between stored blood with sorbitol and xylitol as a substrate or glucose + xylitol respectively. The quick decrease of the ATP level to zero and the simultaneous strong increase of haemolysis in the sorbitol blood within the fourth week of storage is discussed in connection with a lowering of the NAD/NADH2 quotient. For the purpose of keeping the 2.3 DPG level of erythrocytes a storage solution with sorbite and xylitol (ASCX-AG-Pyr 10mM) seems to be well suited for a storing time of 2---3 weeks at first.     

An expanded two-state allosteric model for interactions of human hemoglobin A with nonsaturating concentrations of 2,3-diphosphoglycerate

Oxygen binding curves (OEC) for red cell suspensions have a biphasic shape and reduced n50 values when the concentration of 2,3-diphosphoglycerate (DPG) is lowered by aging or experimental procedures. The mechanism for the abnormal shape of the OEC has been related to variations in the activity of free DPG. DPG binds to tetrameric Hb at a single site, and in red cells its normal concentration is equivalent to that of tetrameric Hb. This equivalence renders the oxygen affinity of Hb and the shape of the OEC very sensitive to small changes in the activity of DPG. The OEC for stripped Hb solutions in the presence of nonsaturating concentrations of DPG also exhibit a biphasic shape but with much larger changes in the n values than observed for red cells. Upon addition of chloride, a known competitor of DPG binding to Hb, the shape of the OEC becomes similar to that of red cell suspensions with the same DPG/Hb ratio. Studies on Hb solutions in the presence of varying concentrations of DPG, but without chloride, have revealed that the cofactor shifts the entire OEC to the right, including both its upper and lower asymptotes. This finding indicates that DPG lowers the intrinsic oxygen affinity for both the T and R states. Theoretical considerations leading to a successful modeling of OEC obtained under varying conditions of DPG and chloride require an expanded two-state allosteric model in which allowance is made for DPG-dependent variations in the dissociation constants of oxygen for both the T and R conformations.     

Biochemical and phenotypical correction of an envelope mutant of Salmonella typhimurium

Salmonella typhimurium DA 361 bears an env D1 mutation with the following abnormal phenotypical and biochemical characteristics: a) it autolyses at stationary phase in nutrient broth; b) it grows in chains of short rods; c) it is a poor maltose fermenter and d) it has a diphosphatidylglycerol (DPG) content twice as high than its isogenic non-lytic pair DA 362 (env D+) and LT2, of which both are derivatives. Growth of DA 361 in the presence of 400 mM ethanol leads on a 50% decrease of DPG level, thereby equalling its PG/DPG ratio with those of the control strain. Consequently, a correction on the other phenotypical and biochemical anomalies are induced since the DA 361 strain decreases its autolytic activity, ferments normally maltose and appear as rods undifferentiated from DA 362.     

Influence of allosteric effectors and temperature on oxygen binding properties and the Bohr effect of bovine hemoglobin

The O2 binding properties of bovine Hb were examined. The increase in Cl- and DPG concentration enhanced P50. A reduction in n(max) was observed at high Cl- concentration, while DPG had little effect on n(max). An increase in Cl- concentration enhanced the Bohr effect, the magnitude of which reached a maximum at 0.1 M Cl- and 20 degrees C. This concentration is nearly equal to that at the highest slope of the log P50 vs. log [Cl-] plot, and also equal to the physiological Cl- concentration (0.1 M) of bovine blood. Furthermore, the influence of Cl- concentration on the Bohr effect is independent of temperature. On the other hand, in the absence of Cl-, bovine Hb is sensitive to DPG; an increase in DPG concentration enhanced the Bohr effect, which reached a maximum at 3 mM DPG and 20 degrees C. This concentration is nearly equal to that at the highest slope of the log P50 vs. log [DPG] plot. At low DPG concentrations, the DPG effect on the Bohr effect became small with increasing temperature, whereas at high DPG concentrations, the DPG effect was insensitive to temperature changes. At the physiological concentration of DPG (0.5 mM), increases in both Cl- concentration and temperature diminished the DPG effect. At the physiological concentrations of Cl- and DPG, the Bohr effect was -0.36 at 37 degrees C. The deltaH value at the physiological concentrations of Cl- and DPG was approximately -5.8 kcal/mol at pH 7.4. These results indicate that Cl- and temperature are important determinants of the O2 binding properties of bovine Hb.     

Organic phosphates increase the solubility of avian haemoglobin D and embryonic chicken haemoglobin.

