2001 Lemieux Award Lecture Organic chemistry and hemoglobin: Benefits from controlled alteration

2002 ◽  
Vol 80 (3) ◽  
pp. 217-221 ◽  
Author(s):  
Ronald Kluger
Keyword(s):  
Organic Chemistry ◽  
Nitric Oxide ◽  
Blood Pressure ◽  
Oxygen Affinity ◽  
Red Cells ◽  
Red Cell ◽  
Native Protein ◽  
Cross Link ◽  
Cross Links ◽  
Internal Electron

Hemoglobin carries oxygen in circulation within red cells but does not function outside the cells because it fails not only to release oxygen but also dissociates into dimers that make up the tetrameric protein. Bifunctional anionic acylating agents that contain a structurally rigid bridge introduce cross-links that stabilize hemoglobin and alter its oxygen affinity so that it could be used to carry oxygen outside cells. Nitric oxide binds to hemoglobin and in circulation this causes undesirable increases in blood pressure. It had been reported that higher weight collections of hemoglobin do not cause vasoconstriction. Reagents with two pairs of reaction sites joined by a rigid link connect and cross-link two hemoglobins. The resulting bis-tetramers lack the cooperativity of the native protein and bind oxygen too tightly to be useful; occupation by oxygen blocks the sites from nitric oxide. Nitric oxide may be delivered from thionitrosyl groups, which occur in hemoglobin in the red cell. Cross-linked hemoglobin can be specifically nitrosylated. These species can then serve as circulating sources of nitric oxide resulting from an internal electron transfer.Key words: proteins, hemoglobin, cross-link, red cells, cooperativity, connecting.

Nitrite and Red Cell Function in Carp: Control Factors for Nitrite Entry, Membrane Potassium Ion Permeation, Oxygen Affinity and Methaemoglobin Formation

1990 ◽  
Vol 152 (1) ◽  
pp. 149-166 ◽  
Author(s):  
FRANK B. JENSEN
Keyword(s):  
Cell Function ◽  
Oxygen Affinity ◽  
Red Cells ◽  
Red Cell ◽  
In Vitro Experiments ◽  
K Uptake ◽  
The Mean ◽  
K Release

Red cell function was studied in carp by a combination of in vivo and in vitro experiments with nitrite as the perturbing agent. In vivo accumulation of nitrite caused a marked increase in the red cell methaemoglobin content, and reduced the mean cellular volume. The oxygen affinity of unoxidized haemoglobin was strongly decreased, partly as result of the elevated concentration of cellular nucleoside triphosphates and haemoglobin associated with red cell shrinkage. Red cell pH was unchanged compared to controls, but reduced when referred to constant extracellular pH and O2 saturation. The mean cellular K+ content decreased, reflecting a K+ loss from the red cells during their shrinkage. This K+ loss contributed significantly to the large plasma hyperkalaemia during nitrite exposure. In vitro experiments revealed that nitrite influx into deoxygenated red cells was much larger than into oxygenated red cells. Nitrite permeation of the red cell membrane was not inhibited by DIDS and did not change extracellular pH. Methaemoglobin (MetHb) formation was more pronounced in deoxygenated blood than in oxygenated blood, but quasi-steady states were reached, reflecting a balance between nitrite-induced MetHb formation and the action of MetHb reductase. Red cells incubated in the oxygenated state released K+, whereas a net K+ uptake occurred in deoxygenated cells. Nitrite did not change the K+ loss from oxygenated cells, but shifted the K+ uptake in deoxygenated cells to a pronounced K+ release by the time high MetHb levels were reached. Both types of red cell K+ release were inhibited by DIDS and appeared to occur via a route involving Band 3. The data are consistent with the hypothesis that a significant DIDS-sensitive K+ efflux from the red cells occurs whenever a large fraction of the haemoglobin molecules assumes an R-like quaternary structure.

