Strong Allosteric Effectors Research Articles

Intradimeric but not Interdimeric Interface Control the Function of Hemoglobin

Chemical modifications of the sulhydryl groups located at the α1β1 interface in tetrameric human hemoglobin -- a region usually considered inert-- altered significantly its oxygenation properties: both the affinity for oxygen and cooperative ligation were substantially reduced. Still, these derivatives responded to allosteric effectors, such as pH and organic phosphates. Electron paramagnetic resonance of the nitrosyl derivatives showed a hyperfine splitting around g ∼ 2 indicating the cleavage of the bond between the α-heme iron and the proximal histidine, which indicates the presence of a low affinity conformation of hemoglobin. Compared to native hemoglobin, a reduced dimerization was also confirmed by isothermal titration calorimetry.1H-NMR measurements, on the other hand, showed unexpected results. Both the O2-ligated and CO-ligated forms of the derivatives exhibited spectra typical of the R-conformation, i.e., the absence of the typical T-makers around 11 and 14 ppm even in the presence of strong allosteric effectors, whereas the unligated form of this derivative showed the typical T-makers (these signals originate from specific interdimeric interactions.)These 1H-NMR results stand diametrically opposed to the measured oxygenation characteristics and are entirely inconsistent with the classical T - R dichotomic view that seemed to describe satisfactorily the correlation structure-function of hemoglobin for decades. The studied derivatives represent a case in which there is a complete divorce between the 1H-NMR-determined “structure” and oxygenation characterized “function”.

The Intradimeric Alpha1beta1 Interface Holds the Key to Allosteric Control in Hemoglobin

The major quaternary structural change responsible for the allosteric transition in human adult hemoglobin (Hb) from low-affinity T- conformation to high-affinity R-conformation upon ligand binding to hemes has been described as a 15-degree rotation of one alpha1beta1 dimer over the pairing one. This interdimeric event has been in the limelight for decades. However, since very few changes, if any, have been reported for the alpha1beta1 intradimeric interface, its contribution to allostery has been neglected. Here, dramatic changes in allosteric characteristics that are caused by a specific steric alteration of the alpha1beta1 interface by chemical modification are reported.The chemically-modified alpha1beta1 interface Hb showed an extremely low affinity for oxygen and an almost complete absence of cooperativity, comparable to those caused in native Hb by a combination of strong allosteric effectors. The functional characterization is supported by structural evidence revealed by EPR using the nitrosyl derivative, which resembles that one characterized by an extremely low affinity for the ligand showing a triplet superhyperfine around g = 2. Sedimentation experiments showed that modified dimers associated tightly into tetramers. Calorimetric experiments, on the other hand, revealed that the stabilizing interactions were mainly hydrophobic.The above-mentioned findings suggest that the alpha1beta1 interface is not inert, but rather an extremely important interface playing a pivotal role on the control of oxygen affinity.

Quaternary Structures of Intermediately Ligated Human Hemoglobin A and Influences from Strong Allosteric Effectors: Resonance Raman Investigation

The Fe-histidine stretching ( ν Fe-His) frequency was determined for deoxy subunits of intermediately ligated human hemoglobin A in equilibrium and CO-photodissociated picosecond transient species in the presence and absence of strong allosteric effectors like inositol(hexakis)phosphate, bezafibrate, and 2,3-bisphosphoglycerate. The ν Fe-His frequency of deoxyHb A was unaltered by the effectors. The T-to-R transition occurred around m = 2–3 in the absence of effectors but m > 3.5 in their presence, where m is the average number of ligands bound to Hb and was determined from the intensity of the ν 4 band measured in the same experiment. The α1- β2 subunit contacts revealed by ultraviolet resonance Raman spectra, which were distinctly different between the T and R states, remained unchanged by the effectors. This observation would solve the recent discrepancy that the strong effectors remove the cooperativity of oxygen binding in the low-affinity limit, whereas the 1H NMR spectrum of fully ligated form exhibits the pattern of the R state.

High and low oxygen affinity conformations of T state hemoglobin.

To understand the interplay between tertiary and quaternary transitions associated with hemoglobin function and regulation, oxygen binding curves were obtained for hemoglobin A fixed in the T quaternary state by encapsulation in wet porous silica gels. At pH 7.0 and 15 degrees C, the oxygen pressure at half saturation (p50) was measured to be 12.4 +/- 0.2 and 139 +/- 4 torr for hemoglobin gels prepared in the absence and presence of the strong allosteric effectors inositol hexaphosphate and bezafibrate, respectively. Both values are in excellent agreement with those found for the binding of the first oxygen to hemoglobin in solution under similar experimental conditions. The corresponding Hill coefficients of hemoglobin gels were 0.94 +/- 0.02 and 0.93 +/- 0.03, indicating, in the frame of the Monod, Wyman, and Changeux model, that high and low oxygen-affinity tertiary T-state conformations have been isolated in a pure form. The values, slightly lower than unity, reflect the different oxygen affinity of alpha- and beta-hemes. Significantly, hemoglobin encapsulated in the presence of the weak effector phosphate led to gels that show intermediate oxygen affinity and Hill coefficients of 0.7 to 0.8. The heterogeneous oxygen binding results from the presence of a mixture of the high and low oxygen-affinity T states. The Bohr effect was measured for hemoglobin gels containing the pure conformations and found to be more pronounced for the high-affinity T state and almost absent for the low-affinity T state. These findings indicate that the functional properties of the T quaternary state result from the contribution of two distinct, interconverting conformations, characterized by a 10-fold difference in oxygen affinity and a different extent of tertiary Bohr effect. The very small degree of T-state cooperativity observed in solution and in the crystalline state might arise from a ligand-induced perturbation of the distribution between the high- and low-affinity T-state conformations.

