The pH dependence of the affinity,kinetics and cooperativity of ligand binding to the root effect haemoglobin of the goldfish Carassius auratus

• 1.1. The binding of O₂ to goldfish haemoglobin showed a strong pH dependence P₅₀=5.5 mmHg; n = 2.4 at pH 8.0 and P₅₀ = 170 mmHg; n = 1.0 at pH 5.5 such that the protein is only 50% saturated in a solution of air equilibrated buffer at pH 5.5.
• 2.2. The binding of CO is cooperative at high pH (n = 2.8; L = 1000; K_R = 0.1 μM; K_T = 4 μM) and non-cooperative (n = 1) at pH 5.5.
• 3.3. The rate of O₂ dissociation is extremely fast and pH dependent; being 30 sec⁻¹ at pH 8.0 and 400 sec⁻¹ at pH 6.0 at 1°C. At 23°C the rate of this process is too fast to obtain accurate data using stopped-flow techniques.
• 4.4. Partial photolysis of the oxyhaemoglobin species leads to homogeneous recombination kinetics at pH 8.0 with an associated rate constant of 4.7 × 10⁷ M⁻¹ sec⁻¹. At pH < 7.5 the recombination process occurs in two steps. One rate is equal to that observed at pH 8.0. The slower process is favoured at low pH.
• 5.5. Photolysis of the CO haemoglobin complex indicates that, at high pH, combination of CO with deoxyhaemoglobin is cooperative, whilst recombination with Hb(CO)₃ is non-cooperative and occurs at a rate of 1.2 × 10⁶ M⁻¹ sec⁻¹.
• 6.6. At neutral pH recombination of CO with partially linganded haemoglobin occurs in a two-step process. The proportion contributed by each of these two steps in pH dependent.
• 7.7. The functioning of this Root effect haemoglobin is discussed in terms of the two state-model of cooperativity in which the αβ chain heterogeneity is minimal

     

Thermodynamic aspects of the co-operativity in four-step oxygenation equilibria of haemoglobin

Accurate oxygen equilibrium curves of human haemoglobin (concentration, 600 μm as haem) were determined by an automatic recording method (Imai et al., 1970) under a variety of conditions combining six different temperatures with seven sets of solute conditions, producing wide-ranging structural constraints on haemoglobin. The heat and entropy change of oxygenation for four individual steps (ΔH_tand ΔS_i, i = 1 to 4) were evaluated by a least-squares method directly from each set of six equilibrium curves without knowing the values of the four equilibrium constants k_t. As shown in previous studies with dilute haemoglobin solutions (Imai &; Tyuma, 1973; Imai &; Yonetani, 1975b) ΔH_i depended strongly on i; small amounts of heat were liberated at oxygenation steps involving the release of H⁺ and anions such as Cl^?, 2,3-diphosphoglycerate, and inositol hexaphosphate, while large amounts of heat were liberated on the oxygenation of the R state or highly constrained T state, from which no or few non-haem ligands are released. The observed amounts of heat, when corrected for the heat of H⁺ and anion release associated with oxygenation, became uniform to a good approximation, indicating that the intrinsic heat of haem oxygenation is essentially equal for the four oxygenation steps, and a large part of the non-uniformity of ΔH_i may be ascribed to the oxygen-linked release of the non-haem ligands. ΔS_i exhibited similar behaviour. The relation, k₁ ? k₂ ? k₃ ? k₄ which usually holds under physiological conditions, is a consequence of the presence of an enthalpy-entropy compensation process at the first three steps and its absence at the fourth step. The compensation temperature was around 300 K. The origin of the co-operativity cannot be specified as either an enthalpic or entropic effect. In the presence of 0.1 m-Cl^? and 2 mm-2,3-diphosphoglycerate, the T to R transition at any oxygenation step is an endothermic process and haemoglobin gains entropy on the transition. The deoxy T structure is stabilised by the enthalpy term, while the oxy R structure is stabilised by the entropy term, so that the T to R transition occurs at a stop where the entropy contribution exceeds the enthalpy contribution. The present study shows that the oxygen-linked binding of non-haem ligands is very important in co-operative oxygen binding by haemoglobin, as predicted by Perutz, (1970).     

