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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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 ◽

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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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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A constant-volume ultrafiltration technique for the calculation of equilibrium binding data

Biochimica et Biophysica Acta (BBA) - General Subjects ◽

10.1016/0304-4165(78)90057-0 ◽

1978 ◽

Vol 543 (3) ◽

pp. 397-402 ◽

Cited By ~ 5

Author(s):

Barry W.A. Williamson ◽

Richard C. Strange ◽

Iain W. Percy-Robb

Keyword(s):

Constant Volume ◽

Equilibrium Binding ◽

Binding Data

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Conformation of the sebacyl β1Lys82–β2Lys82 crosslink in T-state human hemoglobin

Proteins Structure Function and Bioinformatics ◽

10.1002/(sici)1097-0134(19980215)30:3<309::aid-prot10>3.0.co;2-j ◽

1998 ◽

Vol 30 (3) ◽

pp. 309-320 ◽

Cited By ~ 4

Author(s):

Xinhua Ji ◽

Michael Braxenthaler ◽

John Moult ◽

Clara Fronticelli ◽

Enrico Bucci ◽

...

Keyword(s):

Human Hemoglobin ◽

T State

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Temperature-dependent binding of cyclosporine to an erythrocyte protein.

Clinical Chemistry ◽

10.1093/clinchem/33.4.481 ◽

1987 ◽

Vol 33 (4) ◽

pp. 481-485 ◽

Cited By ~ 11

Author(s):

R P Agarwal ◽

G A Threatte ◽

R A McPherson

Keyword(s):

Binding Constant ◽

Binding Capacity ◽

Equilibrium Dialysis ◽

Temperature Dependent ◽

Fixed Amount ◽

Competitive Binding Assay ◽

Plot Analysis ◽

Binding Data ◽

Saturation Capacity ◽

Increasing Temperature

Abstract In this competitive binding assay to measure endogenous binding capacity for cyclosporine (CsA) in erythrocyte lysates, a fixed amount of [3H]CsA plus various concentrations of unlabeled CsA is incubated with aliquots of a test hemolysate. Free CsA is then adsorbed onto charcoal and removed by centrifugation; CsA complexed with a cyclosporine-binding protein (CsBP) remains in the supernate. We confirmed the validity of this charcoal-separation mode of binding analysis by comparison with equilibrium dialysis. Scatchard plot analysis of the results at 4 degrees C yielded a straight line with slope corresponding to a binding constant of 1.9 X 10(7) L/mol and a saturation capacity of approximately 4 mumol per liter of packed erythrocytes. Similar analysis of binding data at 24 degrees C and 37 degrees C showed that the binding constant decreased with increasing temperature, but the saturation capacity did not change. CsBP was not membrane bound but appeared to be freely distributed within erythrocytes. 125I-labeled CsA did not complex with the erythrocyte CsBP. Several antibiotics and other drugs did not inhibit binding between CsA and CsBP. These findings may explain the temperature-dependent uptake of CsA by erythrocytes in whole blood and suggest that measurement of CsBP in erythrocytes or lymphocytes may help predict therapeutic response or toxicity after administration of CsA.

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The Binding Of 125I-Von Willebrand Factor To Human Platelets In The Presence Of Ristocetin

10.1055/s-0038-1652985 ◽

1981 ◽

Author(s):

P Silber ◽

T H Finlay

Keyword(s):

Von Willebrand Factor ◽

Binding Sites ◽

Binding Constant ◽

Specific Binding ◽

Human Platelets ◽

Scatchard Analysis ◽

Binding Data ◽

Number Of Binding Sites ◽

Von Willebrand ◽

Willebrand Factor

The effect of ristocetin on the binding of 125I-porcine von Willebrand factor to human platelets was studied. Previously, we had shown that 125I-porcine von Willebrand factor binds to human platelets in the absence of ristocetin. The present work demonstrates that binding is stimulated by ristocetin and this stimulation is maximal at a ristocetin concentration of 2 mg/ml. At a ristocetin concentration of 0.5 mg/ml, Scatchard analysis indicates a binding constant of 5.18 × 10-9M and the presence of 105,000 binding sites. This compares with our previous finding, in the absence of ristocetin, of a binding constant of 2.92 × 10-7M and 4760 binding sites. These binding data assume the porcine von Willebrand factor to be a tetramer with a molecular weight of 9 × 105. This study indicates that ristocetin causes tighter binding and increases the number of binding sites on human platelets for porcine von Willebrand factor. Unlabelled porcine von Willebrand factor competitively inhibits the specific binding of the labelled protein and gives a binding constant of 0.17 × 10-9M. Similar results were obtained using human von Willebrand factor.

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