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Stereochemistry of Cooperative Effects in Hemoglobin
... The identification of the groups involved has been a matter of long scientific debates, but by-andlarge the interpretation proposed by M.F. Perutz (1970Perutz ( , 1998) based on a comparison of the 3D structure of oxy-and deoxyHb is generally accepted. Other effective heterotropic ligands (such as chloride and 2,3 glycerate-bis-phosphate BPG) have been discovered and characterized. ...
... Changeux has published the story of the discovery of the nonoverlapping mechanism of control linked to binding of a small molecule at a site removed from the active site of enzymes ( Changeux 2011). The sigmoidal O 2-binding curve and the Bohr effect, which had been quantitatively characterized and extensively analyzed, were therefore the best examples of allosteric behavior; moreover, the 3D structure of Hb solved by M. F. Perutz and coworkers showed the hemes to be separated by large distances (approximately 30 A ˚ ) and the structure to change upon ligand binding, with tertiary and quaternary conformational changes ( Perutz 1970;Perutz et al. 1998). In 1951, Wyman and Allen had already proposed that the Bohr effect implied a ligand-linked conformational change of the protein; Perutz's direct structural data proved this hypothesis. ...
... This model assumes that there are only two different quaternary structures ( Fig. 5): one with fewer and weaker interactions among the subunits (fully ligated Hb or relaxed: R), and the other with more numerous and stronger bonds between the subunits (deoxy Hb, or tense: T) ( Monod et al. 1965;Eaton et al. 1999;Cui and Karplus 2008). The overstabilization of the T state has been attributed by Perutz (1970Perutz ( , 1987) to eight salt bridges (either in between or within the subunits) which are broken upon O 2 binding at the heme. In the R state, Hb binds O 2 with high affinity, while in the T state, the O 2 affinity is reduced and the protein binds more strongly to heterotropic ligands (H + , Cl À , BPG, CO 2 ). ...
... S2andTable 1). It was reported that in normal hemoglobin (HbA), salt bridge between aVal1 and aArg141 of deoxy HbA disappear on oxygenation (Perutz, 1970aPerutz, , 1970b). As a result pK a of the N-terminal aNH 2 is reduced resulting in release of Bohr proton (Perutz, 1970aPerutz, , 1970b) with concomitant increase in the conformational flexibility across residues in the oxy state (Narayanan et al., 2015). ...
... It was reported that in normal hemoglobin (HbA), salt bridge between aVal1 and aArg141 of deoxy HbA disappear on oxygenation (Perutz, 1970aPerutz, , 1970b). As a result pK a of the N-terminal aNH 2 is reduced resulting in release of Bohr proton (Perutz, 1970aPerutz, , 1970b) with concomitant increase in the conformational flexibility across residues in the oxy state (Narayanan et al., 2015). Compared to normal hemoglobin, sickle hemoglobin was observed to have larger Bohr effect (Ueda et al., 1979). ...
... In case of normal human hemoglobin the constituent residues of this peptide play a crucial role in the structurefunction relationship of hemoglobin. For example, on oxygenation the salt bridges between bHis146-bAsp94, bHis146-aLys40, Hbond between bTyr145-bVal98 and interaction between bTyr145aThr41 disappear (Baldwin and Chothia, 1979;Perutz, 1970aPerutz, , 1970b). In addition, interaction between 2,3-DPG and bHis143 was also reported to dissociate in oxy form. ...
Sickle hemoglobin (HbS) polymerization initiates in the deoxy state with the binding of hydrophobic patch formed by isopropyl group of βVal6 residue of a hemoglobin tetramer with the hydrophobic pocket of another tetramer, whose hydrophobic patch binds to the hydrophobic groove of a third molecule. Subsequent elongation of a single stranded polymer followed by the formation of a double strand and finally combination of seven such pairs of double strands results in a fourteen stranded fibrous polymer. Precipitation of this fiber inside the erythrocytes results in sickling of red blood cells. Surprisingly, the polymerization does not occur in the oxy state of HbS. Due to the unavailability of crystal structure of oxy form of HbS, the molecular basis of inhibition of polymerization in the oxy state is unknown to date.
... The overall findings of the gel experiments are that Viappiani et al. discovered a fraction of the subunits in the r tertiary conformation in the liganded T quaternary structure and a fraction of the subunits in the t tertiary conformation in the unliganded R quaternary structure. Interestingly, according to Perutz, salt bridges are responsible for the low affinity of T and break when ligands bind [57]. Consequently the partition function of Attila Szabo and Martin Karplus describing the Perutz stereochemical predicts that all subunits trapped in the liganded T conformation should show R-state rebinding kinetics [53]. ...
... A major outstanding problem for understanding the function of hemoglobin is to provide an atomistic structural explanation for the 1000-fold difference in oxygen affinity between the t and r subunits [32,52]. An atomistic explanation was first attempted by Max Perutz in his groundbreaking paper on the difference of R and T affinity and the origin of the Bohr effect, with the corresponding partition function developed by Szabo and Karplus, that marked the beginning of the investigation of detailed structure-function relations in proteins [53,57]. Structures are known for unliganded and liganded t in T and r in liganded R, but a complete structural explanation will have to await the determination of the structures of r in fully liganded T and t in fully unliganded R. ...
Hans Frauenfelder’s discovery of conformational substates in studies of myoglobin carbon monoxide geminate rebinding kinetics at cryogenic temperatures (Austin RH, Beeson KW, Eisenstein L, Frauenfelder H, & Gunsalus IC (1975) Dynamics of Ligand Binding to Myoglobin. Biochemistry 14(24):5355–5373) followed by his introduction of energy landscape theory with Peter Wolynes (Frauenfelder H, Sligar SG, & Wolynes PG (1991) The Energy Landscapes and Motions of Proteins. Science 254(5038):1598–1603) marked the beginning of a new era in the physics and physical chemistry of proteins. Their work played a major role in demonstrating the power and importance of dynamics and of Kramers reaction rate theory for understanding protein function. The biggest impact of energy landscape theory has been in the protein folding field, which is well-known and has been documented in numerous articles and reviews, including a recent one of my own (Eaton WA (2021) Modern Kinetics and Mechanism of Protein Folding: a Retrospective. J. Phys. Chem. B. 125(14):3452–3467). Here I will describe the much less well-known impact of their modern view of proteins on both experimental and theoretical studies of hemoglobin kinetics and function. I will first describe how Frauenfelder’s experiments motivated and influenced my own research on myoglobin, which were key ingredients to my work on understanding hemoglobin.
... The two states concerted MWC model (Monod-Wyman-Changeux) [37] proposed an allosteric mechanism which in stereochemical basis was assumed to implicate that the relative ratio of the equilibrium between low vs. high O2 affinity forms of Hb, could represent conformational forms: tense (T) versus a relax (R) [114]. ...
... The 2,3-DPG-Hb complex of much lower affinity for O2 than isolated Hb could be regarded as the physiological T structure [114] [120]. Deoxygenation increase the concentration of free Mg 2+ ion showing that 2,3-DPG and Mg 2+ were mutually excluding ligand of Hb. ...
... The first classic model has been the "two-state allosteric model" (or MWC model) of Monod, Wyman, and Changeau [7][8][9] which was structurally interpreted by Perutz in his "ste- reochemical model" [10]. The "sequential model" (or KNF model) of Koshland, Nemethy and Filmer [11] meant another approach to the interpretation of allostery. ...
... He proposed a stereochemical model of Hb to structurally describe the mechanistic details of the cooperativity on the basis of the molecular structures of deoxy-and oxy-Hbs. He assumed a structure-function correlation and assigned the T and R quaternary structures to low-affinity and high-affinity functional states [10]. For decades, the other dominant model for allostery was the 'sequential', or KNF (Koshland-Nemethy-Filmer), model [11]. ...
The general question by what mechanism an “effector” molecule and the hemes of hemoglobin interact over widely separated intramolecular distances to change the oxygen affinity has been extensively investigated, and still has remained of central interest. In the present work we were interested in clarifying the general role of the protein matrix and its dynamics in the regulation of human adult hemoglobin (HbA). We used a spectroscopy approach that yields the compressibility (κ) of the protein matrix around the hemes of the subunits in HbA and studied how the binding of heterotropic allosteric effectors modify this parameter. κ is directly related to the variance of volume fluctuation, therefore it characterizes the molecular dynamics of the protein structure. For the experiments the heme groups either in the α or in the β subunits of HbA were replaced by fluorescent Zn-protoporphyrinIX, and series of fluorescence line narrowed spectra were measured at varied pressures. The evaluation of the spectra yielded the compressibility that showed significant dynamic asymmetry between the subunits: κ of the α subunit was 0.17±0.05/GPa, while for the β subunit it was much higher, 0.36±0.07/GPa. The heterotropic effectors, chloride ions, inositol hexaphosphate and bezafibrate did not cause significant changes in κ of the α subunits, while in the β subunits the effectors lead to a significant reduction down to 0.15±0.04/GPa. We relate our results to structural data, to results of recent functional studies and to those of molecular dynamics simulations, and find good agreements. The observed asymmetry in the flexibility suggests a distinct role of the subunits in the regulation of Hb that results in the observed changes of the oxygen binding capability.
... The two states concerted MWC model (Monod-Wyman-Changeux) [37] proposed an allosteric mechanism which in stereochemical basis was assumed to implicate that the relative ratio of the equilibrium between low vs. high O2 affinity forms of Hb, could represent conformational forms: tense (T) versus a relax (R) [114]. ...
... The 2,3-DPG-Hb complex of much lower affinity for O2 than isolated Hb could be regarded as the physiological T structure [114] [120]. Deoxygenation increase the concentration of free Mg 2+ ion showing that 2,3-DPG and Mg 2+ were mutually excluding ligand of Hb. ...