Non‐synergistic interactions between strong allosteric effectors and human embryonic and adult haemoglobins

The binding of two strong allosteric effectors (2,3 Diphosphoglycerate D.P.G., and Bezafibrate, Bzf) to both adult and the three human embryonic haemoglobins, either individually or in combination, have been studied in detail. The adult protein exhibits one binding site for D.P.G and two for Bzf. When both effectors are present simultaneously their effects are simply additive. The same qualitative pattern of binding is observed in the case of the three human embryonic haemoglobins, although with different binding constants. The lack of synergism between these effectors and the different binding affinity expressed by these proteins are discussed in terms of the known amino acid sequence differences.

Carboxylate ions are strong allosteric ligands for the HisB10 sites of the R-state insulin hexamer.

The insulin hexamer is an allosteric protein which displays positive and negative cooperativity and half-site reactivity that is modulated by strong homotropic and heterotropic ligand binding interactions at two different loci. These loci consist of phenolic pockets situated on the dimer-dimer interfaces of T-R and R-R subunit pairs and of anion sites comprising the HisB10 metal ion sites of the R3 units of the T3R3 and R6 states. In this study, we show that suitably tailored organic carboxylates are strong allosteric effectors with relatively high affinities for the R-state HisB10 metal sites. Methods of quantifying the relative affinities of ligands for these sites in both Co(II)- and Zn(II)-substituted insulin hexamers are presented. These analyses show that, in addition to the electron density on the ion, the carboxylate affinity is influenced by polar, nonpolar, and hydrophobic interactions between substituents on the carboxylate and the amphipathic protein surface of the narrow tunnel which controls ligand access to the metal ion. Since the binding of anions to the HisB10 site makes a critically important contribution to the stability of the T3R3 and R6 forms of the insulin hexamer, the design of high-affinity ligands with a carboxylate donor for coordination to the metal ion provides an opportunity for constructing insulin formulations with improved pharmaceutical properties.

22] Hemoglobin-oxygen equilibrium binding: Rapid-scanning spectrophotometry and singular value decomposition

The knowledge of the interaction between oxygen (O2) and hemoglobin (Hb) is essential both for understanding oxygen transport phenomena and for testing theories of protein structure and function. The equilibrium reaction is measured routinely simply to estimate the position and shape of the O2-binding curve from, respectively, the O2 pressure at half saturation (P50) and the index of cooperativity (i.e., the Hill number, n), but thermodynamic analysis of individual binding steps is more difficult because of inherent complexities in the reaction. The Hb conformational transition is another area under study. Strong allosteric effectors, such as inositol hexaphosphate and bezafibrate, are being used to test this by possibly enhancing the signal. In the future, mathematical models will include calculations of tetramer dissociation and subunit differences in ligand binding. As instruments and theory improve, the critical components of the reaction will be resolved better and, once limiting assumptions are no longer required for experimental analyses, the evaluation of Hb-O2 equilibrium binding will become a simpler and more reliable process.

Enhanced O2 Transportation During Cardiopulmonary Bypass in Piglets by the use of Inositol Hexaphosphate Loaded Red Blood Cells

A continuous lysing and resealing of erythrocytes permitted internalization of inositol hexaphosphate (IHP), a strong allosteric effector of Hb, leading to significant rightward shifts of the HbO2 dissociation curve. Twelve piglets were put on cardiopulmonary bypass (CPB) with the heart beating, cooled to 25 degrees C then rewarmed to 37 degrees C before weaning off CPB. AoP, LV pressure, PAP, and cardiac output (CO) were monitored. Blood samples were taken before CPB, at 25 degrees C, at 30 degrees C, at 37 degrees C and after CPB for assessment of blood gases, arterio-venous difference in O2 content, lactates, P50 (partial pressure of O2 at 50% Hb saturation), and ionogram. Control group I included five pigs where the CPB circuit was primed with Ringer's lactate solution and porcine blood. In group II (n = 5), priming was done with Ringer's lactate solution and IHP loaded erythrocytes. P50 was significantly higher during CPB than before surgery in group II (20%), but not in group I (1%). There was a significant increase in VO2 in group II (6.02 ml/min) compared to group I (4.03 ml/min) (p less than 0.05) after CPB. Hemodynamics improved after CPB in group II (mean AoP 42 mmHg and syst LVP 70 mmHg) compared to group I (AoP 25 mmHg and syst LVP 22.5 mmHg). These preliminary results show that O2 transportation at the end of CPB is enhanced and myocardial function is improved in piglets with the use of IHP erythrocytes.

