ATP release from erythrocytes: A role of adenosine1.

BACKGROUND: The oxygen required to meet metabolic needs of all tissues is delivered by the red blood cell (RBC), a small, flexible cell which, in mammals, is devoid of a nucleus and mitochondria. Despite its simple appearance, this cell has an important role in its own distribution, enabling the delivery of oxygen to precisely meet localized metabolic need. When red blood cells enter in hypoxic area, a signalling pathway is activated within the cell, resulting in the release of ATP in amounts adequate to activate purinergic receptors on vascular endothelium, which trigger secretion of nitric oxide and other factors resulting in vasodilatation. OBJECTIVE: The present study investigates the effect of adenosine exposure on this molecular mechanism. METHODS AND RESULTS: We report that RBC in the presence of adenosine in low oxygen conditions, ATP release increase after 24 h exposure. Adenosine induced-ATP release in deoxygenated red blood cell show data similar to that of RBC in high oxygen conditions: (1) RBC after band 3 modification by 4,4'- diisothio-cyanatostilbene- 2,2'-disulphonic acid; (2) CO-treated RBC. In the presence of Sphingosine kinase (SphK1) inhibitor, adenosine mediated effects on ATP release were abolished. Activity of adenylate cyclase increase following to adenosine exposure, on the contrary red cell phosphofructokinase is not modified within the RBC in the presence of adenosine. CONCLUSION: Our data support involvement of band 3/deoxyHb binding and adenylate cyclase in the pathway responsible for ATP release from RBC following exposure to adenosine.

Morphological changes induced in erythrocyte by amyloid beta peptide and glucose depletion: A combined atomic force microscopy and biochemical study.

Circulating red blood cells (RBCs) undergo aging, a fundamental physiological phenomenon that regulates their turnover. We show that treatment with beta amyloid peptide 1-42 (Aß) accelerates the occurrence of morphological and biochemical aging markers in human RBCs and influences the cell metabolism leading to intracellular ATP depletion. The morphological pattern has been monitored using Atomic Force Microscopy (AFM) imaging and measuring the RBCs' plasma membrane roughness employed as a morphological parameter capable to provide information on the structure and integrity of the membrane-skeleton. Results evidence that Aß boosts the development of crenatures and proto-spicules simultaneously to acceleration in the weakening of the cell-cytoskeleton contacts and to the induction of peculiar nanoscale features on the cell membrane. Incubation in the presence of glucose can remove all but the latter Aß-induced effects. Biochemical data demonstrate that contemporaneously to morphological and structural alterations, Aß and glucose depletion trigger a complex signaling pathway involving caspase 3, protein kinase C (PKC) and nitric oxide derived metabolites. As a whole, the collected data revealed that, the damaging path induced by Aß in RBC provide a sequence of morphological and functional intermediates following one another along RBC life span, including: (i) an acceleration in the development of shape alteration typically observed along the RBC's aging; (ii) the development of characteristic membrane features on the plasma membrane and (iii) triggering a complex signaling pathway involving caspase 3, PKC and nitric oxide derived metabolites.

Erythrocytes as Potential Link between Diabetes and Alzheimer's Disease.

Many studies support the existence of an association between type 2 diabetes (T2DM) and Alzheimer's disease (AD). In AD, in addition to brain, a number of peripheral tissues and cells are affected, including red blood cell (RBC) and because there are currently no reliable diagnostic biomarkers of AD in the blood, a gradually increasing attention has been given to the study of RBC's alterations. Recently it has been evidenced in diabetes, RBC alterations superimposable to the ones occurring in AD RBC. Furthermore, growing evidence suggests that oxidative stress plays a pivotal role in the development of RBC's alterations and vice versa. Once again this represents a further evidence of a shared pathway between AD and T2DM. The present review summarizes the two disorders, highlighting the role of RBC in the postulated common biochemical links, and suggests RBC as a possible target for clinical trials.

Methionine 35 sulphoxide reduces toxicity of Aß in red blood cell.