This book reviews experimental contributions from many sources to search for a model of structure and molecular function of the blood-astrocytes-neuronal-system. The initial premise involved the dynamics of the hydrated shells of ions and proteins. An oxy-Hb of lower pka compared to deoxy-Hb allows for a higher dissociation of protons. This functioning for the mutual inclusion of O2 and Mg2+ is surrounded by a hydration shell of about 60 molecules. 2,3 DPG-dependent deoxygenation involves the tetramer chains of hydrophobic attractions. Their favorable thermodynamics allows for the dissociation of O2 and Mg2+ by breaking the H-bonds between the protein and the water shell. The turnover between hydrated versus hydrophobic forms of proteins involved in enzyme kinetics requires energy expenditures during the turnover of [ES], changing the enzyme hydration states into its [EP] form. A divalent metal (Mg++) when chelated by a protein loses its hydration sphere. It then releases its hydration (which is incomplete) and shows an intrinsic stronger charge. This is the denominated Mg2+ nascent, which functions by capturing water from Na+ and K+, allowing for sieve effects operating as intermediates of the physical open system. The dissipative energy potential is controlled within astrocytes by decreasing the number of H-bonds through rapid circulation. This is made possible by decreasing the number of H-bonds to reach the vapor state associated with air breathing, which could also operate through the vomeronasal organ that experiences direct contact with the brain. The breakdown of MgATP by the Na+/K+ ATPase of MgATP is involved in the release of ADP3-, and Pi2- and nascent Mg2+ that decrease ATP4-. Mg2+ could be the generator of an action potential via the activation of a Na+/K+ ATPase pump, which opens the gates for Na+ in and K+. The free [Mg2+] up-regulates responsiveness of the post synaptic AC (adenylyl cyclase) NA (noradrenaline) released by the long axons of the corpus coerellus into the synaptic junctions, and also contributes to additional up-regulation by increasing the CAMP. The up-regulation of AC by Mg2+ is turned off by Ca+2. Stressors trigger the Mg2+ response, which results in emotional pain. The zipping-out of DNA by the CAMP results in an Mg2+-CAMP-DNA complex that up-regulates gene expression for every participant in the synthesis of proteins during development, and eventually the formation of long-term memory occurs. Genes that relate to the synthesis of microtubules may participate in the formation of short-term memory. The regenerative capacity of CAMP could be involved in Alzheimer’s treatment by using the vomeronasal pathway to reach specific brain areas.
... Human hemoglobin is an allosteric protein that carries molecular oxygen (O 2 ) [1]. Human hemoglobin is a tetramer consisting of two α and two β subunits [2]. Each subunit carries one identical heme group, which can reversibly bind one diatomic ligand such as O 2 or CO [3]. ...
... Each subunit carries one identical heme group, which can reversibly bind one diatomic ligand such as O 2 or CO [3]. The deoxygenated hemoglobin has a low-affinity structure (T-state) and the oxygenated one has a high-affinity structure (R-state) [2]. As tetrameric hemoglobin is oxygenated, its quaternary structure is changed and the O 2 affinity is increased [4,5]. ...
A nanosecond laser flash-photolysis technique was used to study bimolecular and geminate molecular oxygen (O2) rebinding to tetrameric human hemoglobin and its isolated α and β chains in buffer solutions equilibrated with 1 atm of air and up to 25 atm of xenon. Xenon binding to the isolated α chains and to the α subunits within tetrameric hemoglobin was found to cause a decrease in the efficiency of O2 escape by a factor of ~ 1.30 and 3.3, respectively. A kinetic model for O2 dissociation, rebinding, and migration through two alternative pathways in the hemoglobin subunits was introduced and discussed. It was shown that, in the isolated α chains and α subunits within tetrameric hemoglobin, nearly one- and two-third escaping molecules of O2 leave the protein via xenon docking sites, respectively. The present experimental data support the idea that O2 molecule escapes from the β subunits mainly through the His(E7) gate, and show unambiguously that, in the α subunits, in addition to the direct E7 channel, there is at least one alternative escape route leading to the exterior via the xenon docking sites.
... the first and the second reaction induces the positive cooperative effect. It should be noted that no impacts of Hb's spatial and structural properties have been assumed (5,11,(23)(24)(25). Such effects may, however, contribute to the explanation for the binding energy variation observed but considered outside the main scope of this work. ...
Uptake of oxygen by hemoglobin (Hb), described by the oxygen-Hb dissociation curve, is obviously important for the existence of all vertebrates. Its sigmoidal curve shape indicates that oxygen binds more tightly if sites already are occupied, commonly referred to as the cooperative effect. The effect has been challenging to understand and quantify ever since its experimental demonstration in 1904. Here, we derive an ab initio analytical expression for the dissociation curve based on the fundamental principle of uniform oxygen chemical potential and absolute activity throughout the system at equilibrium using the grand partition function. The resulting analytical dissociation expression therefore only has four molecular oxygen-Hb binding energies as free variables, which are determined by fitting the analytical expression to measured data. The corresponding resulting negative reaction enthalpies identified in increasing magnitude are, 〖∆H〗_1=-41.6, 〖∆H〗_2=-48.8, 〖∆H〗_3=-51.2, 〖and ∆H〗_4=-51.8 kJ/mol, in the range observed experimentally. The difference between 〖∆H〗_1 and 〖∆H〗_4 is ~ 10 kJ/mol, smaller than the maximum entalphy difference measured experimentally, ~ 16.7 kJ/mol. Hence, the cooperative effect can therefore be explained, from an energy point of view, as caused by the reaction entalphy difference between 〖∆H〗_1 and the three subsequent entalphy values. No impact of Hb’s spatial- and structural properties is assumed. The finding highlights the importance of identifying the ligand-receptor molecular binding energies, and thereby the reaction enthalpies, under different conditions as a way for calculating not only the oxygen-Hb but ligand-receptor dissociation curves in general under various conditions, apriori, since the procedure for determining these curves ab initio has been established.
... Mammalian Hbs are tetrameric molecules composed of two α -type and two β -type globin chains that switch between the low-affinity, deoxygenated taut/tense (T) and the high-affinity, oxygenated relaxed (R) states. Increased blood O 2 affinity in species subjected to altitudinal hypoxia commonly results from reductions in intra-erythrocytic levels of allosteric effectors [mainly protons, chloride, CO 2 and the organic phosphate 2,3-diphosphoglycerate (DPG)] that reduce affinity by binding at specific sites of deoxyHb, or from gene-based changes (amino acid substitutions) that increase the intrinsic affinity of Hb for O 2 or lower its sensitivity to these effectors (Perutz & TenEyck 1972;Weber & Fago 2004;Storz et al. 2010a;Mairbaurl & Weber 2012). Adaptive increases in Hb's intrinsic O 2 affinity at altitude have commonly been attributed to single amino acid substitutions, as exemplified by Andean llamas, where it results from the loss of one DPG binding site, and Himalayan and Andean geese, where it correlates with single amino acid substitutions that eliminate the same intramolecular, T-statestabilizing hydrogen bond (Jessen et al. 1991;Weber et al. 1993). ...
... The crystallography x-ray analysis by Max Perutz [ 15 ] was able to determine the quaternary structure of oxyHb, without characterization of the role for a hydrophilic site. ...
Prigogine proposed a coupling between larger sources of enthalpy to allow an open system to operate life. The sun flow of energy is coupled to water cluster thermogenic breakdown of H-bonds to vapor. The position of proline in a polypeptide chain allows sliding between segments, in the tertiary folding structure response to electrostatic attractions, could differentiate positive vs negative domains. Thus, bypass the microscopic reversibility principle, illustrated as a single door, vectorial kinetic only made possible by the jokingly Maxwell proposed operator demons. The physiological function of Hb oxygenation by pO2 shows a microscopic thermogenesis biological vector, functioning by the enthalpy potential of the large mass action of surrounding air and releasing entropy. The mechanism shows the H-bonds breakdown required for changes in the structure-function levels by the proline mediated folding. The tense (T) to relax (R) forms shows vectorial microscopic dynamics, during Hb oxygenation. Thus involves a sliding by H-bonds breakdown, distancing between subunits and. Thus, open a larger entrance to a fully hydrated Mg 2+ to coordinate amphoteric and negative R groups characterizable to a hydrophilic site. The transition of R to T allows positive R groups to bind 2,3-DPG 5-to form deoxyHb. Thus, a microscopic smaller entrance by decreasing its opening size does not allow entrance of the fully hydrated Mg 2+ , but allow the exit of nitric oxide (NO) and a poorly hydrated Mg 2+ , denominated nascent. This one acts for competitive hydration sieving on the shells of Na + , which in terms take water from the K + shell, potentiating a K + /Na +-translocation operating the electrogenic transmembrane potential. The deoxygenation in the reverse transition of R to T binds NO, protecting against a premature decrease of the chromosome's telomeres size by stressing factors such as depression, anxiety and physic traumatisms, over endothelial cells delaying premature senescence. The arginine metabolism produces NO, dilating blood vessels, improving the circulatory systems and the muscular recovery-development. A diet rich in arginine by producing a high sustainable level of NO may prevent the resistance to treatment by the consolidation of large vascular masses. The H-bonds donor potential by their breakdown leads to randomness (or entropy) decreasing the kinetic energy of solvation, scaling down the polarity on the thermogenic dissipation of oxy vs deoxyHb and choroid plexus epithelium on plasma generation of cerebrospinal fluid (CSF). The enthalpy of photosynthesis-metabolism releases CO2, whereas the water cluster mediated thermostatic function releases vapor. In both systems, the entropy release maintains a high potential of enthalpy. Hence, overcomes the thermic and electric noises by an irreversible dissipative kinetics, facilitating a clear development of a meditative level of reasoning and learning. Thus, the brain acquires an autonomous function, beyond behavioral genetic conditioning.
