Architecture of Allosteric Structure. Equation of State Containing Three Unknown Quantities for Fractional Saturation of whole Human Blood with O2: Formatting the Adair Equation to Accommodate Ordered Equivalent Sequences of O2-Binding Reactions and a Single Conformation Change Not Concerted with O2 Binding

10.26434/chemrxiv.10721087.v1 ◽

2019 ◽

Author(s):

Francis Knowles ◽

Douglas Magde

Keyword(s):

Equation Of State ◽

Equilibrium Constant ◽

Oxygen Affinity ◽

Conformation Change ◽

Binding Model ◽

High Affinity ◽

Binding Data ◽

Sequential Binding ◽

Cell Acid ◽

O2 Binding

O2-Equilibrium binding data of hemoglobin in whole blood under standard conditions (Kernohan JC. & Roughton FJW (1972) in Oxygen Affinity of Hemoglobin and Red Cell Acid Base Status, ed Rorth and Astrup, Copenhagen, Munksgaard, pp 65-72; Severinghaus JW in ibid pp. xx-xx) was fitted to an equation of state comprised of three unknown quantities: Kα, the equilibrium constant for binding O2 by equivalent low affinity α-chains; KΔC, a dimensionless equilibrium constant describing the conformation change between low- and high-affinity conformations of hemoglobin, Tstate and Rstate; Kβ, the equilibrium constant for binding O2 by equivalent high affinity β-chains, the Perutz/Adair Equation. Values of the unknown quantities at pH 7.4 and 37oC are: Kα = 15,090 L/mol; KΔC = 0.0260; Kβ = 393,900 L/mol. A graph of predicted versus observed values of fractional saturation, F, is linear: FPRE = 0.9998 FOBS – 0.0005, r2 =0.9997. The Perutz/Adair equation of state is defined as such insofar as all aspects of the stereochemical model (Perutz MF (1970) Nature London 228, 726-739) are imposed on the earlier sequential binding model of Adair (1925) JBC 63, 493-545.

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Hemoglobin Non-equilibrium Oxygen Dissociation Curve

10.1101/2020.01.09.900001 ◽

2020 ◽

Author(s):

Rosella Scrima ◽

Sabino Fugetto ◽

Nazzareno Capitanio ◽

Domenico L. Gatti

Keyword(s):

Partial Pressure ◽

Correct Diagnosis ◽

Oxygen Affinity ◽

Oxygen Dissociation Curve ◽

Dissociation Curve ◽

High Affinity ◽

Oxygen Dissociation ◽

Sequential Binding ◽

Non Equilibrium

AbstractAbnormal hemoglobins can have major consequences for tissue delivery of oxygen. Correct diagnosis of hemoglobinopathies with altered oxygen affinity requires a determination of hemoglobin oxygen dissociation curve (ODC), which relates the hemoglobin oxygen saturation to the partial pressure of oxygen in the blood. Determination of the ODC of human hemoglobin is typically carried out under conditions in which hemoglobin is in equilibrium with O2 at each partial pressure. However, in the human body due to the fast transit of RBCs through tissues hemoglobin oxygen exchanges occur under non-equilibrium conditions. We describe the determination of non-equilibrium ODC, and show that under these conditions Hb cooperativity has two apparent components in the Adair, Perutz, and MWC models of Hb. The first component, which we call sequential cooperativity, accounts for ∼70% of Hb cooperativity, and emerges from the constraint of sequential binding that is shared by the three models. The second component, which we call conformational cooperativity, accounts for ∼30% of Hb cooperativity, and is due either to a conformational equilibrium between low affinity and high affinity tetramers (as in the MWC model), or to a conformational change from low to high affinity once two of the tetramer sites are occupied (Perutz model).

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Resolution of the Equilibrium Constant for the T State → RState Conformational Change of Human Hemoglobin into Endothermic and Exothermic Component Reactions

10.26434/chemrxiv.10838249.v1 ◽

2019 ◽

Author(s):

Francis Knowles ◽

Douglas Magde

Keyword(s):