The isolated minor haemoglobin fractions (haemoglobin D) of ostrich, chicken and duck haemoglobin, which constitute about 30% of total intracellular haemoglobin, form crystalline aggregates upon deoxygenation at physiological temperature, ionic strength and pH and at haemoglobin concentrations even well below those present in the red cell. The aggregation is reversed by oxygenation, and can be inhibited by addition of organic phosphates or the corresponding major haemoglobin fraction in a stoichiometric ratio of 1:1. Embryonic haemoglobin from chicken has similar characteristics with respect to its solubility. The results indicate close functional homology of alpha D and embryonic pi-chains as well as a novel role for organic phosphates in the regulation of haemoglobin function.     

Low-temperature sensitivity and enhanced Bohr effect in red deer (Cervus elaphus) haemoglobin: a molecular adaptive strategy to life at high altitude and low temperature.

A study of the functional properties of haemoglobin from red deer (Cervus elaphus) whose habitat varies over a wide range of latitude, was performed. The oxygen-binding properties of the most common haemoglobin phenotype from the species living in Sardinia were examined with particular attention to the effect of pH, chloride, 2, 3-bisphosphoglycerate and temperature. Results indicate that red deer haemoglobin, like all haemoglobins from ruminants so far examined, is characterized by a low intrinsic oxygen affinity, with chloride being its main physiological modulator in vivo. The functional results and the low temperature sensitivity of the oxygen affinity are discussed in the light of the amino acid sequence of closely related ruminant haemoglobins.     

Organic phosphates in the red blood cells of fish

Fish are dependent on aerobic metabolism. They respond to changes in oxygen availability by a wide spectrum of compensatory and respiratory adjustments to safeguard tissue oxygenation. Such adjustments are directed to facilitate both oxygen uptake at the gas exchange surfaces and oxygen unloading to tissues. The importance of erythrocytic organic phosphates as regards oxygen transfer has been recognised since 1967 when the 'dramatic' effect of 2,3DPG on human haemoglobin was first reported. The present review examines the appearance of all the major erythrocytic organic phosphates during the evolutionary radiation of fish. In addition, it provides examples illustrating qualitative and quantitative ontogenetic changes of organic phosphates in the red blood cell of several fish species and describes their effects on oxygen affinities. The interaction of the organic phosphates with haemoglobins and divalent cations are also examined. Of particular interest is the regulation of erythrocytic organic phosphates according to both environmental and physiological conditions.     

Phospholipid composition and metabolism of crustacean gills as related to changes in environmental salinities: relationship between Na+-K+-ATPase activity and phospholipids

The phospholipid composition and metabolism are studied in crustacean gills. It is reported that branchiae are rich in PC, PE and DPG and abundant in long chain polyunsaturated fatty acids (20:4 omega 6 and 20:5 omega 3 acids). The pathways of phospholipids synthesis appear similar to those described for vertebrates. It is demonstrated that there exist significant differences in the level of phosphatides between the anterior and posterior gills of Eriocheir sinensis. No matter what the salinity, the three more posterior located gills of Chinese crabs are shown to contain much more unsaturated phospholipids (PE and DPG). This is particularly true when animals are acclimated to dilute media. Moreover, lipids of posterior gills appear more fluid than the anterior ones as reported by the values of the degree of fluorescence polarization and the index of unsaturation of fatty acids. It is suggested that these lipid changes may indicate the existence of a functional difference between the various branchiae of euryhaline Eriocheir sinensis with respect to their ability to transport salt. It is shown that the renewal of DPG and PS is increased in posterior gills isolated from freshwater Chinese crabs. It is postulated that the enhanced formation of DPG in posterior gills is an indicator of an increased synthesis of mitochondria having as principal function to produce the necessary energy for the Na+ uptake. An attempt is made to correlate the PS metabolism and the Na+-K+-ATPase activity which is particularly located in the mitochondrial fractions of the three pairs of posterior gills.     

Changes of P50 in hypoxaemia, hypercapnia and polycythaemia: multivariate analysis

Multivariate analysis of P50 changes in hypoxia, hypercapnia and polycythaemia was performed in an heterogeneous group of forty three patients: hypoxic subjects with or without hypercapnia, with or without polycythaemia and polycythaemic subjects without hypoxia. A statistical analysis was undertaken using comparison of the means, study of the correlations, principal component analysis, multiple regression and correspondence analysis. In the patients studied, P50 changes were not wholly explained by those of 2-3 DPG and pH; PaCO2, per se, did not play an important part. Haemoglobin concentration and P50 value would represent an adaptative mechanism to hypoxia: when hypoxia is moderate (80 greater than PaO2 greater than or equal to 65 torr) and isolated, oxygen haemoglobin affinity decreases (P50 increases); when hypoxia is severe (PaO2 less than 65 torr) and combined with hypercapnia and disturbed acid-base equilibrium, P50 comes back to normal range but haemoglobin increases, restoring thus, the normal blood oxygen content.     