... The crystallography x-ray analysis by Max Perutz [ 7 ] was able to determine the quaternary structure of oxyHb, without characterization of the role for a hydrophilic site. The oxyHb has a topology of two hydrophilic interphase and that coordinate one each. ...
Prigogine proposed a coupling between larger sources of enthalpy to allow an open system to operate life. The sun flow of energy is coupled to water cluster thermogenic breakdown of H-bonds to vapor. The position of proline in a polypeptide chain allows sliding between segments, in the tertiary folding structure response to electrostatic attractions, could differentiate positive vs negative domains. Thus, bypass the microscopic reversibility principle, illustrated as a single door, vectorial kinetic only made possible by the jokingly Maxwell proposed operator demons. The physiological function of Hb oxygenation by pO2 shows a microscopic thermogenesis biological vector, functioning by the enthalpy potential of the large mass action of surrounding air and releasing entropy. The mechanism shows the H-bonds breakdown required for changes in the structure-function levels by the proline mediated folding. The tense (T) to relax (R) forms shows vectorial microscopic dynamics, during Hb oxygenation. Thus involves a sliding by H-bonds breakdown, distancing between subunits and. Thus, open a larger entrance to a fully hydrated Mg 2+ to coordinate amphoteric and negative R groups characterizable to a hydrophilic site. The transition of R to T allows positive R groups to bind 2,3-DPG 5-to form deoxyHb. Thus, a microscopic smaller entrance by decreasing its opening size does not allow entrance of the fully hydrated Mg 2+ , but allow the exit of nitric oxide (NO) and a poorly hydrated Mg 2+ , denominated nascent. This one acts for competitive hydration sieving on the shells of Na + , which in terms take water from the K + shell, potentiating a K + /Na +-translocation operating the electrogenic transmembrane potential. The deoxygenation in the reverse transition of R to T binds NO, protecting against a premature decrease of the chromosome's telomeres size by stressing factors such as depression, anxiety and physic traumatisms, over endothelial cells delaying premature senescence. The arginine metabolism produces NO, dilating blood vessels, improving the circulatory systems and the muscular recovery-development. In vascular niches, endothelial cells of blood vessels produce NO, which activates the Notch signaling pathway of neighboring cancer stem cells, thus regulating their self-replication. A diet rich in arginine by producing a high sustainable level of NO may prevent the resistance to treatment by the consolidation of large vascular masses. The H-bonds donor potential by their breakdown leads to randomness (or entropy) decreasing the kinetic energy of solvation, scaling down the polarity on the thermogenic dissipation of oxy vs deoxyHb and choroid plexus epithelium on plasma generation of cerebrospinal fluid (CSF). The enthalpy of photosynthesis-metabolism releases CO2, whereas the water cluster mediated thermostatic function releases vapor. In both systems, the entropy release maintains a high potential of enthalpy. Hence, overcomes the thermic and electric noises by an irreversible dissipative kinetics, facilitating a clear development of a meditative level of reasoning and learning. Thus, the brain acquires an autonomous function, beyond behavioral genetic conditioning.
... The crystallography x-ray analysis by Max Perutz [ 25 ] was able to determine the quaternary structure of oxyHb and deoxyHb. The latter showed that the tetramer subunits link to a cavity accommodating a single molecule of 2,3-DPG 5-by the positivity charged are amino acid residues liming a central pocket. ...
7-transmembrane (7TM)-hormonal receptors with by noradrenaline (NA) bind activate adenylate cyclase (AC). The dendrites at the oral-cavity of NA-AC-Hypothalamic (OC-NA-AC-HT) axis have axons across the blood-brain barrier (BBB). Its function is to modulate the hypothalamic-pituitary-adrenal (HTPA) axis and its secretions into the capillary arterioles irrigating the oral cavity. The atrophy of the mammalian olfactory bulb lead humans into motor and re-adaptive brain needs. Thus, an emotional brain develops at human nurturing age. Thus, saliva hormonal communication of infants conditions by dopamine exchanges as behavioral reward. The cAMP response element binding (CREB) protein via stimulatory or inhibitory receptors (Rs and Ri) on G protein phosphorylation could stimulate signaling rewards during stages of neuronal plasticity, allowing mechanisms for conectomas development. Moreover, nurturing as a basal period allows reconfiguration of the structures of the brain itself like to 3 times improvement of the velocity of impulse transmission by axons myelination at important pathways. Infants manifest a functional amygdala and hippocampus when still have an under develop frontal brain. Emotional learning allows to bypass genetic constrains to obtain a self-cognitive level, which links to family and society experience at the unconscious level. Conformational changes release O2 from the 4 Hemes and 2Mg 2+ during deoxygenation. Mutual exclusion allows the movement of His 143 to the new domain by the confluence of the positive R groups surrounding the single cavity for 2,3-DPG 5-of the deoxyHb structure. Nascent Mg 2+ dynamics drive Na + /K + membrane translocation for electrogenic action potential. The H-bonds breakdown and reconstitution involved in the conformational change, showed the energy flow sequence by mutual exclusion in oxy to deoxyHb, blood-plasma to cerebrospinal fluid (CSF) and AC conformational change from hydrophilic to hydrophobic domains. A mechanism by a switch on/off by Mg 2+-cAMP coordinative insertion to open DNA in a 3 helix transitory structure with bases pointed externally. Hence, allowing a localized transcription for induce gene and phenotype expression. The initial 16.33 molarity of () by H-bonds exhaustion retains randomness. The kinetics energy of solvation provides a polarity scale for unidirectional unitary sense circulatory flow for the thermogenic transitions dissipation to exclude organismal entropy. The direct link to oral cavity allows an autonomous emotional human brain. The non-polar sides tends at microtubules to generate a coherence water superposition as dimers (), with kinetic energy when reach the oral cavity is exhaled as a 5% vapor.
... Examination of the 3D structure of lth b showed th at among the haemoglobin entries, only this haem group is oxygenated. This observation is consistent with the early finding of Max Perutz (Perutz et al., 1968, Perutz, 1970, Perutz Sz TenEyck, 1972, and of other research groups (Baldwin, 1979) that the iron atom is displaced from the porphyrin plane and only upon oxygenation, the tilt of the haem group becomes less steep as the central iron moves into the core and almost lies in the plane of the four nitrogen atoms. Therefore, consequently, conformational changes induced by the movement of the iron atom are expected to occur in the ligand skeleton. ...
Many different proteins bind and utilise haem to perform important biological functions; the aim of this work was to gain an understanding of some of the underlying molecular recognition processes. We considered how the protein environment determines haem binding, and what are the consequences for ligand conformation. Sets of homologous globins and of non-homologous haem-binding proteins were chosen on the basis of sequence and structural similarity, allowing us to compare ligand conformation and to explore the factors that modulate its structure. Comparison of bound and un-bound haems revealed a conformational disparity between the data-sets, suggesting that protein structural factors provide the dominant effect over the conformational features of the ligand. Even in the homologous globins, the ligand side-chain conformation is variable; greater variability within the non-homologous group reflects different local amino acid sequences of their binding pockets. Moreover, the haem skeleton can be severely distorted, being particularly sensitive to local environment, to attached molecules, and to ligation state. The haem environment was analysed to understand its role in the functional and structural variability of the ligand. Predominance of mainly-alpha structures characterises haemoproteins. While the binding sites are radically different in topology, there are preferred binding modes, with the ligand side-chains engaged in a network of hydrophobic and hydrogen bonding interactions. The stacking of aromatic side-chains on the haem skeleton also appears to be essential in the formation of protein-ligand complexes. Analysis of the haem interface revealed that specific residues prefer to line the binding site and to form ligand contacts. An attempt to correlate properties of the unrelated protein-ligand complexes with haem redox potential was made. A compendium of these analyses has been developed and made accessible via the WWW, providing a user-friendly interface for analysing protein-haem interactions. A search tool allows on-the-fly analysis of protein-ligand relationships, which should facilitate both the comparison of different binding sites, and prediction and design of novel ones.
... Its classic tetrameric structure, comprising 2 a and 2 p subunits, has been used to study the mechanism of biochemical co-operativity (e.g. Perutz 1970;. Cooperativity in Hb is manifested when the binding of the oxygen ligand increases the affinity for subsequent oxygen binding (Ackers et a i, 1992). ...
Protein-protein interactions are fundamental to many biological processes. This work updates the current knowledge of the nature of these interactions using the 3-dimensional co-ordinates of protein complexes deposited in the Brookhaven Protein Data Bank (PDB). The characterisation of interfaces within a data set of 32 homo-dimers is presented, including the analysis of interface size, shape complementarity, amino acid composition, polarity and segmentation. The interfaces in these complexes were observed to be, in general, circular, hydrophobic patches on the surface of proteins, which were planar, and segmented with respect to the amino acid sequence. Interface properties were also analysed in different types of protein-protein complex, including dimers, trimers, enzyme-inhibitors, and antibody-proteins. These were classified as permanent (where the components only occur and function in a complexed state), and 'optional' (where the components also exist independently). The 'optional' complexes were less hydrophobic, and contained more inter-molecular hydrogen bonds. These complexes also had smaller and less well packed interfaces than those that only exist in the complexed state. Protein-protein interface properties were also used to differentiate the observed interface from other sites on the protein surface. Circular and contiguous patches of amino acid residues were defmed on the surface of protein structures. For all surface patches defined, 6 properties (solvation potential, residue interface propensity, planarity, hydrophobicity, accessible surface area, and protrusion) were calculated, and compared to those of the observed interface. Using this method, homo-dimer interfaces were found to be those patches that were the most planar, hydrophobic, surface accessible and protruding. This method of patch analysis was used in a predictive algorithm, to locate putative interaction sites on the surface of isolated protomers. The algorithm proved successful in the prediction of such sites in homo-dimers and enzyme- inhibitor complexes.