Equilibrium Constant ◽

Whole Blood ◽

Binding Constant ◽

Binary Complex ◽

Human Hemoglobin ◽

Conformation Change ◽

Unknown Quantity ◽

Equilibrium Binding ◽

Binding Data ◽

T State

The dimensionless equilibrium constant for the allosteric conformation change, KΔC = 0.02602 (Knowles & Magde, linked ms 2) following binding of O2 by α-chains in Tstate Hb4/BPG (whole blood under standard conditions) is shown to be comprised of: (i) an endothermic change in conformation, from Tstate to Rstate, of 24.3 kJ/mol; (ii) exothermic conversion of Tstate TαO2-chains to Rstate RαO2-chains of -13.8 kJ/mol; (iii)exothermic binding of BPG by R-states. Eq. (1) defines the component steps whereby the Tstate conformation is converted to the Rstate conformation. ΔGo(R(Hb4), BPG) describes the endothermic decomposition of the binary complex, THb4/BPG into RHb4 and BPG, equal to + 33.7 kJ/mol (DeBruin et al. (1973). J. Biol. Chem. 248, 2774-2777). ΔGo for the equilibrium constant for ΔGO(KΔC) and Σ ΔGo for binding of O2 by the pair of equivalent Tstate α-chains, ΔGO(Tα*O2), + 9.41 kJ/mol and – 49.6 kJ/mol, respectively, are determined by fitting of O2 equilibrium binding data to the Perutz-Adair equation. ΔGo for reaction of a pair of equivalent Rstate α-chains with O2, ΔGO(RαO2), was estimated from the known affinity of myoglobin for O2 at 37oC (Theorell H. (1936). Biochem. Z., 268, 73-81), -63.4 kJ/mol. The unknown quantity, ∆GO(R(HbO2)4/BPG), was obtained by solving Eq. (1), being -10.5 kJ/mol, K (R(HbO2)4/BPG) = 58.4 L/mol. The value of the equilibrium constant for binding BPG to R-state conformations represents 0.0073% of the value of the binding constant of BPG to Tstate conformations: 800,000 L/mol. The value of KΔC; (i) accounts for the ability of O2 to escape, virtually unhindered from rbcs and (ii) provides a biophysical basis for manifestation of high resting rates of metabolism in warm blooded species.

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Anion transport inhibitor binding to band 3 in red blood cell membranes.

The Journal of General Physiology ◽

10.1085/jgp.81.3.421 ◽

1983 ◽

Vol 81 (3) ◽

pp. 421-449 ◽

Cited By ~ 54

Author(s):

A S Verkman ◽

J A Dix ◽

A K Solomon

Keyword(s):

Conformational Change ◽

Temperature Jump ◽

Band 3 ◽

Anion Transport ◽

Stopped Flow ◽

Binding Studies ◽

Binding Model ◽

High Affinity ◽

Sequential Binding ◽

Red Blood Cell Membranes

The inhibitor of anion exchange 4,4'-dibenzoamido-2,2'-disulfonic stilbene (DBDS) binds to band 3, the anion transport protein in human red cell ghost membranes, and undergoes a large increase in fluorescence intensity when bound to band 3. Equilibrium binding studies performed in the absence of transportable anions show that DBDS binds to both a class of high-affinity (65 nM) and low-affinity (820 nM) sites with stoichiometry equivalent to 1.6 nmol/mg ghost protein for each site, which is consistent with one DBDS site on each band 3 monomer. The kinetics of DBDS binding were studied both by stopped-flow and temperature-jump experiments. The stopped-flow data indicate that DBDS binding to the apparent high-affinity site involves association with a low-affinity site (3 microM) followed by a slow (4 s-1) conformational change that locks the DBDS molecule in place. A detailed, quantitative fit of the temperature-jump data to several binding mechanisms supports a sequential-binding model, in which a first DBDS molecule binds to one monomer and induces a conformational change. A second DBDS molecule then binds to the second monomer. If the two monomers are assumed to be initially identical, thermodynamic characterization of the binding sites shows that the conformational change induces an interaction between the two monomers that modifies the characteristics of the second DBDS binding site.

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Resolution of the Equilibrium Constant for the T State → RState Conformational Change of Human Hemoglobin into Endothermic and Exothermic Component Reactions

10.26434/chemrxiv.10838249 ◽

2019 ◽

Author(s):

Francis Knowles ◽

Douglas Magde

Keyword(s):