Restoring Oxygen Carrying Capacity of Sickle Erythrocytes with Nitric Oxide Donors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3785-3785
Author(s):  
Borys Hrinczenko
Keyword(s):  
Nitric Oxide ◽  
Oxygen Affinity ◽  
Red Cell ◽  
No Donors ◽  
Sickle Erythrocytes ◽  
Low Levels ◽  
Pre Treatment ◽  
Normal Controls

Abstract Sickle cell anemia (SCA) is an inherited blood disorder of hemoglobin function. A genetic mutation results in the substitution of a valine for glutamic acid residue at position 6 of the beta-globin chain yielding the mutant hemoglobin S (HbS). HbS polymerizes within erythrocytes during deoxygenation resulting in altered affinity of oxygen binding. The slightly different P50 (PO2 at which Hb is half-saturated with oxygen) values of sickle erythrocytes obtained during either oxygenation or deoxygenation (hysteresis) demonstrate HbS polymerization induced inhibition of oxygen affinity. Nitric oxide (NO) has been found to be an important signaling molecule in the circulatory system. NO derivatives of Hb provide insights into the physiological role of Hb. NO can bind to Hb at either the heme moiety forming nitrosylhemoglobin (HbNO) or to the conserved beta-93 cysteine yielding S-nitrosohemoglobin (SNO-Hb). In deoxygenated venous blood NO preferentially binds to the hemes of Hb forming HbNO while in oxygenated arterial blood NO binds to the beta-93 cysteine residues forming SNO-Hb. Increased oxygen affinity is seen in both SNO-Hb (Bonaventura C, et al, 1999) and also with HbNO. Decreasing the HbS P50 inhibits intra-erythrocyte HbS polymerization that may be an effective strategy to treat SCA. Clinical trials of NO breathing effects on oxygen affinity are conflicting. One study found an increased oxygen affinity of blood from SCA patients breathing 80 ppm NO with no effect seen in normal controls (Head A, et al, 1997). Another study found that levels of NO bound to Hb are too low to affect overall oxygen affinity (Gladwin M, et al, 1999). The purpose of this in vitro study was to determine the oxygen affinity of deoxygenated sickle erythrocytes pre-treated with exogenous NO donors. Blood from SCA (HbSS) and normal controls (HbAA) were collected and suspended in PBS buffer and deoxygenated with argon gas. The Hb concentration of each sample was calculated and then was either left untreated (control) or treated with varying concentrations of NO donors. The NO donors included: 2-(N, N-diethylamino)-diazenolate-2-oxide (DEANO), S-nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside (SNP), an aqueous solution of NO, and sodium trioxodinitrate (Angeli’s salt, AS). Methemoglobin and protein degradation were negligible. Samples were then transferred via airtight syringes into a stirred and temperature controlled (37°C) chamber of PBS solution at ambient oxygen pressure fitted with a very sensitive oxygen electrode. Oxygen levels were measured in real time. The amount of oxygen extracted from the PBS medium followed first order kinetics. Studies with HbSS red cell suspensions showed that the largest increment in oxygen extraction from the medium was obtained with DEANO pre-treatment. Calculations indicated that low levels of NO treatment, at approximately a 1:1000 ratio of [NO]/[heme], yielded the largest oxygen consumption. The effects of pre-treatment with the other NO donors on sickle erythrocytes (HbSS) were not as pronounced. DEANO is an NO donor yielding a “pure” NO radical as opposed to other redox forms. Similar studies with HbAA and HbSC did not show increases in oxygen extraction. Taken together the data suggest that low levels of NO perturb the quaternary structure of intraerythrocyte HbS polymer allowing depolymerization and increased oxygen affinity. The hope is that these in vitro studies will better characterize the role of NO in its interactions with Hb and the red cell and to use this knowledge for potential therapies in diseases such as SCA.

scholarly journals Influence of intramolecular cross-links on the molecular, structural and functional properties of PEGylated haemoglobin

2007 ◽  
Vol 402 (1) ◽  
pp. 143-151 ◽  
Author(s):  
Tao Hu ◽  
Belur N. Manjula ◽  
Dongxia Li ◽  
Michael Brenowitz ◽  
Seetharama A. Acharya
Keyword(s):  
Functional Properties ◽  
Oxygen Affinity ◽  
Direct Consequence ◽  
Molecular Properties ◽  
Cross Linking ◽  
Hydrodynamic Drag ◽  
Cross Link ◽  
Central Cavity ◽  
Structural And Functional Properties ◽  
Cross Links