... While pathways have been identified using experimental techniques in well-studied proteins such as hemoglobin, 10,[33][34][35] the combination of NMR spectroscopy and computational techniques have allowed for the most robust description of allosteric pathways. 27,30,31,36,37 Computational techniques used to investigate allostery rely on graph theoretical approaches to identify important residues or connections that convey information from the e↵ector binding pocket to the active site. ...
The long-ranged coupling between sites that gives rise to allostery in a protein is built up from short-ranged physical interactions. Computational tools used to predict this coupling and its functional relevance have relied heavily on the application of graph theoretical metrics to residue-level correlations measured from all-atom molecular dynamics simulations. The short-ranged interactions that yield these residue-level correlations, and thus the appropriate graph Laplacian, are quantified by the effective coarse-grained Hessian. Here we compute an effective harmonic coarse-grained Hessian for a benchmark allosteric protein, IGPS, and demonstrate the improved locality of this Laplacian over two other connectivity matrices. Additionally, two centrality metrics are developed that indicate the direct and indirect importance of each residue at producing the covariance between the effector binding pocket and the active site. The results from this procedure are corroborated by previous mutagenesis experiments and lead to unique functional insights. In contrast to previous computational analyses, our results suggest that fP76-hK181, not fD98-hK181, is the most important contact for conveying direct allosteric paths across the HisF-HisH interface. fD98 is found to play a minor role in paths and contribute greatly to indirect allostery between the effector binding pocket and the glutaminase active site.
... The latter, became surrounded by the terminal amino acid, Lys 82, and the protonated imidazole rings of histidines His 143 and His 2, in the 1 and 2 chains, which bear two NH bonds and show overall positive charge. The 2,3-DPG-Hb complex of much lower affinity for O 2 than isolated Hb could be regarded as the physiological T structure [48] [49]. Upon deoxygenation increase the concentration of free Mg 2+ ion [50] showing that deoxyHb and Mg 2+ were competitors binding reciprocally with organic phosphates [50]. ...
... Each subunit contains a heme group consisting of a charged iron (Fe) ion held in an heterocyclic ring called porphyrin. It had long been believed that binding of O2 to one monomer triggered the transition of the tetramer from the tense (T) state to the relaxed (R) state, a mechanism at the core of cooperative binding [Per73]. Along this process, one pair of subunits rotates of an angle ∼ 15 deg about the other ([BC79] and SI Fig.A.3). ...
In this thesis, the focus is set on reconciling the realms of structure and sequence for protein analysis. Sequence analysis tools shine when faced with proteins presenting high sequence identity (≤ 30\%), but are lack - luster when it comes to remote homolog detection. Structural analysis tools present an interesting alternative, but solving structures - when at all possible- is a tedious and expensive process. These observations make the need for hybrid methods - which inject information obtained from available structures in a sequence model - quite clear. This thesis makes four main contributions toward this goal. First we present a novel structural measure, the RMSDcomb, based on local structural conservation patterns - the so called structural motifs. Second, we developed a method to identify structural motifs between two structures using a bootstrap method which relies on filtrations. Our approach is not a direct competitor to flexible aligners but can provide useful to perform a multiscale analysis of structural similarities. Third, we build upon the previous methods to design hybrid Hidden Markov Models which are biased towards regions of increased structural conservation between sets of proteins. We test this tool on the class II fusion viral proteins - particularly challenging because of their low sequence identity and mild structural homology. We find that we are able to recover known remote homologs of the viral proteins in the Drosophila and other organisms. Finally, formalizing a sub - problem encountered when comparing filtrations, we present a new theoretical problem - the D-family matching - on which we present various algorithmic results. We show - in a manner that is analogous to comparing parts of two protein conformations - how it is possible to compare two clusterings of the same data set using such a theoretical model.
... Each subunit contains a heme group consisting of a charged iron (Fe) ion held in an heterocyclic ring called porphyrin. It had long been believed that binding of O2 to one monomer triggered the transition of the tetramer from the tense (T) state to the relaxed (R) state, a mechanism at the core of cooperative binding [28]. Along this process, one pair of subunits rotates of an angle ∼ 15 deg about the other ( [29] and SI Fig.9). ...
The root mean square deviation (RMSD) and the least RMSD are two
widely used similarity measures in structural bioinformatics. Yet,
they stem from global comparisons, possibly obliterating locally
conserved motifs. We correct these limitations with the so-called
combined RMSD , which mixes independent lRMSD measures, each computed
with its own rigid motion. The combined RMSD can be used to compare
(quaternary) structures based on motifs defined from the sequence
(domains, SSE), or to compare structures based on structural motifs
yielded by local structural alignment methods. We illustrate the
benefits of combined RMSD over the usual RMSD on three problems,
namely (i) the analysis of conformational changes based on combined
RMSD of rigid structural motifs (case study: a class II fusion
protein), (ii) the calculation of structural phylogenies (case study:
class II fusion proteins), and (iii) the assignment of quaternary
structures for hemoglobin. Using these, we argue that the combined
RMSD is a tool a choice to perform positive and negative
discrimination of degree of freedom, with applications to the design
of move sets and collective coordinates. Combined RMSD are available
within the Structural Bioinformatics Library (http://sbl.inria.fr).
... Decreases the Magnitude of the Bohr Effect. The Bohr effect is the pH dependency of the affinity of Hb for oxygen, a property that results from effects of protons on the equilibrium between the T-state and R-state structures.[17][18][36][37][38] The equation r = │∆logP 50 / ∆logpH│ is a measure of the magnitude of the Bohr effect38 . ...
Sickle cell disease is an inherited disorder of hemoglobin (Hb). During a sickle cell crisis, deoxygenated sickle hemoglobin (deoxyHbS) polymerizes to form fibers in red blood cells (RBCs), causing the cells to adopt “sickled” shapes. Using small molecules to increase the affinity of Hb for oxygen is a potential approach to treating sickle cell disease, because oxygenated Hb interferes with the polymerization of deoxyHbS. We have identified a triazole disulfide compound (4,4'-Di(1,2,3-triazolyl)disulfide, designated TD-3), which increases the affinity of Hb for oxygen. The crystal structures of carboxy- and deoxy- forms of human adult Hb (HbA), each complexed with TD-3, revealed that one molecule of the monomeric thiol form of TD-3 (5-mercapto-1H-1,2,3-triazole, designated MT-3) forms a disulfide bond with β-Cys93, which inhibits the salt-bridge formation between β-Asp94 and β-His146. This inhibition of salt bridge formation stabilizes the R-state and destabilizes the T-state of Hb, resulting in reduced magnitude of the Bohr effect and increased affinity of Hb for oxygen. Intravenous administration of TD-3 (100 mg/kg) to C57BL/6 mice increased the affinity of murine Hb for oxygen and the mice did not appear to be adversely affected by the drug. TD-3 reduced in vitro hypoxia-induced sickling of human sickle RBCs. The percentage of sickled RBCs and the P50 of human SS RBCs by TD-3 were inversely correlated with the fraction of Hb modified by TD-3. Our study shows that TD-3, and possibly other triazole disulfide compounds that bind to Hb β-Cys93, may provide new treatment options for patients with sickle cell disease.
... The modification in the tertiary structure upon ligand binding at the heme, as predicted by Koshland et ai. (1966), progressively loosens the noncovalent interactions that hold the tetramer in the T conformation, allowing a switch to the high-affinity R state ( Perutz, 1970). The tetrameric Hb can be considered as being made of two rigid dimeric units (c>:,f31 and c>:,f32) held together by looser C>:1f32 and C>:2f3, contacts. ...
... Previous methodological efforts were engaged toward the identification of clusters or chains of residues mediating long-range communication in proteins (Chiappori et al., 2012;Papaleo et al., 2012;Laine et al., 2012;Raimondi et al., 2013;Pandini et al., 2013;Blacklock and Verkhivker, 2013;McClendon et al., 2014; and most of these methods construct a graph to represent the protein. b) The proposed allosteric transition of tetrameric haemoglobin by Perutz (Perutz and TenEyck, 1972;Perutz et al., 1998). The α-subunits and β-subunits are shown for each state with blue and purple colors, respectively and the haem groups are shown in light blue. ...
We presented COMMA, a method to describe and compare the dynamical architectures of different proteins or different variants of the same protein. COMMA extracts dynamical properties from conformational ensembles to identify communication pathways, chains of residues linked by stable interactions that move together, and independent cliques, clusters of residues that fluctuate in a concerted way. It provides a description of the infostery of a protein or protein complex that goes beyond the notions of chain, domain and secondary structure element/motif, and beyond classical measures of how a protein moves and/or changes its shape. We showed the efficiency of our approach in providing mechanistic insights on the effects of deleterious mutations by pinpointing residues playing key roles in the propagation of these effects. In addition COMMA reveals a link between clusters of coevolving residues and networks of dynamical correlations. It enables to contrast the different types of communication occurring between residues and to hierarchise the different regions of a protein depending on their communication efficiency. Furthermore, we presented an approach to exploit both the sequences and structural dynamics to predict a mutational landscape. The discussion of examples, revealed physical interpretation on how the study of conservation brings significant insights on the sensitivity of conserved positions to mutations. Our proposed method, can detect protein regions that are prone to disorder or substantial conformational rearrangements. Moreover, it enabled us to suggest mutations that regulate the stability of the disordered coiled-coils.