BACKGROUND: The oxidation of methionine residue in position 35 of Ab to sulphoxide (Ab-sulphoxide) has the ability to deeply modify wild-type Ab 1-42 (Ab) neurotoxic action. Our previous studies suggest that in nucleated cells, lower toxicity of Ab-sulphoxide might result not from structural alteration, but from elevation of methionine sulphoxide reductase A (MsrA) activity and mRNA levels. DESIGN: On this basis, we hypothesised that red blood cell (RBC), a cell devoid almost completely of MsrA activity, shares with nucleated cells an antioxidant system induced by methionine 35 sulphoxide, responsible for the lower toxicity of Ab-sulphoxide in RBC. (Results) Supporting this hypothesis, we found that the low toxicity of Ab-sulphoxide in RBC correlated with pentose phosphate pathway (PPP) flux increase, and this event was associated with a low level of methionine oxidation in total proteins. None of these effects were observed when cells were exposed to Ab native. DISCUSSION: These results outline the importance of the redox state of methionine 35 in the modulation of Ab-mediated events and suggest an important protective role for PPP in RBC of patients affected by Alzheimer's disease.

Vascular dysfunction-associated with Alzheimer's disease.

Our attention is focused on the study of a new model based on the red blood cell (RBC) and on its interaction with amyloid beta peptide 1-42 (Aß). RBC are highly deformable to assist blood flow in the microcirculation. For this reasons RBC abnormalities could contribute to Alzheimer's disease (AD) by obstructing oxygen delivery to brain, causing hypoxia. In our work, considering that RBC membrane contains, among blood elements, higher acetylcholinesterase (AChE) levels, we can assume that in blood occurs a mechanism similar to the one which occurs at the neuronal level leading to an increase of Aß toxicity mediated by its binding with AChE, located on the RBC external face. Furthermore, since mechanical properties of RBC membrane are regulated by a number of molecular components of signalling and/or regulatory pathways, of these, particular interest has been addressed toward Nitric Oxide (NO) metabolism, due to its dependence to AChE.

Involvement of acetylcholinesterase and protein kinase C in the protective effect of caffeine against ß-amyloid-induced alterations in red blood cells.

It is well known the role of oxidative stress in the pathophysiology of Alzheimer's disease (AD) and of other neurodegenerative pathologies. We have previously documented that Amyloid beta peptide (1-42) (Abeta) dependent-oxidative modifications affect red blood cell (RBC) morphology and function. Experimental studies show that caffeine (CF) consumption is inversely correlated with AD. In this study, we investigated the role played by RBC in the protective mechanism elicited by CF against Abeta mediated toxicity. PS exposure levels by FACS analysis, as well as protein band 3 functionality analysis, indicated that CF at 100 µM protected against Abeta-mediated membrane alterations, which are known to occur in AD. Moreover, CF counteracts inhibition of ATP release from RBC by Abeta, restoring its ability to modulate vasodilation. Concurrently, analysis of protein kinase C (PKC) and caspase 3 activities, responsible for cytoskeleton alterations, revealed that unlike to caspase 3, PKCα activation induced by Abeta was fully abolished by CF through a mechanism involving Acetylcholinesterase (AChE), located on external face of RBC plasma membrane. These results provide support for the hypothesis concerning the protective role of CF in AD patients could include also a peripheral mechanism involving RBC.

Amyloid beta peptide (1-42)-mediated antioxidant imbalance is associated with activation of protein kinase C in red blood cells.

Glycolysis and pentose phosphate pathway (PPP) in red blood cell (RBC) are modulated by the cell oxygenation state. This metabolic modulation is connected to variations in intracellular nicotinamide adenine dinucleotide phosphate-reduced form (NADPH) and adenosine triphosphate (ATP) levels as a function of the oxygenation state of the cell, and, consequently, it should have physiologic relevance. In the present study, we analysed the effects of amyloid beta peptide (1-42) (Abeta) on RBC metabolism and its relationship with the activity of protein kinase C (PKC). Our results showed that metabolic response to Abeta depended on the degree of cell oxygenation. In particular, under high O2 pressure, in Abeta-treated RBC, glucose metabolized through PPP approached that metabolized by RBC under low O2 pressure, differently to that observed in untreated cells. The effect of Abeta on RBC metabolism was paralleled by increase in PKC enzyme activity, but cytosolic Ca2+ concentration does not seem to be involved in this mechanism. Incubation of Abeta-treated RBC with a specific inhibitor of PKC partially restores PPP flux. A possible rationalization of the different metabolic behaviours shown by RBC following Abeta treatment is proposed. It takes into account the known post-translational modifications to cytoskeleton proteins induced by PKC. The reduction in PPP flux may lead to a weakened defence system of antioxidant reserve in RBC, becoming a source of reactive species, and, consequently, its typical, structural and functional features are lost. Therefore, oxidative stress may outflow from the RBC and trigger damage events in adjacent cells and tissue, thus contributing to vascular damage.