Equilibrium Constant ◽

Whole Blood ◽

Binding Constant ◽

Binary Complex ◽

Human Hemoglobin ◽

Conformation Change ◽

Unknown Quantity ◽

Equilibrium Binding ◽

Binding Data ◽

T State

The dimensionless equilibrium constant for the allosteric conformation change, KΔC = 0.02602 (Knowles & Magde, linked ms 2) following binding of O2 by α-chains in Tstate Hb4/BPG (whole blood under standard conditions) is shown to be comprised of: (i) an endothermic change in conformation, from Tstate to Rstate, of 24.3 kJ/mol; (ii) exothermic conversion of Tstate TαO2-chains to Rstate RαO2-chains of -13.8 kJ/mol; (iii)exothermic binding of BPG by R-states. Eq. (1) defines the component steps whereby the Tstate conformation is converted to the Rstate conformation. ΔGo(R(Hb4), BPG) describes the endothermic decomposition of the binary complex, THb4/BPG into RHb4 and BPG, equal to + 33.7 kJ/mol (DeBruin et al. (1973). J. Biol. Chem. 248, 2774-2777). ΔGo for the equilibrium constant for ΔGO(KΔC) and Σ ΔGo for binding of O2 by the pair of equivalent Tstate α-chains, ΔGO(Tα*O2), + 9.41 kJ/mol and – 49.6 kJ/mol, respectively, are determined by fitting of O2 equilibrium binding data to the Perutz-Adair equation. ΔGo for reaction of a pair of equivalent Rstate α-chains with O2, ΔGO(RαO2), was estimated from the known affinity of myoglobin for O2 at 37oC (Theorell H. (1936). Biochem. Z., 268, 73-81), -63.4 kJ/mol. The unknown quantity, ∆GO(R(HbO2)4/BPG), was obtained by solving Eq. (1), being -10.5 kJ/mol, K (R(HbO2)4/BPG) = 58.4 L/mol. The value of the equilibrium constant for binding BPG to R-state conformations represents 0.0073% of the value of the binding constant of BPG to Tstate conformations: 800,000 L/mol. The value of KΔC; (i) accounts for the ability of O2 to escape, virtually unhindered from rbcs and (ii) provides a biophysical basis for manifestation of high resting rates of metabolism in warm blooded species.

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The Binding of Calcium Ions to Bovine Factor X by Rate Dialysis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648668 ◽

1978 ◽

Vol 40 (02) ◽

pp. 350-357

Author(s):

Robert H Yue ◽

Menard M Gertler

Keyword(s):

Molecular Weight ◽

Dissociation Constant ◽

Calcium Ions ◽

Activation Process ◽

Factor X ◽

The Real ◽

High Affinity ◽

Binding Data ◽

Sequential Mechanism ◽

Bovine Factor

SummaryThe binding of Ca+2 to bovine factor X (molecular weight of 74,000) (Yue und Gertler 1977) was studied by the technique of rate dialysis and with the use of 45Ca+2. The binding data are consistent with a model of sequential mechanism. One mole of Ca+2 binds to the glycoprotein with a dissociation constant of 5.2 × 10-5 M and an additional 39 ± 4 moles of Ca+2 bind to this zymogen with a dissociation constant of 3.7 × 10-3M. The binding of the high affinity Ca+2 causes a functionally significant change in the zymogen, and (calcium) (factor X) complex is the real substrate in the activation process by the protease in Russell’s viper venom.

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Hb Potomac (101 Glu replaced by Asp): speculations on placental oxygen transport in carriers of high-affinity hemoglobins

Blood ◽

10.1182/blood.v51.2.331.331 ◽

1978 ◽

Vol 51 (2) ◽

pp. 331-338 ◽

Cited By ~ 38

Author(s):

S Charache ◽

R Jacobson ◽

B Brimhall ◽

EA Murphy ◽

P Hathaway ◽

...

Keyword(s):

Oxygen Transport ◽

Oxygen Affinity ◽

Normal Children ◽

Tryptic Peptides ◽

High Affinity ◽

Female Carriers

Abstract Blood from a woman with unexplained erythrocytosis had increased oxygen affinity, but no abnormality could be detected by electrophoresis or chromatography of her hemolysate. Separation of the tryptic peptides of her beta chains disclosed two half-sized peaks in the regions of beta T- 11. The faster of these was abnormal, with the structure beta 101 Glu replaced by Asp. The new hemoglobin was called “Potomac.” Three of the proband's four surviving siblings and both of her children were carriers. Differences in the ratio of carrier: normal children born to male of female carriers of 23 other high-affinity hemoglobins were not significant. The high proportion of carriers in this kindred was probably due to chance alone, and not because high maternal oxygen affinity interfered with oxygen transport to fetuses with normal hemoglobin.