... Mammalian Hbs are tetrameric molecules composed of two α-type and two β-type globin chains that switch between the lowaffinity, deoxygenated tense (T) and the high-affinity, oxygenated relaxed (R) states. Increased blood O 2 affinity in species subjected to altitudinal hypoxia commonly results from reductions in intraerythrocytic levels of allosteric effectors [mainly protons, chloride, CO 2 and the organic phosphate 2,3-diphosphoglycerate (DPG)] that reduce affinity by binding at specific sites of deoxyHb, or from gene-based changes (amino acid substitutions) that increase the intrinsic affinity of Hb for O 2 or lower its sensitivity to these effectors (Mairbaurl and Weber, 2012;Perutz and TenEyck, 1972;Storz et al., 2010a;Weber and Fago, 2004). In human Hb, DPG binds at four β-chain amino acid residues (β1Val, β2His, β82Lys and β143His), chloride ions mainly at one α-chain site (between 131Ser and 1Val) and one β-chain site (between 82Lys and 1Val), protons mainly to histidine residues ( primarily β146His) and α1Val (the N-terminal residues of the α-chains), and CO 2 binds at the free NH 2 groups of N-terminal (Val) residues of both chains (Berenbrink, 2006;Lukin and Ho, 2004;Perutz, 1983;Weber et al., 2013). ...
Inhabiting deep and sealed subterranean burrows, mole rats exhibit a remarkable suite of specializations, including eusociality (living in colonies with single breeding queens), extraordinary longevity, cancer immunity and poikilothermy, and extreme tolerance of hypoxia and hypercapnia.With little information available on adjustments in hemoglobin (Hb) function that may mitigate the impact of exogenous and endogenous constraints on the uptake and internal transport of O2, we measured hematological characteristics, as well as Hb-O2 binding affinities and their sensitivities to pH (Bohr effect), CO2, temperature and 2,3 diphosphoglycerate (DPG, the major allosteric modulator of Hb-O2 affinity in the red cells) in four social and two solitary species of African mole rats (family Bathyergidae) originating from different biomes and soil types across Central and Southern Africa. We find no consistent patterns in hematocrit (Hct) and blood and red cell DPG and Hb concentrations or in intrinsic Hb-O2 affinity and its sensitivity to pH and DPG that correlate with burrowing, sociality and soil-type. However, the results reveal low specific (pH-independent) effects of CO2 on Hb-O2 affinity compared to humans that predictably safeguard pulmonary loading under hypoxic and hypercapnic burrow conditions. The O2-binding characteristics are discussed in relation to available information on the primary structure of Hbs from adult and developmental stages of mammals subjected to hypoxia and hypercapnia and the molecular mechanisms underlying functional variation in rodent Hbs.
... The figure 15 illustrates that because these ligands were at an invariant concentration, could be displaced from Hb by a competitive mechanism involving pO 2 increment. The 2,3-DPG-Hb complex of much lower affinity for O 2 than isolated Hb could be regarded as the physiological T structure [156,157]. Deoxygenation increase the concentration of free Mg 2+ ion showing that 2,3-DPG and Mg 2+ were mutually excluding ligand of Hb. Mg 2+ and O 2 were mutually inclusive by each one increasing affinity for the other [134]. ...
Sensorial neurons activate the locus coeruleus long axons for a presynaptic Ca²⁺-dependent release of noradrenaline (NA). Postsynaptic adenylyl cyclase (AC) of several areas of the brain shows Mg²⁺-dependent responsiveness to the neurotransmitter in the absence of Ca²⁺ when the free Mg²⁺ is present in excess of substrate Mg-ATP. The brain capillary glucose uptake by red-cells leads to the endogenous generation of 2,3-DPG, and promotes the oxyHb-deoxygenation to discharge Mg²⁺ at the noradrenergic synapses. Since adrenaline cannot cross the blood brain barrier, it could not have a feedback; however, cortisol does. cAMP controls the activation of the initial voltage-gated state of the action potential, configuring nerve impulse, transcription-plasticity mechanism and long-term memory. Overall, this molecular mechanism could be used to propose a model linking environmental stimulus resulting in cAMP and the rate of gene expression. DNA-dependent RNA polymerase activation is inducible, expressed under the conditions of adaptive value by cAMP. The zipper-closure role of the enzyme functions with divalent metal for inclusion of the cyclic nucleotide cAMP and cGMP in DNA. cAMP by binding to Mg²⁺ ion interacts with the negatively charged phosphate groups. The latter chelated by Mg²⁺ opens the double chain in DNA for binding DNA-dependent RNA polymerase. The greater activity of cAMP-Me²⁺-DNA complex with regard to a stabilized double helix allows for the introduction of a mechanism for genetic induction vs constitutive state. The cAMP-Me²⁺-DNA complex by increasing a turn-on activity could consolidate cAMP stimulus, allowing genetic variance by insertion of cAMP, by adhesion of cAMP to stimulate a similar area of the DNA at different stages on the overall evolutionary/adaptive response to environmental stress. Charles Darwin discovers evolution relating the adaptive strength of the birds’ beaks conditioned by the hardness of environmental nuts. The nutritional stress increases free Mg²⁺. Noradrenaline-stimulated adenylyl cyclase stimulates the hypothalamus, which involves additional metabolic resources to increase the strength of the bird beak. Specific stimulus for the DNA location controlling the bird’s beak development allows for a complementary configuration for cAMP insertion into the near specific bird-beak-DNA segment involved in beak growing and development. cAMP-Me²⁺-DNA complex inserted in the DNA complex is continuously open for DNA transcript involved in developing a new, stronger beak. A genetic link between the induction mechanism and a transitory modification of DNA expression could enrich the relationship of induced vs constitutive templates in the reproductive cells. The balance would favor the tendency toinduce activation and increase the DNA response to external stimuli over the constitutive remaining and more stable DNA.
... Hemoglobin is a heterotetrameric protein consisting of two α subunits and two β subunits, and each subunit has an iron-centered heme that reversibly binds oxygen [1]. Hemoglobin transports heme-bound oxygen from the lungs to all of the tissues in the body via oxygenation-linked shifts in the conformational equilibrium between the tense state (T state; deoxygenated) and the relaxed state (R state; oxygenated) [1][2][3]. This structural transition is controlled allosterically, as the binding of oxygen to one subunit affects the oxygen-affinity of PLOS the other subunits in the same tetrameric assembly [3]. ...
Background
Deer mice (Peromyscus maniculatus) that are native to high altitudes in the Rocky Mountains have evolved hemoglobins with an increased oxygen-binding affinity relative to those of lowland conspecifics. To elucidate the molecular mechanisms responsible for the evolved increase in hemoglobin-oxygen affinity, the crystal structure of the highland hemoglobin variant was solved and compared with the previously reported structure for the lowland variant.
Results
Highland hemoglobin yielded at least two crystal types, in which the longest axes were 507 and 230 Å. Using the smaller unit cell crystal, the structure was solved at 2.2 Å resolution. The asymmetric unit contained two tetrameric hemoglobin molecules.
Conclusions
The analyses revealed that αPro50 in the highland hemoglobin variant promoted a stable interaction between αHis45 and heme that was not seen in the αHis50 lowland variant. The αPro50 mutation also altered the nature of atomic contacts at the α1β2/α2β1 intersubunit interfaces. These results demonstrate how affinity-altering changes in intersubunit interactions can be produced by mutations at structurally remote sites.
... A molecular switch is a packing arrangement, within a monomer or between monomers in an oligomer, that assumes multiple configurations separated by a significant barrier. For example, hemoglobin alternates between relaxed (R) and tense (T) conformations in different crystal structures without populating intermediate states (76,81). Alternative packing of β97 His occurs either between α38 Thr and α41 Thr in the R state or between α41 Thr and α44 Pro in the T state (8, figures 10a and 13). ...
Understanding how distinct parts of proteins produce coordinated behavior has driven and continues to drive advances in protein science and enzymology. However, despite consensus about the conceptual basis for allostery, the idiosyncratic nature of allosteric mechanisms resists general approaches. Computational methods can identify conformational transition states from structural changes, revealing common switching mechanisms that impose multistate behavior. Thermodynamic cycles use factorial perturbations to measure coupling energies between side chains in molecular switches that mediate shear during domain motion. Such cycles have now been complemented by modular cycles that measure energetic coupling between separable domains. For one model system, energetic coupling between domains has been shown to be quantitatively equivalent to that between dynamic side chains. Linkages between domain motion, switching residues, and catalysis make nucleoside triphosphate hydrolysis conditional on domain movement, confirming an essential yet neglected aspect of free energy transduction and suggesting the potential generality of these studies. Expected final online publication date for the Annual Review of Biophysics Volume 46 is May 20, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... The main physiological role of Hb is to act as oxygen carrier from the lungs to the tissues; in order to perform its task in an efficient way, Hb has to react with oxygen in a cooperative way. 3 Thanks to the conceptual framework introduced by the Monod-Wyman-Changeux model 4 and the seminal crystallographic work of Perutz, it is widely recognized that the "secret" of Hb cooperativity lies in the fact that this molecule is able to adopt at least two different quaternary structures in solution: a "relaxed" (R) structure stabilized by the presence of ligands, and a "tense" (T) structure that is stable when the protein is unligated. 5 The ligated-to-unligated transition in Hb involves both conformational changes within the subunits (tertiary structure transition) and changes in the relative disposition of the subunits (quaternary structure transition). This last transition consists mainly in a ∼15°rotation of one αβ dimer with respect to the other in which interdimer interfaces are rearranged (whereas intradimer interfaces remain largely unchanged). ...