Cytochrome f and plastocyanin kinetics in Chlorella pyrenoidosa. I. Oxidation kinetics after a flash

P-700, plastocyanin and cytochrome f redox kinetics were measured after one flash, using dark-adapted Chlorella in the presence of hydroxylamine and 3(3,4-dichlorophenyl)-1,1-dimethylurea. Plastocyanin becomes increasingly oxidized with a half-time of 70 μs, then undergoes reduction with a half-time of 7 ms. Cytochrome f oxidation has a sigmoidal time-course and a half-time of 100 μs. Its reduction exhibits a half-time of 4 ms. These results are interpreted in a linear scheme:

An equilibrium constant of 2 between cytochrome f and plastocyanin (PC), which contrasts with the large equilibrium constant between PC and P-700 is computed.The presence of cytochrome b₆ in a cyclic path around Photosystem I is confirmed under these conditions.     

Molecular structure and linkage isomerization of isothiocyanato(3-thiapentane-1,5-dithiolato)oxorhenium(V) complex

The properties of isothiocyanato(3-thiapentane-1,5-dithiolato)oxorhenium(V) [ReO(SSS)NCS, (1a), (3+1) type], where isothiocyanato occupies the fifth position, have been studied. Two linkage isomers, i.e., ReO(SSS)NCS (1a) and ReO(SSS)SCN (1b), were found to be formed during syntheses. The sufficient quantities of 1a were isolated in the solid state, and characterized by X-ray crystallography and IR spectroscopy. From ¹H and ¹⁵N NMR measurements, it was found that 1a is in equilibrium with 1b in liquid state. In the solvents with low dielectric constant such as CH₂Cl₂, only 1a isomer was detected, while in the solvents with high such as CH₃CN, both 1a and 1b isomers were observed. We have obtained the equilibrium constant (K_iso) for the linkage isomerization reaction in CD₃CN by measuring ¹⁵N NMR spectra at various temperatures. The values of K_iso at 25 °C, the standard enthalpy (ΔH^°), and the standard entropy (ΔS^°) for the isomerization equilibrium were evaluated as 0.409, 14.4 kJ mol⁻¹, and 40.9 J K⁻¹ mol⁻¹, respectively.     

Spectrin interactions with globin chains in the presence of phosphate metabolites and hydrogen peroxide: implications for thalassaemia

We have shown the differential interactions of the erythroid skeletal protein spectrin with the globin subunits of adult haemoglobin (HbA); these indicate a preference for α-globin over that for β-globin and intact HbA in an adenosine 5′-triphosphate (ATP)-dependent manner. The presence of Mg/ATP led to an appreciable decrease in the binding affinity of the α-globin chain to spectrin and the overall yield of globin-spectrin cross-linked complexes formed in the presence of hydrogen peroxide. Similar effects were also seen in the presence of 2-,3-diphosphoglycerate (2,3 DPG), the other important phosphate metabolite of erythrocytes. The binding affinity and yield of cross-linked high molecular weight complexes (HMWCs) formed under oxidative conditions were significantly higher in α-globin compared with intact haemoglobin, HbA and the β-globin chain. The results of this study indicate a possible correlation of the preferential spectrin binding of the α-globin chain over that of the β-globin in the haemoglobin disorder β-thalassaemia.     

The basic flow equations of electrophysiology in the presence of chemical reactions: I. Development of the equations

Starting from the basic flux equation, it is possible to obtain an integral form relating the current componentsI _i at an arbitrary pointr ₂ to the distribution of mobilities and concentrationsc _i, potential forces\(\bar \mu \), and chemical productivityp _i without any restrictive assumptions such as constant mobilities, constant field, steady state, or electrical neutrality. The equation is

$$\begin{gathered} I_i (r_2 ) = G_i (r_2 )\left[ {\Delta \bar \mu _i - \int_{r_1 }^{r_2 } {z_i } FA\left( {p_i - dc_i /dt} \right)\left( {\frac{1}{{G_i (r)}}} \right)dr} \right]; \hfill \\ G_i (r) = 1/\int_{r_1 }^r {\frac{{dr}}{{z_i^2 F^2 c_i u_i }}.} \hfill \\ \end{gathered} $$     

Modulation of the Voltage-gated Potassium Channel (Kv4.3) and the Auxiliary Protein (KChIP3) Interactions by the Current Activator NS5806