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Chloroquine augments the binding of insulin to its receptor

Biochemical Journal ◽

10.1042/bj3110787 ◽

1995 ◽

Vol 311 (3) ◽

pp. 787-795 ◽

Cited By ~ 10

Author(s):

A P Bevan ◽

J R Christensen ◽

J Tikerpae ◽

G D Smith

Keyword(s):

Binding Sites ◽

Slow Component ◽

Insulin Binding ◽

Dissociation Rate ◽

Receptor Interaction ◽

Dependent Manner ◽

Binding Model ◽

Binding Data ◽

Plasma Membrane Receptor ◽

Dissociation Rate Constants

The effect of chloroquine on the interaction of insulin with its receptor has been investigated under both equilibrium and non-equilibrium conditions. Chloroquine was found to augment insulin binding in a pH-dependent manner between pH 6.0 and pH 8.5, with the maximum occurring at approximately pH 7.0. Analysis of the equilibrium binding data in terms of independent binding sites gave equivocal results but suggested an increase in the high-affinity component. Analysis using the negative co-operativity binding model of De Meyts, Bianco and Roth [J. Biol. Chem. (1976) 251, 1877-1888] suggested that the affinity at both high and low occupancy was increased equally. The kinetics of association of insulin with the plasma-membrane receptor indicated that, although the net rate of association increased in the presence of chloroquine, this was due to a reduction in the dissociation rate rather than an increase in the association rate. This was confirmed by direct measurement of the rates of dissociation. Dissociation was found to be distinctly biphasic, with fast and slow components. Curve fitting suggested that the decrease in dissociation rate in the presence of chloroquine was not due to a decrease in either of the two dissociation rate constants, but rather to an increase in the amount of insulin dissociating by the slow component. It was also found that the increase in dissociation rate in the presence of excess insulin, ascribed to negative co-operativity, could be accounted for by an increase in the amount of insulin dissociating by the faster pathway, rather than by an increase in the dissociation rate constant. Thus chloroquine appears to have the opposite effect to excess insulin, and evidence was found for the induction of positive co-operativity in the insulin-receptor interaction at high chloroquine concentrations. Evidence was also found for the presence of low-affinity chloroquine binding sites with binding parameters similar to the concentration dependence of the chloroquine-induced augmentation of insulin binding.

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Discrimination of chiral copper(ii) complexes upon binding of galactonoamidine ligands

Dalton Transactions ◽

10.1039/c6dt02153k ◽

2016 ◽

Vol 45 (38) ◽

pp. 15203-15210 ◽

Cited By ~ 5

Author(s):

Susanne Striegler ◽

Jessica B. Pickens

Keyword(s):

Binding Model ◽

Coordination Sites ◽

Sequential Binding

Chiral binuclear Cu(ii) complexes are differentiated upon binding top-methylbenzyl-d-galactonoamidine. A sequential binding model is elaborated reflecting the altered coordination sites.

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Hemoglobin Crete (beta 129 ala leads to pro): a new high-affinity variant interacting with beta o -and delta beta o -thalassemia

Blood ◽

10.1182/blood.v54.1.54.54 ◽

1979 ◽

Vol 54 (1) ◽

pp. 54-63 ◽

Cited By ~ 34

Author(s):

A Maniatis ◽

T Bousios ◽

RL Nagel ◽

T Balazs ◽

Y Ueda ◽

...

Keyword(s):

Cell Morphology ◽

Clinical Picture ◽

Oxygen Affinity ◽

High Oxygen ◽

Red Cell ◽

High Affinity ◽

Contact Sites ◽

Erythroid Hyperplasia ◽

Proline Substitution ◽

High Oxygen Affinity

Abstract Hemoglobin Crete, beta129 (h7)ala leads to pro, is a new mutant hemoglobin (Hb) with high oxygen affinity that was discovered in a Greek family in various combinations with beta- and deltabeta- thalassemia. The propositus, who presented an unusual clinical picture of an “overcompensated” hemolytic state, with erythrocytosis, splenomegaly, abnormal red cell morphology, and marked erythroid hyperplasia, appeared doubly heterozygous for Hb Crete and deltabeta- thalassemia. His red cells contained 67% Hb Crete and 30% Hb F, and the combination of these two hemoglobins resulted in a blood P50O2 of 11.2 mm Hg. A brother with Hb Crete trait (38% Hb Crete, 56% Hb A, blood P50O2 23.0 mm Hg) did not have significant erythrocytosis. Purified Hb Crete was heat-unstable and exhibited a high oxygen affinity, and a normal Bohr effect. We postulate that the beta 129 proline substitution disrupts the H helix, perturbing nearby residues involved in alpha 1 beta 1 contact sites of the Hb tetramer.

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