Allosteric effectors play an important role in regulating the oxygen supply efficiency of hemoglobin for blood storage and disease treatment. However, allosteric effectors that are approved by the US FDA are limited. In this study, cefmetazole sodium (CS) was found to bind adult hemoglobin (HbA) from FDA library (1338 compounds) using surface plasmon resonance imaging high-throughput screening. Using surface plasmon resonance (SPR), the interaction between CS and HbA was verified. The oxygen dissociation curve of HbA after CS interaction showed a significant increase in P50 and theoretical oxygen-release capacity. Acid-base sensitivity (SI) exhibited a decreasing trend, although not significantly different. An oxygen dissociation assay indicated that CS accelerated HbA deoxygenation. Microfluidic modulated spectroscopy showed that CS changed the ratio of the alpha-helix to the beta-sheet of HbA. Molecular docking suggested CS bound to HbA's β-chains via hydrogen bonds, with key amino acids being N282, K225, H545, K625, K675, and V544.The results of molecular dynamics simulations (MD) revealed a stable orientation of the HbA-CS complex. CS did not significantly affect the P50 of bovine hemoglobin, possibly due to the lack of Valβ1 and Hisβ2, indicating that these were the crucial amino acids involved in HbA's oxygen affinity. Competition between the 2,3-Diphosphoglycerate (2,3-DPG) and CS in the HbA interaction was also determined by SPR, molecular docking and MD. In summary, CS could interact with HbA and regulate the oxygen supply efficiency via forming stable hydrogen bonds with the β-chains of HbA, and showed competition with 2,3-DPG.
In this paper, we studied the structure, dipole moment, polarizability, and static first hyperpolarizability of linear phosphazenes. The relationship between the nonlinear optical (NLO) properties and the energy gaps was also taken into account. The structure of these phosphazenes has been performed using the B3LYP, ωB97X–D and M06–2X functionals. The linear and NLO properties of these compounds have shown selective responses to the different DFT levels that are in good agreement with the experimental values of similar compounds. The ωB97X–D functional gives a high static first hyperpolarizability compared to the B3LYP and M06–2X levels. An inverse relationship has been obtained between the HOMO–LUMO gaps and βtot. This theoretical study confirms the suitability of these phosphazenes for optoelectronic and optical devices.
The kinetics of oxygen binding to human hemoglobin at pH 7, 0.1 m phosphate, 20°, 2.5 x 10⁻⁵ to 10⁻³m heme has been investigated by chemical relaxation methods in order to obtain information about the elementary steps and the mechanism of the cooperative process of ligand binding. The temperature jump relaxation method allows the oxygen-binding reaction to be followed over its entire time range. The relaxation process is characterized by two phases which are well separated in time. Both the time constants and the relative amplitudes are dependent on protein and ligand concentration. The two relaxation phases can be understood qualitatively. The fast phase corresponds at low oxygen saturation mainly to the binding of the first ligand molecule and at high saturation mainly to the binding of the last ligand molecule. At intermediate oxygen saturation the fast phase is determined predominantly by the kinetics of both the first and the last step—the intermediate reaction steps do not contribute significantly. The slow relaxation phase involves all elementary binding reactions, but is determined mainly by the kinetics of the intermediate oxygen-binding steps.
The relative amplitudes of the fast and the slow relaxation phases reflect strongly the population of the reaction species involved. It is concluded that hemoglobin species with one and three ligands bound must be populated measurably at equilibrium. Reaction models which assume negligible concentrations of these intermediates are therefore not valid.
Three reactions have been characterized directly by their kinetic properties: (a) a fast reaction step at low oxygen saturation yielding an apparent off-rate constant of about 1000 s⁻¹ and an apparent on-rate constant (per tetramer) of about 4 x 10⁷m⁻¹s⁻¹; (b) a fast reaction step at high oxygen saturation, yielding an apparent on-rate constant of about 4 x 10⁷m⁻¹s⁻¹; (c) a slow reaction, which at high oxygen concentration yields an apparent rate constant of 5 x 10⁶m⁻¹s⁻¹.
It thus appears that the binding of the first oxygen molecule to deoxyhemoglobin is a very rapid process which is characterized by an apparent rate constant as large as that found for the binding of the last oxygen molecule. The low oxygen affinity of deoxyhemoglobin arises mainly from the high dissociation rate constant, i.e. short lifetime, of Hb4O2. It cannot be decided if these kinetic properties apply equally to both the α and β chains or if only one type of chain is involved in the fast phase of the relaxation spectrum.
A four-step binding model (Adair scheme) explains quantitatively the principal observations of the relaxation kinetics. Adair rate parameters have been obtained which describe the appearance of two relaxation phases only, the ligand concentration dependence (from 0 to 250 µm O2), the protein concentration dependence (from 2 x 10⁻⁵ to 10⁻³m heme), and the relative and absolute relaxation amplitudes of both phases. The calculated stopped flow kinetic progress curve, using these values, is in agreement with the available data (which are in the saturation range greater than 50%) and predicts a biphasic time course, in contrast to the kinetics of CO-binding.
A decision between mechanisms based upon the relaxation kinetics data can be made only with certain assumptions. The values of the Adair-recombination rate constants exclude a “concerted” mechanism in which there are two very rapidly interconverting structure forms of the protein, each exhibiting equal or nearly equal intrinsic kinetic and equilibrium properties for the four subunits.
A concerted mechanism might apply if structure changes are not very fast compared to ligand binding or, if the kinetic properties of the α and β chains are markedly different. However, no consistent fit of the relaxation spectrum has been obtained so far with these assumptions.
Photodynamic therapy (PDT) has been shown to effectively treat cancer by producing cytotoxic reactive oxygen species (ROS) via excitation of photosensitizers (PS). However, most PS lack tumor cell specificity, possess poor aqueous solubility, and cause systemic photosensitivity. Removing heme from hemoglobin (Hb) yields an apoprotein called apohemoglobin (apoHb) with a vacant heme-binding pocket that can efficiently bind to hydrophobic molecules such as PS. In this study, the PS aluminum phthalocyanine (Al-PC) was bound to the apoHb-haptoglobin (apoHb-Hp) protein complex, forming an apoHb-Al-PC-Hp (APH) complex. The reaction of Al-PC with apoHb prevented Al-PC aggregation in aqueous solution, retaining the characteristic spectral properties of Al-PC. The stability of apoHb-Al-PC was enhanced via binding with Hp to form the APH complex which allowed for repeated Al-PC additions to maximize Al-PC encapsulation. The final APH product had 65% of the active heme-binding sites of apoHb bound to Al-PC and hydrodynamic diameter of 18 nm, that could potentially reduce extravasation of the molecule through the blood vessel wall and prevent kidney accumulation of Al-PC. Furthermore, more than 80% of APH’s absorbance spectra was retained when incubated for over a day in plasma at 37 °C. Heme displacement assays confirmed Al-PC was bound within the heme-binding pocket of apoHb and binding specificity was demonstrated by ineffective Al-PC binding to human serum albumin, Hp, or Hb. In vitro studies confirmed enhanced singlet oxygen generation of APH over Al-PC in aqueous solution and demonstrated effective PDT on human and murine cancer cells. Taken together, this study provides a method to produce APH for enhanced PDT via improved PS solubility and potential targeted therapy via uptake by CD163+ macrophages and monocytes in the tumor (i.e. tumor associated macrophages). Moreover, this scalable method for site-specific encapsulation of Al-PC into apoHb and apoHb-Hp may be used for other hydrophobic therapeutic agents.
The role of magnetism in the biological functioning of hemoglobin has been debated since its discovery by Pauling and Coryell in 1936. The hemoglobin molecule contains four heme groups each having a porphyrin layer with a Fe ion at the center. Here, we present combined density-functional theory and quantum Monte Carlo calculations for an effective model of Fe in a heme cluster. In comparison with these calculations, we analyze the experimental data on human adult hemoglobin (HbA) from the magnetic susceptibility, Mössbauer and magnetic circular dichroism (MCD) measurements. In both the deoxygenated (deoxy) and the oxygenated (oxy) cases, we show that local magnetic moments develop in the porphyrin layer with antiferromagnetic coupling to the Fe moment. Our calculations reproduce the magnetic susceptibility measurements on deoxy and oxy-HbA. For deoxy-HbA, we show that the anomalous MCD signal in the UV region is an experimental evidence for the presence of antiferromagnetic Fe-porphyrin correlations. The functional properties of hemoglobin such as the binding of O2, the Bohr effect and the cooperativity are explained based on the magnetic correlations. This analysis suggests that magnetism could be involved in the functioning of hemoglobin.
Aging is associated with increased prevalence of skeletal and cardiac muscle disorders, such as sarcopenia and cardiac infarction. In this study, we constructed a compendium of purified ginsenoside compounds from Panax ginseng C.A. Meyer, which is a traditional Korean medicinal plant used to treat for muscle weakness. Skeletal muscle progenitor cell-based screening identified three compounds that enhance cell viability, of which 20(R)-ginsenoside Rh2 showed the most robust response. 20(R)-ginsenoside Rh2 increased viability in myoblasts and cardiomyocytes, but not fibroblasts or disease-related cells. The cellular mechanism was identified as downregulation of cyclin-dependent kinase inhibitor 1B (p27Kip1) via upregulation of Akt1/PKB phosphorylation at serine 473, with the orientation of the 20 carbon epimer being crucially important for biological activity. In zebrafish and mammalian models, 20(R)-ginsenoside Rh2 enhanced muscle cell proliferation and accelerated recovery from degeneration. Thus, we have identified 20(R)-ginsenoside Rh2 as a p27Kip1 inhibitor that may be developed as a natural therapeutic for muscle degeneration.