Protein kinase C mediates caspase 3 activation: A role for erythrocyte morphology changes.

We have previously showed that morphological alterations in Red Blood Cells (RBCs) are correlated to an impaired eNOS enzymatic activity and a concomitant reduced NO derived metabolites formation. Here we extend our previous observations, reporting that RBC morphology is regulated by a series of specific cell signaling events linked to Protein Kinase C (PKC)-mediated activation of caspase 3. Pretreatment of RBCs with the PKC inhibitor chelerythrine, prior to the addition of phorbol-12-myristate-13-acetate (PMA), an activator of PKC, blocks the appearance of the morphology alterations and the sustained decrease in nitrates and nitrites levels induced by PMA. Inhibition of PKC also completely inhibits PMA mediated caspase-3 activation. On the other hand, caspase 3 inhibition, lessens the PMA induced-effects on the appearance of RBC morphology alterations, although it enhances PMA-mediated effects on nitric oxide (NO) derived metabolites levels. These data demonstrate that PKC-mediated activation of caspase 3 is an integral and essential part of signaling pathway in RBCs, that may be a regulatory factor of RBC mechanical properties, through regulation of NO metabolism.

NO Metabolites Levels in Human Red Blood Cells are Affected by Palytoxin, an Inhibitor of Na(+)/K(+)-ATPase Pump.

Palytoxin (PTX), a marine toxin, represents an increasing hazard for human health. Despite its high toxicity for biological systems, the mechanisms triggered by PTX, are not well understood. The high affinity of PTX for erythrocyte Na(+)/K(+)-ATPase pump is largely known, and it indicates PTX as a sensitive tool to characterize the signal transducer role for Na(+)/K(+)-ATPase pump. Previously, it has been reported that in red blood cells (RBC), probably via a signal transduction generated by the formation of a PTX-Na(+)/K(+)-ATPase complex, PTX alters band 3 functions and glucose metabolism. The present study addresses the question of which other signaling pathways are regulated by Na(+)/K(+)-ATPase in RBC. Here it has been evidenced that PTX following its interaction with Na(+)/K(+)-ATPase pump, alters RBC morphology and this event is correlated to decreases by 30% in nitrites and nitrates levels, known as markers of plasma membrane eNOS activity. Orthovanadate (OV), an antagonist of PTX binding to Na(+)/K(+)-ATPase pump, was able to reverse the effects elicited by PTX. Finally, current investigation firstly suggests that Na(+)/K(+)-ATPase pump, following its interaction with PTX, triggers a signal transduction involved in NO metabolism regulation.

Antiepileptic carbamazepine drug treatment induces alteration of membrane in red blood cells: possible positive effects on metabolism and oxidative stress.

Carbamazepine (CBZ) is an iminostilbene derivative commonly used for treatment of neuralgic pain and bipolar affective disorders. CBZ blood levels of treated patients are within the range of micromolar concentrations and therefore, significant interactions of this drug with erythrocytes are very likely. Moreover, the lipid domains of the cell membrane are believed to be one of the sites where iminostilbene derivatives exert their effects. The present study aimed to deeply characterize CBZ effects on erythrocytes, in order to identify extra and/or cytosolic cell targets. Our results indicate that erythrocyte morphological changes promoted by the drug, may be triggered by an alteration in band 3 functionality i.e. at the level of anionic flux. In addition, from a metabolic point of view this perturbation could be considered, at least in part, as a beneficial event because it could favour the CO2 elimination. Since lipid peroxidation, superoxide and free radical scavenging activities, caspase 3 activity and hemoglobin (Hb) functionality were not modified within the CBZ treated red blood cell (RBC), band 3 protein (B3) may well be a specific membrane target for CBZ and responsible for CBZ-induced toxic effects in erythrocytes. However some beneficial effects of this drug have been evidenced; among them an increased release of ATP and nitric oxide (NO) derived metabolites from erythrocytes to lumen, leading to an increased NO pool in the vasculature. In conclusion, these results indicate that CBZ, though considered responsible for toxic effects on erythrocytes, can also exhibit effects that at least in some conditions may be seen as beneficial.