KChIP3 (potassium channel interacting protein 3) is a calcium-binding protein that binds at the N terminus of the Kv4 voltage-gated potassium channel through interactions at two contact sites and has been shown to regulate potassium current gating kinetics as well as channel trafficking in cardiac and neuronal cells. Using fluorescence spectroscopy, isothermal calorimetry, and docking simulations we show that the novel potassium current activator, NS5806, binds at a hydrophobic site on the C terminus of KChIP3 in a calcium-dependent manner, with an equilibrium dissociation constant of 2–5 μm in the calcium-bound form. We further determined that the association between KChIP3 and the hydrophobic N terminus of Kv4.3 is calcium-dependent, with an equilibrium dissociation constant in the apo-state of 70 ± 3 μm and 2.7 ± 0.1 μm in the calcium-bound form. NS5806 increases the affinity between KChIP3 and the N terminus of Kv4.3 (K_d = 1.9 ± 0.1 μm) in the presence and absence of calcium. Mutation of Tyr-174 or Phe-218 on KChIP3 abolished the enhancement of Kv4.3 site 1 binding in the apo-state, highlighting the role of these residues in drug and K4.3 binding. Kinetic studies show that NS5806 decreases the rate of dissociation between KChIP3 and the N terminus of K_V4.3. Overall, these studies support the idea that NS5806 directly interacts with KChIP3 and modulates the interactions between this calcium-binding protein and the T1 domain of the Kv4.3 channels through reorientation of helix 10 on KChIP3.     

Construction of a series of mercury(II) complexes based on a bis-pyridyl-bis-amide ligand: Effect of counter anions, interactions on the supermolecular structures

Four new complexes, [Hg(L)Cl₂]₂ (1), [Hg(L)Br₂]₂ (2), [Hg(L)I₂(DMF)₂]_n (3), and [HgLCl(SCN)]_n (4) (L = N,N-bis-(3-pyridyl)isophthalamide) were obtained through the self-assembly of a rigid conjugated clamp-like bis-pyridyl-bis-amide ligand L with HgX₂ (X = Cl for 1, Br for 2, I for 3, and Cl for 4 with the addition of KSCN) and characterized by single crystal X-ray diffraction, elemental analysis, IR spectrum, etc. Employments of different anions result in different structures. Complexes 1 and 2 feature bimetallic macrocycle formed by coordinating two Hg(II) metal centers by two ligands which are in syn-syn conformation. The macrocyclic subunits further self-assemble into a porous macrocycle structure via the hydrogen-bonding and π-π stacking interactions. Introduction of I⁻ and SCN⁻ ions bring about stronger steric hindrance effect. Complexes 3 and 4 are polymers with infinite 1D polymeric chain in herringbone fashion and the hydrogen-bonding interactions and π-π stacking interactions between the parallel benzene rings and the pyridyl rings stabilize the supromolecular framework. Furthermore, we measured their fluorescent properties in the solid state at room temperature and XRD properties also have been determined.     

Assembly of metal coordination framework, [M(C5O5)(dpe)], with a 2D bi-layer architecture: Thermal stability and magnetic properties (M = Mn, Fe, Cd and Co; dpe = 1,2-bis(4-pyridyl)ethane)

A series of coordination polymers, [M(C₅O₅)(dpe)] (M = Mn 1, Fe 2, Cd 3 and Co 4; dpe = 1,2-bis(4-pyridyl)ethane) with a 2D metal-organic framework (MOF) has been synthesized and characterized by single-crystal X-ray diffraction studies. Structural determination reveals that compounds 1-4 are isostructural and possess a 2D brick-wall-like layered framework with a rectangle grid as the basic building unit through the connectivity of metal ions with μ₃-, μ₄-croconates and anti-dpe ligands. Two 2D layers are then cross-linkaged by gauche-dpe ligands to complete a 2D bi-layered MOF and then extended to a 3D supramolecular architecture through π-π and C-H?O interactions between the croconate and pyridyl rings of dpe, which exhibit high thermo-stability and keep their crystalline forms up to 350 °C. The magnetic exchange coupling between the metal centers for compounds 1, 2 and 4 was analyzed and based on the Curie-Weiss expression and a binuclear magnetic model. The negative values of the Weiss constant and the magnetic exchange coupling constant indicate the antiferromagnetic nature between the metal ions via the bridges of bis-bidentate adjacent μ₃-croconate ligands.     