The role of magnetism in the biological functioning of hemoglobin has been debated since its discovery by Pauling and Coryell in 1936. The hemoglobin molecule contains four heme groups each having a porphyrin layer with a Fe ion at the center. Here, we present combined density-functional theory and quantum Monte Carlo calculations for an effective model of Fe in a heme cluster. In comparison with these calculations, we analyze the experimental data on human adult hemoglobin (HbA) from the magnetic susceptibility, Mossbauer and magnetic circular dichroism (MCD) measurements. In both the deoxygenated (deoxy) and the oxygenated (oxy) cases, we show that local magnetic moments develop in the porphyrin layer with antiferromagnetic coupling to the Fe moment. Our calculations reproduce the magnetic susceptibility measurements on deoxy and oxy-HbA. For deoxy-HbA, we show that the anomalous MCD signal in the UV region is an experimental evidence for the presence of antiferromagnetic Fe-porphyrin correlations. The various important properties of hemoglobin are explained based on the antiferromagnetic correlations including the Bohr effect and cooperativity. This analysis shows that magnetism is involved in a fundamental way in the functioning of hemoglobin.
Many ligand-binding proteins possess multiple binding sites for the same ligand, having binding stoichiometries higher than 1:1. Homotropic cooperativity is defined as the influence of one ligand upon the binding affinity of another ligand of the same type. This chapter analyzes ligand binding to proteins having multiple binding sites. It analyzes the functional behaviour of proteins capable of binding ligands with stoichiometry higher that 1:1. The chapter demonstrates that the slopes of their ligand binding isotherms may be increased, decreased or unchanged with respect to that of monomeric, single-binding site proteins. A protein possessing multiple ligand-binding sites may be made up of non-identical subunits, or the binding sites may be non-equivalent if they reside on the same polypeptide chain. Cooperativity occurs because each ligation step changes the structure of one subunit by an induced fit mechanism and favors the formation or breakage of intersubunit bonds.
The presence of multiple hemoglobins in fish has been well documented. However, the molecular basis for this multiplicity is scarcely elucidated. The present paper describes the separation of multiple hemoglobins in the hemolysate of the Japanese eel, Anguilla japonica, and the results of analyses on the subunit compositions of these hemoglobins.
Electrophoresis on a cellulose acetate membrane reveals that the Japanese eel has six hemoglobin components: three cathodally migrating components (E I a, E I b and E I c) and three anodally migrating components (E II a, EII b and Elk).Each of these multiple hemoglobins is isolated by column chromatographies.
Structural analyses of the globin chains and hybridization experiments between the isolated hemoglobins show that the six hemoglobins have the following globin compositions E I a (αI 2 βI 2), EIb (αI 2 βI γI), EIc (αI 2 γI2), EIIa (αII 2 βII 2), EIIb (αII 2 βII γII) and Ellc (αII 2γII2). Either hemoglobin EIb or Ell b is found to be a unique asymmetric hybrid tetramer consisting of the two unlike dimers.The former is derived from the two parent symmetric tetramers, El a and El c, and the latter from Ella and El Ic.These asymmetric hemoglobins exist in an equilibrium with their parent hemoglobins.
The formation of asymmetric hybrid hemoglobins in vitro from their two parent symmetric tetramers can be monitored by electrophoresis.From the kinetic study, it is found that the three hemoglobins E I have the same tetramer-dimer equilibrium constant and the rate constant of dissociation of hemoglobins El into their dimers is about 0.1 h⁻¹ at 25°C, p H 7.4. On the other hand, the dissociation rate of hemoglobins Ell is considerably faster than that of hemoglobins El.
In seeking to account for kinetic and affinity differences between ligands in structural terms, it would be desirable to compare liganded and unliganded forms of the same structure, which are in principle crystallographically observable, and to infer the structure of the transient, unobservable transition state for the reaction. For example, the affinity of hemoglobin (Hb) for CO could be examined through a comparison of the structures of deoxyHb (T state) and CO Hb (also T state), yielding information about KT, and a comparison of the structures of deoxyHb (R state) and CO Hb (also R state), yielding information about KR. Unfortunately, only the first and last of these four structures have been determined (Fermi, 1975; Baldwin, 1980). Ingenious attempts by Anderson (1973) to prepare T-state CO Hb led to disorder of the crystals; R-state deoxyHb may, however, be accessible by similar experimental strategies, to supplement related results on chemically modified and mutant hemoglobins (Perutz and TenEyck, 1971).
All hemoproteins that transport dioxygen contain at their active site a protoheme molecule attached to an imidazole of the histidine in the protein (Antonini and Brunori, 1971, p. 226). Yet the affinities of the different hemoproteins for dioxygen or carbon monoxide vary over a large range (Ibid, p. 351). Based upon the differences in the porphyrin geometries and the location of the proximal imidazole and distal residues in these hemoproteins (Perutz et al., 1976; Weber et al., 1978; Takano, 1977; Baldwin, 1980; Baldwin and Chothia, 1979), two steric effects have been proposed to account for affinity variations.
The interaction of human hemoglobin (Hb) with surface active ionic liquids (SAILs), 1-dodecyl-3-methylimidazolium chloride [C12mim][Cl] and 1-hexyl-3-methylimidazolium dodecylsulfate [C6mim][SDS], has been studied in aqueous medium through various techniques like surface tension, UV-vis spectroscopy, fluorescence spectroscopy, isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). The interactional behavior of SAILs toward Hb at the air/solution interface is investigated and various interfacial and thermodynamic parameters have also been calculated. The conformational changes in Hb upon interacting with SAILs have been illustrated from UV-visible measurements in combination with fluorescence spectroscopy. These results indicate that at lower concentration the [C6mim][SDS] monomer forms stronger Hb–[C6mim][SDS] monomer complexes as compared to [C12mim][Cl], whereas at higher concentration [C12mim][Cl] denatures Hb more and induces the release of heme from the hydrophobic pocket of Hb. The enthalpy changes were also investigated by using isothermal titration calorimetry (ITC). The dynamic light scattering (DLS) measurements revealed the effect of SAILs on the hydrodynamic diameter (Dh) of Hb.
X-ray analysis of the natural valency hybrid α+ M Boston2 βdeoxy2 shows that the ferric iron atoms in the abnormal α subunits are bonded to the phenolate side chains of the tyrosines that have replaced the distal histidines; the iron atoms are displaced to the distal side of the porphyrin ring and are not bonded to the proximal histidines. The resulting changes in tertiary structure of the α subunits stabilize the haemoglobin tetramer in the quaternary deoxy structure, which lowers the oxygen affinity of the normal β subunits and causes cyanosis. The strength of the bond from the ferric iron to the phenolate oxygen appears to be the main factor responsible for the many abnormal properties of haemoglobin M Boston.
Polycythemia in man is defined as an increase in the hematocrit and hemoglobin concentration above accepted normal values. As such, the finding of polycythemia implies the increased production of erythrocytes. Since red cell production is governed normally by interaction of oxygen (O2) availability, the elaboration of the regulatory hormone, erythropoietin, and marrow function, it is not surprising that derangements in these interrelationships account for virtually all causes of polycythemia. This chapter summarizes briefly various aspects of the regulation of red cell production and focuses on those identified mechanisms which lead to polycythemia.
Hemoglobin (Hb) is an extensively studied paradigm of proteins that alter their function in response to allosteric effectors. Models of its action have been used as prototypes for structure-function relationships in many proteins, and models for the molecular basis of its function have been deeply studied and extensively argued. Recent reports suggest that dynamics may play an important role in its function. Relatively little is known about the slow, correlated motions of hemoglobin subunits in various structural states because experimental and computational strategies for their characterization are challenging. Allosteric effectors such as inositol hexaphosphate (IHP) bind to both deoxy-Hb and HbCO, albeit at different sites, leading to a lowered oxygen affinity. The manner in which these effectors impact oxygen binding is unclear and may involve changes in structure, dynamics or both. Here we use neutron spin echo (NSE) measurements accompanied by wide-angle x-ray scattering (WAXS) to show that binding of IHP to HbCO results in an increase in the rate of coordinated motions of Hb subunits relative to one another with little if any change in large scale structure. This increase of large-scale dynamics seems to be coupled with a decrease in the average magnitude of higher frequency modes of individual residues. These observations indicate that enhanced dynamic motions contribute to the functional changes induced by IHP and suggest that they may be responsible for the lowered oxygen affinity triggered by these effectors. This article is protected by copyright. All rights reserved.
Key Points
The reversible association of deoxyHb with band 3 acts as an O2-triggered molecular switch to regulate erythrocyte properties. Transgenic mice lacking the deoxyHb site on band 3 fail to respond to changes in O2 with changes in erythrocyte properties.
Significance
Regulation of biochemical activity is essential for proper cell growth and metabolism. Many proteins’ activities are regulated by interactions with other molecules binding some distance away from the proteins’ active sites. In such allosteric proteins, active sites should thus be mechanically coupled to spatially removed regulatory regions. We studied crystal and NMR structures of proteins in various regulatory and ligand-binding states. We calculated and analyzed distributions of strains throughout several proteins. Strains reveal allosteric and active sites and suggest that quasi-two-dimensional strained surfaces mediate mechanical couplings between them. Strain analysis of widely available structural data can illuminate protein function and guide future experimental investigation.
Liganded hemoglobin is a stronger acid than ligand-free hemoglobin. The kinetics of the transformation between these states was studied in the pH range 6.9 to 9.0 in 0.3 m NaCl with a pH indicator (phenol red or m-cresol purple) to follow the pH changes accompanying flash photolysis of human carbon monoxide hemoglobin. At least three distinct processes involving protons were detected kinetically: a rapid uptake of H⁺ presumably reflecting the shift from the ligand-bound to ligand-free comformation, followed by a slower biphasic release of protons accompanying the dark reaction of CO and hemoglobin.