The influence of intramolecular cross-links on the molecular, structural and functional properties of PEGylated {PEG [poly(ethylene glycol)]-conjugated} haemoglobin has been investigated. The sites and the extent of PEGylation of haemoglobin by reductive alkylation are not influenced by the presence of an αα-fumaryl cross-link at Lys-99(α). The propylated hexaPEGylated cross-linked haemoglobin, (propyl-PEG5K)6-αα-Hb, exhibits a larger molecular radius and lower colloidal osmotic pressure than propylated hexaPEGylated non-cross-linked haemoglobin, (propyl-PEG5K)6-Hb. Perturbation of the haem microenvironment and the α1β2 interface by PEGylation of haemoglobin is reduced by intramolecular cross-linking. Sedimentation velocity analysis established that PEGylation destabilizes the tetrameric structure of haemoglobin. (Propyl-PEG5K)6-Hb and (propyl-PEG5K)6-αα-Hb sediment as stable dimeric and tetrameric molecules, respectively. The ββ-succinimidophenyl PEG-2000 cross-link at Cys-93(β) outside the central cavity also influences the molecular properties of haemoglobin, comparable to that by the αα-fumaryl cross-link within the central cavity. However, the influence of the two cross-links on the oxygen affinity of PEGylated haemoglobin are very distinct, indicating that the high oxygen affinity of PEGylated haemoglobin is not a direct consequence of the dissociation of the haemoglobin tetramers into dimers. αα-Fumaryl cross-linking is preferred to modulate both oxygen affinity and molecular properties of PEGylated haemoglobin, and cross-linking outside the central cavity could only modulate molecular properties of PEGylated haemoglobin. It is suggested that PEGylation induces a hydrodynamic drag on haemoglobin and this plays a role in the microcirculatory properties of PEGylated haemoglobin.

Arginine Supplementation of Sickle Transgenic Mice Leads to a Rapid Decrease in MCHC and Percent Dense Cells and a Slow Increase When Supplementation Is Discontinued.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3579-3579
Author(s):  
Mary E. Fabry ◽  
Zipora Etzion ◽  
Robert M. Bookchin ◽  
Sandra M. Suzuka ◽  
Ronald L. Nagel
Keyword(s):  
Nitric Oxide ◽  
Transgenic Mice ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
High Density ◽  
Red Cells ◽  
Red Cell ◽  
Red Cell Indices ◽  
Plasma Arginine ◽  
Arginine Supplementation

Abstract Nitric oxide (NO) is produced from arginine by nitric oxide synthase (NOS) and is essential to the maintenance of vascular tone. Plasma arginine levels are reduced in sickle cell anemia patients, and we have previously reported that S+S− Antilles mice have lower plasma arginine than control mice (C57BL). Long term arginine supplementation (5% arginine in chow) restored plasma arginine levels in S+S− Antilles mice to that found in C57BL mice, and, in S+S− Antilles mice, MCHC and the percent high density red cells were reduced. Our observation that Ca++-activated K+ channel [K(Ca) channel or Gardos channel] activity is reduced in supplemented vs non-supplemented S+S− Antilles mice can account for reduced red cell density and dense cell formation (Blood99: 1103, 2002). The time course of arginine induced changes in red cell indices in sickle transgenic mice has not yet been studied. Using the Advia 120 Hematology System, we found that, within 7 days after the onset of arginine supplementation, MCHC* (CHCM in the Advia system) fell from 33.4±0.5 g/dL to 31.0±0.2 (mean ±SD, P<0.000002, N=6) and the percent high density red cells (MCHC>37 g/dL) decreased from 10.5±4.3 % to 4.5±1.6 (P<0.01, N=6). MCV rose from 43.6±0.8 fL to 46.4±0.5 (P<0.00003, N=6). Reticulocyte counts and mean corpuscular hemoglobin did not vary significantly. Although plasma arginine levels increase within a few hours of oral administration and persist for only a few hours, no change in red cell indices was observed 7 days after discontinuing arginine supplementation, and the indices slowly returned to baseline levels after 40 days. In summary, MCHC* and the percent high density RBCs decrease significantly within 7 days of the onset of arginine supplementation. When arginine supplementation is withdrawn, the return to baseline values is much slower and required more than 40 days to complete. These observations have important implications for arginine supplementation in sickle cell anemia patients. In particular, the persistence of decreased MCHC* and reduced percent high density RBCs for an extended period of time after supplementation was withdrawn parallels our previously reported observation of persistent improvement of perfusion in sickle transgenic mice after supplementation was withdrawn and suggests that daily administration of arginine may not be necessary once an induction period has passed.