Blood presence of circulating oncofetal fibronectin mRNA, by RT-PCR, does not represent a useful specific marker for the management and follow-up of thyroid cancer patients.

BACKGROUND: Recent studies strongly suggest the use of oncofetal fibronectin (onfFN) mRNA in diagnostic follow-up and staging due to its very high specificity for thyroid cancers. Since the use of this marker has not been well established yet, particularly in the monitoring of minimal residual disease, we have tried to verify the diagnostic power of onfFN and its usefulness as a prognostic molecular marker. For this reason, we evaluated (by RT-PCR) the presence of onfFN mRNAs, not only in blood samples and thyroid tissues (both normal and neoplastic), but also in different biological fluids (such as K3-EDTA blood samples, saliva and urine) belonging to healthy individuals. METHODS: Molecular investigations, such as RT-PCR protocol, and sequencing of onfFN cDNAs evaluation of the above-mentioned samples were performed. RESULTS: The onfFN transcript was largely expressed in all benign and malignant thyroid tissues [differentiated thyroid carcinomas (DTCs)] tested as well as in a large number of biological fluids; in particular, 100% urine samples were positive for onfFN transcript as compared to the thyroglobulin (Tg) mRNA (75%), while saliva was always positive for onfFN and never for Tg. These findings indicate that onfFN cannot be considered a marker specific for thyroid cancer presence. Finally, Tg results were positive in a large part of the samples, but not always in concomitance with onfFN. CONCLUSIONS: We underline how the complexity of onfFN transcripts could affect the RT-PCR procedure. In addition, the presence of onfFN transcripts in several normal and cancer tissues, along with non-thyroid biological fluids or cells, does not allow the use of this marker for cancer monitoring.

ß-amyloid decreases detectable endothelial nitric oxide synthase in human erythrocytes: a role for membrane acetylcholinesterase.

Until few years ago, many studies of Alzheimer's disease investigated the effects of this syndrome in the central nervous system. Only recently, the detection of amyloid beta peptide (Aß) in the blood has evidenced the necessity to extend studies on extraneuronal cells, particularly on erythrocytes. Aß is also present in brain capillaries, where it interacts with the erythrocytes, inducing several metabolic and functional alterations. Recently, functionally active endothelial type nitric oxide synthase (eNOS) was discovered in human erythrocytes. The goal of the present study was to evidence the effect of Aß on erythrocyte eNOS. We found that Aß following to 24-h exposure causes a decrease in the immune staining of erythrocyte eNOS. Concurrently, Aß alters erythrocyte cell morphology, decreases nitrites and nitrates levels, and affects membrane acetylcholinesterase activity. Propidium, an acetylcholinesterase inhibitor, was able to reverse the effects elicited by Aß. These events could contribute to the vascular alterations associated with Alzheimer's disease disease.

Modeling the ternary complex TCR-Vbeta/CollagenII(261-273)/HLA-DR4 associated with rheumatoid arthritis.

BACKGROUND: It is known that genetic predisposition to rheumatoid arthritis (RA) is associated with the MHC class II allele HLA-DR4 and that residues 261-273 of type II collagen (huCollp261) represent an immunodominant T cell epitope restricted by the DR4 molecule. Despite recent advances in characterization of MHC and T cell receptor (TCR) contacts to this epitope, the atomic details of TCR/huCollp261/HLA-DR4 ternary complex are not known. METHODOLOGY/PRINCIPAL FINDINGS: Here we have used computational modeling to get insight into this interaction. A three-dimensional model of the TCR Vbeta domain from a DR4(+) patient affected by RA has been derived by homology modeling techniques. Subsequently, the structure of the TCR Vbeta domain in complex with huCollp261/HLA-DR4 was obtained from a docking approach in conjunction with a filtering procedure based on biochemical information. The best complex from the docking experiments was then refined by 20 ns of molecular dynamics simulation in explicit water. The predicted model is consistent with available experimental data. Our results indicate that residues 97-101 of CDR3beta are critical for recognition of huCollp261/HLA-DR4 by TCR. We also show that TCR contacts on p/MHC surface affect the conformation of the shared epitope expressed by DR alleles associated with RA susceptibility. CONCLUSIONS/SIGNIFICANCE: This work presents a three-dimensional model for the ternary complex TCR-Vbeta/collagenII(261-273)/HLA-DR4 associated with rheumatoid arthritis that can provide insights into the molecular mechanisms of self reactivity.