The fast proton uptake occurred with a rate constant k = 8000 sec⁻¹ (20°, pH 7.8), whereas the rate of the transition was greater both at pH 6.9 and 9.0 (k > 10,000 sec⁻¹). The transition state parameters were also obtained: ΔH* = 11.2 ± 0.4 kcal per mole and ΔS* = -3.2 ± 1.5 e.u.
The biphasic CO-binding and H⁺ release reactions following the flash were analyzed by treating the results as the sum of two simple exponential expressions; this procedure revealed that 27% of the reaction involving protons occurred at the higher rate. The rapidly reacting hemoglobin observed (50%) under these conditions (21 µm Hb, pH 7.6, 0.3 m NaCl, 60 µm CO, 25°) is attributed, on the basis of its dependence on carbon monoxide hemoglobin concentration, to the α1β1 dimer.
The kinetic properties of both ligand binding and proton release were identical also at pH 6.95, 0.3 m NaCl, when carbon monoxide hemoglobin was subjected to flash photolysis in the presence of O2 (rate constant = 1850 sec⁻¹). At pH 7.9, however, there was a distinct lag in release of hydrogen ions.
The results are consistent with the recent structural interpretation of the Bohr effect (Perutz, M. F., Muirhead, H., Mazzarella, L., Crother, R. A., Greer, J., and Kilmartin, J. V., Nature, 222, 1240 (1969)), as well as the hypothesis that the α1β1 dimer is devoid of cooperative behavior.
Horse methaemoglobin crystallizes with two molecules in a face-centred monoclinic unit cell (space group C2), in which rigid layers of molecules parallel to (001) alternate with layers of liquid. The crystals can be made to swell and shrink in a series of steps involving changes in d(001) and in the angle beta . It appears that only the distances between the molecular layers change, but not their internal structure. The lattice changes allow the modulus of the molecular Fourier transform to be sampled along lines of constant h and k. When k=0 the transform is real and the sampled values of |F| describe a series of loops and nodes. Part I of this series dealt with the principles of deciphering these and established the absolute signs of the 00l reflexions. In part II the absolute signs of certain 20l reflexions were derived from the changes in intensity produced by the substitution of salt solution for water as the liquid of crystallization. In this paper the transform is measured for all values of h and l up to lambda /d = 0\cdot 24, comprising nine layer lines in all. The absolute signs of layer lines with h > 2 are left in doubt, but many sign relations are established within each of them. It is difficult to assess exactly the number of sign relations found by the transform method, but it is estimated that the number of alternative sign combinations is reduced from 296 to 213. The remaining uncertainties are cleared up by the isomorphous replacement method described in part IV.
The magnitude of the Bohr effect, as measured by the number of moles of hydrogen ion produced per mole of oxygen reacting with haemoglobin, Δh+, has been measured as a function of pH for five vertebrate haemoglobins. In the absence of 2,3-diphosphoglyceric acid (DPG) the alkaline Bohr effect is the same for the mammalian haemoglobins and slightly lower for pigeon haemoglobin. However the acid Bohr effect varies by a factor of 5·5 over the various haemoglobins and there is a correlation between the characteristic pH, pHch, of a haemoglobin and the magnitude of its acid Bohr effect, which implies that there is a correlation between the content of charged amino-acids in a haemoglobin and its acid Bohr effect. In the presence of DPG the alkaline Bohr effect for the mammalian haemoglobins is approximately the same and is 50–60% greater than in the absence of DPG. It is concluded that the variable magnitude of the alkaline Bohr effect measured on haemolystates from which DPG has not been removed arises from the variable affinity of different haemoglobins for DPG.
The composition and swelling properties of horse methæmoglobin crystals were investigated by combining density measurements with X-ray diffraction studies. The following conclusions were reached. (1) 52.4% of the volume of normal wet methæmoglobin crystals consists of liquid whose composition can be varied within wide limits. (2) It was shown by immersing crystals in a series of ammonium sulphate solutions at pH 7 that the unit cell dimensions and the general intensity distribution in the X-ray diffraction pattern are independent of the concentration of neutral electrolyte in the suspension medium. Similarly, they are independent of pH if the salt concentration is kept constant, except at pH 5.4, when the unit cell swells by a definite amount on acidification. In pure ammonium sulphate solutions any change in concentration involves a change in pH; more complex swelling effects were therefore observed. (3) In methæmoglobin crystals layers of hæmoglobin molecules alternate with layers of liquid. Swelling and shrinkage produce variations in the layer spacing and shearing of the layers. All lattice changes proceed in definite, reproducible steps, involving changes in the layer spacing of the order of 4 A. at a time. The thickness and structure of the protein layers remain unaltered during swelling and shrinkage. (4) At pH 7 the salt concentration in the liquid of crystallisation is a linear function of the salt concentration in the suspension medium; in ammonium sulphate solution the former is about two-thirds of the latter. If it is assumed that the liquid of crystallisation consists of two components, namely, "bound water" which is not available as solvent to mobile ions and "free liquid" through which ions can diffuse and which has the same composition as the suspension medium, it can be shown that protein hydration ("bound water") amounts to 0.3 g. H2O per g. protein. At the isolectric point hydration is largely independent of electrolyte concentration; it is, however, a function of pH and decreases on both sides of the iso-electric point. It is shown that this hydration is of the right order of magnitude for a monomolecular layer of water molecules covering the surface of the hæmoglobin molecule.
The oxygenation of haemoglobin is accompanied by structural changes in the subunits triggered by shifts of the iron atoms relative to the porphyrin and, in the β-subunits, also by the steric effect of oxygen itself. The oxygen-free form is constrained by salt-bridges which are broken by the energy of haem–haem interaction with the release of H+. 2,3-Diphosphoglycerate may add to the constraints by being stereochemically complementary to a site between the β-chains ; this complementarity is lost on oxygenation.
The model has been constructed by combining information from the three-dimensional Fourier syntheses of horse oxyhaemoglobin at 5·5 Å and of sperm whale myoglobin at 1·4 Å resolution. Between them, these two sets of experimental data were sufficient to locate the positions of each of the haem groups and amino acid residues in haemoglobin within narrow limits. The nature of the residues was known from the chemical sequence. The accuracy of atomic positions is estimated to be of the order of 1 to 2 Å. The model has not yet been checked by comparison of observed and calculated intensities of X-ray reflexions.The model is sufficiently accurate, however, to show how the different kinds of residues are distributed between the interior and the surface of the four subunits; to locate the residues lying at the interfaces between the subunits and at the contacts between neighbouring molecules in the crystal; to determine the surroundings of the haem groups; and to reveal the existence of an internal cavity which is populated by a variety of polar side-chains and is filled with water.The nature of the intra- and intermolecular forces and the dissociation properties of haemoglobin are discussed in the light of the model. Relations between structure and function are dealt with in subsequent papers.
The structures of the deoxy and carbonmonoxy derivatives of the insect haemoglobin erythrocruorin have been determined by difference Fourier syntheses. In deoxyerythrocruorin the iron atom of the haem group was found to be five-coordinated.The carbonmonoxy- versus deoxy- difference map showed that the carbon monoxide molecule is inclined to the haem plane. Slight structural changes could be observed to take place on carbon monoxide binding. These structural changes are probably due to the difference in spin state between deoxy- and carbonmonoxy haemoglobin.
The secondary structure of the haemoglobin chains is similar to that of myoglobin, but some of the helical segments are more irregular and some parts of the non-helical segments have different conformations. The structure of the contacts between unlike subunits suggests that the tetramer, rather than the αβ dimer, is the functional unit of haemoglobin.
The reaction between p-chloromercuribenzoate (PCMB) and haemoglobin has been investigated to define the optimum conditions for dissociation to the subunits. The production of various molecular species has been followed by electrophoresis, gel filtration and ultracentrifugation. The reagent was applied to human haemoglobins A, S, C, Ja (Glasgow), A2, H and F, and also to the haemoglobins of dog, rabbit, horse and pig. Evidence is presented for the occurrence of haemoglobins without α-chains containing dissimilar β-chains, i.e. β2Yβ2X The behaviour of haemoglobins in the presence of PCMB was found to correlate qualitatively and quantitatively with the nature and number of their sulphydryl groups. It is shown that attachment of reagent at position β-93 causes initial dissociation of the tetramer to the dimer: α2β2ag 2αβ. From the location of these reactive sulphydryls it is concluded that the initial dissociation is along the bc plane of the haemoglobin molecule with the formation of two α1β1 dimers. Further dissociation by PCMB at pH 6.0 only occurred with haemoglobins containing a second sulphydryl group on the β-chain (β-112). From the data it is concluded that substitution at both unreactive thiols β-112 and α-104 causes final dissociation to the monomeric species. The contribution of the various sulphydryl groups to dissociation was also indicated by the behaviour of alkylated haemoglobins when treated with PCMB. Evidence is presented that dissociation of haemoglobin to half-molecules in several media occurs across the same plane as that initially affected by PCMB. The connection between the sigmoid oxygen tension curve and dissociation is discussed.
Hemoglobin Yakima. II. High blood oxygen affinity associated with compensatory erythroeytosis and normal hemodynamics Reaction of n-butylisoeyanide with human hemoglobin The magnetic properties and structure of hemoglobin, oxyhemoglobin and carbonmonoxyhemogh)bin
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On the nature of allosteric transitions: implications of non-exclusive ligand binding Studies of the interaction of 2,3-diphosphoglycerate and CO~ with hemoglobins of mouse, man and elephant Effect of
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