Evolution of mammalian hemoglobin function

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 189-197 ◽  
Author(s):  
HF Bunn
Keyword(s):  
Oxygen Affinity ◽  
Red Cells ◽  
Red Cell ◽  
Low Oxygen ◽  
Wide Range ◽  
Blood Oxygen Affinity ◽  
Low Levels ◽  
Major Factors ◽  
2,3 Dpg ◽  
Low Oxygen Affinity

Throughout their evolution, mammalian hemoglobins have acquired a broad repertoire of functional properties well suited to the internal milieu of the red cell. Mammals display a wide range in whole blood oxygen affinity dependent on three major factors: the intrinsic oxygen affinity of the hemoglobin, the level of red cell 2,3-DPG, and the response of the hemoglobin to 2,3-DPG. The concentration of 2,3-DPG varies among groups of mammals. Those animals (cats and ruminants) that have very low levels of this intracellular mediator have hemoglobins of intrinsically low oxygen affinity that fail to respond to the addition of 2,3-DPG. Mammals that have adapted to various types of hypoxia tend to have increased oxygen affinity, primarily mediated through reduced levels of red cell 2,3-DPG. In contrast, mammals who are experimentally subjected to low oxygen tensions develop decreased oxygen affinity owing to increased red cell 2,3-DPG. Mammals employ one of three different mechanisms for the maintenance of higher oxygen affinity of fetal red cells, compared to maternal red cells. Many of these phenomena can be satisfactorily explained at the molecular level but their adaptational significance is less clear.

scholarly journals Evolution of mammalian hemoglobin function

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 189-197 ◽  
Author(s):  
HF Bunn
Keyword(s):  
Oxygen Affinity ◽  
Red Cells ◽  
Red Cell ◽  
Low Oxygen ◽  
Wide Range ◽  
Blood Oxygen Affinity ◽  
Low Levels ◽  
Major Factors ◽  
2,3 Dpg ◽  
Low Oxygen Affinity

Abstract Throughout their evolution, mammalian hemoglobins have acquired a broad repertoire of functional properties well suited to the internal milieu of the red cell. Mammals display a wide range in whole blood oxygen affinity dependent on three major factors: the intrinsic oxygen affinity of the hemoglobin, the level of red cell 2,3-DPG, and the response of the hemoglobin to 2,3-DPG. The concentration of 2,3-DPG varies among groups of mammals. Those animals (cats and ruminants) that have very low levels of this intracellular mediator have hemoglobins of intrinsically low oxygen affinity that fail to respond to the addition of 2,3-DPG. Mammals that have adapted to various types of hypoxia tend to have increased oxygen affinity, primarily mediated through reduced levels of red cell 2,3-DPG. In contrast, mammals who are experimentally subjected to low oxygen tensions develop decreased oxygen affinity owing to increased red cell 2,3-DPG. Mammals employ one of three different mechanisms for the maintenance of higher oxygen affinity of fetal red cells, compared to maternal red cells. Many of these phenomena can be satisfactorily explained at the molecular level but their adaptational significance is less clear.

scholarly journals Hemoglobin Function in the Horse: The Role of 2,3-Diphosphoglycerate in Modifying the Oxygen Affinity of Maternal and Fetal Blood

Blood ◽  
1973 ◽  
Vol 42 (3) ◽  
pp. 471-479 ◽  
Author(s):  
H. Franklin Bunn ◽  
Hyram Kitchen
Keyword(s):  
Oxygen Saturation ◽  
Oxygen Affinity ◽  
Fetal Hemoglobin ◽  
Red Cells ◽  
Red Cell ◽  
Fetal Blood ◽  
Atp Levels ◽  
2,3 Dpg

Abstract The blood of the newborn horse was found to have a higher affinity for oxygen than that of the mother. This difference was due to the fact that the red cells of newborn foals contained 36% lower 2,3-diphosphoglycerate (2,3-DPG) than red cells from their respective mares. The ATP levels of foal and maternal red cells did not differ significantly. Following birth a prompt rise in the foal's red cell 2,3-DPG occurred, approaching normal (maternal) levels within 5 days. Unlike many other species, the hemoglobins of the newborn and adult horse have been shown to be structurally identical. Furthermore, phosphate-free solutions of newborn and maternal hemoglobins had identical oxygen saturation curves in the absence and presence of added 2,3-DPG. This study demonstrates that, in contrast to other species. the increased oxygen affinity of horse fetal red cells is due to a lower level of the cofactor 2,3-DPG rather than to the presence of fetal hemoglobin.