Insight into a novel p53 single point mutation (G389E) by Molecular Dynamics Simulations.

The majority of inactivating mutations of p53 reside in the central core DNA binding domain of the protein. In this computational study, we investigated the structural effects of a novel p53 mutation (G389E), identified in a patient with congenital adrenal hyperplasia, which is located within the extreme C-terminal domain (CTD) of p53, an unstructured, flexible region (residues 367-393) of major importance for the regulation of the protein. Based on the three-dimensional structure of a carboxyl-terminal peptide of p53 in complex with the S100B protein, which is involved in regulation of the tumor suppressor activity, a model of wild type (WT) and mutant extreme CTD was developed by molecular modeling and molecular dynamics simulation. It was found that the G389E amino acid replacement has negligible effects on free p53 in solution whereas it significantly affects the interactions of p53 with the S100B protein. The results suggest that the observed mutation may interfere with p53 transcription activation and provide useful information for site-directed mutagenesis experiments.

A new beta-chain haemoglobin variant with increased oxygen affinity: Hb Roma [beta115(g17)Ala-->Val].

BACKGROUND: Haemoglobin Roma [beta115(G17)Ala-->Val] is a new adult haemoglobin variant found in a patient presenting a mild hypochromia and microcytosis. We studied this previously uncharacterised variant in order to evaluate the effect on the structural and funcional properties of the Ala-->Val substitution at the alpha1beta1 interface. METHODS AND RESULTS: The variant chain was identified by direct DNA sequencing of the beta-globin gene, which revealed a GCC-->GTC mutation in codon 115. This mutation was confirmed by mass spectrometric analysis of the tetramers and peptides. The oxygen-binding properties of the haemoglobin A/haemoglobin Roma mixture, in which the variant makes up 25% of the haemoglobins, showed a significant increase in oxygen affinity with respect to normal haemoglobin A, both in the absence and presence of 2,3-bisphosphoglycerate. The role of the betaG17 position, situated at the alpha(1)beta(1) interface, has been examined using computational models of haemoglobin Roma and other known betaG17 variants, in comparison with normal haemoglobin A. CONCLUSIONS: This study suggests that the beta115(G17)Ala-->Val substitution at the alpha1beta1 interface is responsible for increased oxygen affinity and mild destabilisation of the haemoglobin Roma. GENERAL SIGNIFICANCE: An amino acid substitution at the G17 position of the alpha1beta1 interface may result in stabilisation of the high affinity R-state of the haemoglobin molecule.

Beta2-strand of salivary S cystatins: a "chemeleon sequence".

Secondary structure prediction of salivary cystatins S, SA, and SN carried out by several methods label the 39-58 sequence (beta2-strand) as predominantly alpha-helical. The helical propensity of a peptide corresponding to beta2-strand of salivary SA cystatin analyzed by CD display high helical propensity in aqueous solution, whereas peptides matching the beta2-strand amino acid sequence of cystatins S and SN, display random coil conformation in aqueous solution but acquire alpha-helical conformation in the presence of trifluoroethanol (TFE). Moreover molecular dynamics simulation performed on the homology modeling of cystatin SA constructed on the basis of recently determined three-dimensional structure of salivary cystatin D, suggests that cystatin SA does not significantly deviate from the starting structure over the course of the simulation. The results obtained indicate that the beta2-strand of salivary S cystatins has high helical propensity when isolated from native protein and acquire the final beta structure by interaction with the rest of the polypeptide chain.

Two novel CYP21A2 missense mutations in Italian patients with 21-hydroxylase deficiency: Identification and functional characterisation.