Four metal-organic networks based on benzene-1,4-dioxyacetic acid and dipyrido[3,2-a:2′,3′-c]phenazine ligand

Four structurally diverse complexes, [Cd(dppz)(bdoa)]_n (1), [Zn(dppz)(bdoa)(H₂O)]_n (2), [Fe(dppz)₂(bdoa)]_n·2nH₂O (3), and [Co₂(dppz)₂(bdoa)₂(H₂O)]_n·3nH₂O (4), where H₂bdoa = benzene-1,4-dioxyacetic acid and dppz = dipyrido[3,2-a:2′,3′-c]phenazine, have been hydrothermally synthesized. Compounds 1-4 feature chain structures. There exist π-π interactions in the structures of 1, 2 and 4. Two neighboring chains of 1 are linked through the π-π interactions into a double chain supramolecular structure. The chains of 2 and 4 are further extended by the π-π interactions to form 3D and 2D supramolecular structures, respectively. The structural differences among such complexes show that the transition metals have important influences on their structures. The photoluminescent property of complex 2 and the magnetic property of complex 4 have also been investigated.     

A new β-chain haemoglobin variant with increased oxygen affinity: Hb Roma [β115(g17)Ala → Val]

Background

Haemoglobin Roma [β115(G17)Ala → Val] is a new adult haemoglobin variant found in a patient presenting a mild hypochromia and microcytosis. We studied this previously uncharacterised variant in order to evaluate the effect on the structural and funcional properties of the Ala → Val substitution at the α1β1 interface.

Methods and results

The variant chain was identified by direct DNA sequencing of the β-globin gene, which revealed a GCC → GTC mutation in codon 115. This mutation was confirmed by mass spectrometric analysis of the tetramers and peptides. The oxygen-binding properties of the haemoglobin A/haemoglobin Roma mixture, in which the variant makes up 25% of the haemoglobins, showed a significant increase in oxygen affinity with respect to normal haemoglobin A, both in the absence and presence of 2,3-bisphosphoglycerate. The role of the βG17 position, situated at the α₁β₁ interface, has been examined using computational models of haemoglobin Roma and other known βG17 variants, in comparison with normal haemoglobin A.

Conclusions

This study suggests that the β115(G17)Ala → Val substitution at the α1β1 interface is responsible for increased oxygen affinity and mild destabilisation of the haemoglobin Roma.

General significance

An amino acid substitution at the G17 position of the α1β1 interface may result in stabilisation of the high affinity R-state of the haemoglobin molecule.     

Synthesis, structures and conductivity properties of silver 3-sulfobenzoate coordination polymers

Four coordination polymers based on Ag^I/3-sulfobenzoate/N-donor ligands, [Ag₂Na₂(3-sb)₂(H₂O)₇]_n (1), {[Ag₂(3-sb)(apy)]·(H₂O)}_n (2), {[Ag₂(4,4′-bipy)₂(H₂O)₃]·[Ag₂(4,4′-bipy)₂(H₂O)₂]·2(3-sb)·4(H₂O)}_n (3) and {[Ag(3-sb)(bpe)(H₂O)][Ag(bpe)(H₂O)]·3(H₂O)}_n (4) where 3-sb is 3-sulfobenzoate, apy is 2-aminopyridine, bipy is 4,4′-bipyridine and bpe is 1,2-bis(4-pyridyl)ethylene, were prepared and characterized, and their fluorescence and electric conductivity properties were studied. Complex 1 is a 3D architecture in which 3-sb ligands exhibit μ₄-κ¹(O1,O2-Ag): κ¹(O3,O5-Na) trans-trans coordination mode. The molecular structure of 2 is a 2D layer. Complexes 3 and 4 are cation-anion species and 1D polymers. In these complexes hydrogen bonds provide additional assembly forces, giving 3D hydrogen bonding networks for 1 and 3, and 2D layers for 2 and 4. Abundant weak interactions, such as Ag-Ag interactions in 1-3, Ag-π interactions in 1-4, π-π interactions in 1, 3-4, and C-H···π interactions in 3-4, also can be found. The weak interactions are strongly related to the fluorescence and electric conductivity properties, providing the way for understanding the relationship between structures and properties.