A comparative study of glycosylated haemoglobin level in the Arabian camel (Camelus dromedarius) during different seasons

1. The extent of haemoglobin glycosylation from 60 camels has been determined (4.39%) in blood samples drawn during winter. 2. Phosphate (9.45 mg/dl), DPG (2.9 mumol/ml) and glucose (138 mg/dl) levels were also recorded. 3. In addition the P50 at pH 7.4 was measured (22.8 Torrs). 4. The data obtained compared with human blood levels and with levels reported for camels during summer sampling. 5. Despite the fact that camels have higher blood glucose levels than humans, the extent of glycosylation is much less in camel blood than in human blood.     

Biochemical parameters to assess viability of blood stored for transfusional use

Blood collected in citrate-phosphate-dextrose-adenine (CPDA-1) containing transfusional bags, was weekly tested throughout a 35 day period. Biochemical assays included plasmatic glucose, electrolytes, free Hb, acid-base balance and hemogasanalysis. Intraerythrocytic ATP and 2,3 DPG were also determined. Results show that an almost total 2,3 DPG depletion occurs during the first three weeks, whereas intracellular ATP are about 50% of the initial values, at the same time. Lowering of pH is also maximal at the third week. pCO2 variation pattern is biphasic: an early increase due to HCO3- titration by lactic acid arising from red cells glycolysis, followed by a decrease probably due to plastic bag permeability to CO2 itself. The percentage of O2Hb also rises during blood storage: this might be the combined result of increase in pO2 and decrease in 2,3 DPG content. A rise of free Hb was obtained; extracellular K+ levels underwent a sixfold increase in 35 days. The mechanism of relative variations of these parameters and the gas transport are discussed. Some of these parameters might be used as routine controls to asses viability and functional status of stored red cells for transfusional use.     

Identification of a Major Locus Contributing to Erythrocyte 2,3-Diphosphoglycerate Variability in Hooded (Long-Evans) Rats

The erythrocyte glycolytic intermediate 2,3-diphosphoglycerate (DPG) and adenosine triphosphate (ATP) play an important role in oxygen transport and delivery by binding to hemoglobin (Hb) and reducing its affinity for oxygen. Considerable quantitative variability in the levels of DPG and ATP exists in human populations and in a population of hooded (Long-Evans) rats we have studied. This paper presents the results of studies on the genetic component of DPG-level variation in an outbred population of hooded rats. Beginning with about 100 rats, a two-way selection experiment was initiated. Pairs of rats with the highest DPG levels were mated to produce a High-DPG rat strain and animals with the lowest DPG levels were mated to produce a Low-DPG strain. Mean DPG levels responded rapidly to selection and, from generation 3 on, the differences between strain means were highly significant. Ten High-DPG strain rats were intercrossed with 10 Low-DPG strain rats of generation 10 to produce an F₁ generation in which the DPG levels were almost as high as those of High-DPG animals. This indicates partial dominance of High-DPG alleles. The F₂ DPG-level distribution showed two distinct subpopulations. The high DPG subpopulation contained three times as many animals as the low DPG subpopulation. From these results and the statistical analyses performed, it was concluded that the DPG differences between strains were due to an allelic difference at one major locus, the allele carried by the High-DPG strain showing partial dominance over the allele carried by the Low-DPG strain. It appears that this locus may also effect ATP levels to a large extent and is polymorphic in hooded rat populations. Identification of this locus gives us a useful tool for studies of the physiological effects of DPG variability, as well as providing an example of a major gene effect in a quantitatively varying trait.     

Analysis of the interaction of organic phosphates with hemoglobin.

The interaction of organic phosphates with hemoglobin is studied by use of a simple thermodynamic approach. A model-independent analysis is employed to evaluate the accuracy of Adair constants determined in the presence of 2,3-diphosphoglycerate (DPG). The change of oxygen affinity in the presence of phosphates is related to the macroscopic phosphate binding constants of oxy- and deoxyhemoglobin and used to extract such binding constants from oxygen equilibrium measurements. The change of the Bohr effect in the presence of phosphates and the competitive binding of carbon dioxide and DPG are treated quantitatively. The binding of organic phosphates is incorporated into an allosteric model, in which the effect of phosphate on both tertiary and quaternary structure changes is included. By use of this model, the factors which can be responsible for the increased functional heterogeneity of alpha and beta chains in the presence of phosphates are clarified.