Steroid 21-hydroxylase deficiency is present in more than 90% of patients with congenital adrenal hyperplasia (CAH), an inherited metabolic disorder of adrenal steroidogenesis. Impaired enzymatic activity leads to the accumulation of metabolic intermediates (progesterone and 17-hydroxyprogesterone), which results in excessive androgen production and varied signs of virilisation. CYP21A2 is an active gene and encodes for the steroid 21-hydroxylase enzyme, whereas CYP21A1P is an inactive pseudogene that contains a series of deleterious mutations. The major part of disease- causing mutations in CYP21A2 alleles are CYP21A1P-derived sequence transferred to the active gene by macro or microconversion events. Only around 5% of all disease-causing CYP21A2 alleles harbour rare mutations that do not originate from the pseudogene. In this report, we report the characterisation of two novel CYP21A2 missense mutations (p.H119R and p.I194N) found in two unrelated Italian patients with nonclassic (NC) CAH clinical diagnosis. Functional in vitro assays for mutagenized CYP21 enzymes were performed in transiently transfected mammalian cells to test the residual enzyme activity and the apparent kinetic values. The residual activities obtained for p.H119R and p.I194N mutants allowed to classify them as NC-CAH associated mutations. These results correlate with the rate of severity of the patients' disease. Finally, the new p.H119R and p.I194N mutations should be included in the panel of those already listed for association with the NC form of 21-hydroxylase deficiency.

Haemoglobin polymorphisms affect the oxygen-binding properties in Atlantic cod populations.

A major challenge in evolutionary biology is to identify the genes underlying adaptation. The oxygen-transporting haemoglobins directly link external conditions with metabolic needs and therefore represent a unique system for studying environmental effects on molecular evolution. We have discovered two haemoglobin polymorphisms in Atlantic cod populations inhabiting varying temperature and oxygen regimes in the North Atlantic. Three-dimensional modelling of the tetrameric haemoglobin structure demonstrated that the two amino acid replacements Met55beta1Val and Lys62beta1Ala are located at crucial positions of the alpha1beta1 subunit interface and haem pocket, respectively. The replacements are proposed to affect the oxygen-binding properties by modifying the haemoglobin quaternary structure and electrostatic feature. Intriguingly, the same molecular mechanism for facilitating oxygen binding is found in avian species adapted to high altitudes, illustrating convergent evolution in water- and air-breathing vertebrates to reduction in environmental oxygen availability. Cod populations inhabiting the cold Arctic waters and the low-oxygen Baltic Sea seem well adapted to these conditions by possessing the high oxygen affinity Val55-Ala62 haplotype, while the temperature-insensitive Met55-Lys62 haplotype predominates in the southern populations. The distinct distributions of the functionally different haemoglobin variants indicate that the present biogeography of this ecologically and economically important species might be seriously affected by global warming.

Allosteric properties of hemoglobin and the plasma membrane of the erythrocyte: new insights in gas transport and metabolic modulation.

Within the red blood cell the hemoglobin molecule is subjected to modulation mechanisms, namely homo- and heterotropic interactions, which optimize its functional behavior to the specific physiological requirements. At the cellular level, these modulation mechanisms are utilized to perform a number of other functions that are not minor with respect to the basic function of oxygen transport. Here we report some key examples concerning: (i) the interaction of hemoglobin with band 3 and its influence on glucose metabolism; (ii) the role of the ligand-linked quaternary transition of hemoglobin in the control of "NO bioactivity" and of gas diffusion; (iii) the interaction of plasma membrane with the various oxidative derivatives of the hemoglobin molecule.

Hb Santa Clara (beta 97His-->Asn), a human haemoglobin variant: functional characterization and structure modelling.

This study examines the functional and structural effects of amino acid substitution at alpha(1)beta(2) interface of Hb Santa Clara (beta 97His-->Asn). We have characterized the variation by a combination of electrospray ionisation mass spectrometry and DNA sequence analysis followed by oxygen-binding experiments. Functional studies outlined an increased oxygen affinity, reduced effect of organic phosphates and a reduced Bohr effect with respect to HbA. In view of the primary role of this interface in the cooperative quaternary transition from the T to R conformational state, a theoretical three-dimensional model of Hb Santa Clara was generated. Structural investigations suggest that replacement of Asn for His beta 97 results in a significant stabilization of the high affinity R-state of the haemoglobin molecule with respect to the low affinity T-state. The role of beta FG4 position has been further examined by computational models of known beta FG4 variants, namely Hb Malmö (beta 97His-->Gln), Hb Wood (beta 97His-->Leu), Hb Nagoya (beta 97His-->Pro) and Hb Moriguchi (beta 97His-->Tyr). These findings demonstrate that, among the various residues at the alpha(1)beta(2) (and alpha(2)beta(1)) intersubunit interface, His beta FG4 contributes significantly to the quaternary constraints that are responsible for the low oxygen affinity of human deoxyhaemoglobin.