Arginine supplementation of sickle transgenic mice reduces red cell density and Gardos channel activity

Blood ◽  
2002 ◽  
Vol 99 (4) ◽  
pp. 1103-1108 ◽  
Author(s):  
José R. Romero ◽  
Sandra M. Suzuka ◽  
Ronald L. Nagel ◽  
Mary E. Fabry
Keyword(s):  
Nitric Oxide ◽  
Cell Density ◽  
Sickle Cell ◽  
T Test ◽  
Red Cells ◽  
K Channel ◽  
Mean Corpuscular Hemoglobin ◽  
Red Cell ◽  
Plasma Arginine ◽  
Arginine Supplementation

Nitric oxide (NO), essential for maintaining vascular tone, is produced from arginine by nitric oxide synthase. Plasma arginine levels are low in sickle cell anemia, and it is reported here that low plasma arginine is also found in our sickle transgenic mouse model that expresses human α, human βS, and human βS-Antilles and is homozygous for the mouse βmajor deletion (S+S-Antilles). S+S-Antilles mice were supplemented with a 4-fold increase in arginine that was maintained for several months. Mean corpuscular hemoglobin concentration (MCHC) decreased and the percent high-density red cells was reduced. Deoxy K+ efflux is characteristic of red cells in sickle cell disease and contributes to the disease process by increasing the MCHC and rendering the cells more susceptible to polymer formation. This flux versus the room air flux was reduced in S+S-Antilles red cells from an average value of 1.6 ± 0.3 mmol per liter of red cells × minute (FU) in nonsupplemented mice to 0.9 ± 0.3 FU (n = 4, P &lt; .02, paired t test) in supplemented mice. In room air, Vmax of the Ca++-activated K+ channel (Gardos) was reduced from 4.1 ± 0.6 FU (off diet) to 2.6 ± 0.4 FU (n = 7 and 8,P &lt; .04, t test) in arginine-supplemented mice versus clotrimazole. In conclusion, the major mechanism by which arginine supplementation reduces red cell density (MCHC) in S+S-Antilles mice is by inhibiting the Ca++-activated K+ channel.

Effect of pregnancy and temperature on red cell oxygen-affinity in the viviparous snake Thamnophis elegans

1991 ◽  
Vol 156 (1) ◽  
pp. 399-406
Author(s):  
R. L. Ingermann ◽  
N. J. Berner ◽  
F. R. Ragsdale
Keyword(s):  
Oxygen Affinity ◽  
Oxygen Demand ◽  
Maternal Blood ◽  
Cell Suspensions ◽  
Red Cells ◽  
Nucleoside Triphosphate ◽  
Diurnal Changes ◽  
Red Cell ◽  
Garter Snakes ◽  
Thamnophis Elegans

The oxygen affinity of red cell suspensions from fetal garter snakes was higher than that of cell suspensions from their mothers. This difference appeared to be due to different concentrations of nucleoside triphosphate (NTP, primarily adenosine triphosphate). NTP concentrations were significantly higher, and oxygen affinities were significantly lower, in red cell suspensions from pregnant females compared with those from nonpregnant females or males; there is no precedent for such a pronounced effect of pregnancy on the oxygen affinity of maternal blood. These data indicate that pregnancy may result in an enhanced ability of adult blood to deliver oxygen to the fetus. Since the binding of organic phosphates and oxygen to hemoglobin is sensitive to temperature, and since these animals experience diurnal changes in temperature, we examined the influence of relatively low (20 degrees C) and high (34 degrees C) temperatures on red cell oxygen-affinity. The temperature increase of 14 degrees C resulted in a lowered oxygen-affinity of all red cell suspensions examined. However, this increase in temperature lowered the affinity of maternal red cells to a greater extent than it did the affinity of fetal red cells. This suggests that daytime temperatures may further enhance the ability of maternal blood to deliver oxygen to the fetus at times when fetal oxygen demand is probably greatest.

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