Hydrogen-exchange labeling study of the allosteric R-state to T-state equilibrium in methemoglobin

Hydrogen-exchange labeling methods can be used to identify functionally important changes at positions all through a protein structure, can monitor the effect at these positions of structure changes anywhere in the protein, and can quantify these effects in terms of change in structural-stabilization free energy. These methods were used to study effects at two widely separated positions in human methemoglobin (metHb). The results show that the observed changes in hydrogen-exchange behavior reflect changes in the global R-state to T-state equilibrium, and specifically that stabilizing salt links at the α-chain N-terminus and the β-chain C-terminus are reformed in the R-T transition. The strong allosteric effector, inositol hexaphosphate (IHP), switches R-state methemoglobin to the T-state, but achieves a T/R equilibrium constant of only ≈ 3 (at pH=6.5, 0°C). Addition of the weaker effector, bezafibrate (Bzf), promotes this transition by an additional 0.7 kcal (T/R shifts to ≈ 12). Bzf alone is insufficient to cause the transition, indicating that R/T is 10 or more in stripped metHb under these conditions. However, R/T is small enough, not more than 103, to be reversed by the differential (T versus R) binding energy of IHP. The R-T transition caused by IHP and Bzf acting together can be reversed by some covalent modifications that sever the stabilizing salt links at the chain termini and thus favor transition back to the R-state.

Allosteric modifiers of hemoglobin. 1. Design, synthesis, testing, and structure-allosteric activity relationship of novel hemoglobin oxygen affinity decreasing agents

Three isomeric series of 2-(aryloxy)-2-methylpropionic acids were prepared and studied for their ability to decrease the oxygen affinity of human hemoglobin A. The isomeric aryloxy groups included 4-[[(aryloyl)amino]methyl]phenoxy, 4-(arylacetamido)phenoxy, and 4-[[(arylamino)carbonyl]methyl]phenoxy. A total of 20 compounds were synthesized and tested. Structure-activity relationships are presented. Several of the new compounds were found to be strong allosteric effectors of hemoglobin. The two most active compounds are 2-[4-[[(3,5-dichloroanilino)carbonyl]-methyl]phenoxy]- 2-methylpropionic acid and the corresponding 3,5-dimethyl derivative. The latter two compounds have been compared to other known potent allosteric effectors in the same assay and show greater activity. Both compounds also exhibit a right shift in the oxygen equilibrium curve when incubated with whole blood. The new compounds may be of interest in clinical or biological areas that require or would benefit from a reversal of depleted oxygen supply (i.e., ischemia, stroke, tumor radiotherapy, blood storage, blood substitutes, etc.). They are also structurally related to several marketed antilipidemic agents.

Coupling of tertiary and quaternary changes in human hemoglobin: a 1D and 2D-NMR study of hemoglobin Saint Mande (.beta.N102Y)

Hemoglobin Saint Mandé (beta N102Y) is a low-affinity mutant with the substitution site situated in the quaternary-sensitive alpha 1 beta 2 interface. In adult hemoglobin the Asn102 beta contributes to the stability of the liganded (R) state, forming a hydrogen bond with Asp94 alpha. The quaternary and tertiary perturbations subsequent to the Tyr for Asn substitution in monocarboxylated hemoglobin Saint Mandé have been investigated by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis of the one-dimensional NMR spectra of the liganded and unliganded samples in 1H2O provides evidence that both R and T quaternary structures of Hb Saint Mandé are different from the corresponding ones in HbA. In the monocarboxylated form of the mutant hemoglobin, at acid pH, we have observed the disappearance of an R-type hydrogen bond and the appearance of a new one whose proton resonates like a deoxy T marker. Using two-dimensional NMR methods and on the basis of previous results on the monocarboxylated HbA, we have obtained a significant number of resonance assignments in the spectra of monocarboxylated Hb Saint Mandé at pH 5.6 in the presence or absence of a strong allosteric effector, inositol hexaphosphate. This enabled us to characterize the tertiary conformational changes (relative to the liganded normal hemoglobin) triggered by the quaternary-state modification. The observed structural variations are confined within the heme pocket regions but concern both the alpha and beta subunits. Most of them, localized in the C, F, G, and FG segments, could result directly from the side-chain substitution, while others, such as Leu141 beta, could be explained only by long-range interactions.

Long-term physiological effects of enhanced O2 release by inositol hexaphosphate-loaded erythrocytes.

A continuous lysing and resealing procedure with erythrocytes permitted incorporation in these cells of inositol hexaphosphate (InsP6), a strong allosteric effector of Hb. This leads to significant rightward shifts of the HbO2 dissociation curves with in vitro P50 (partial pressure of O2 at 50% Hb saturation), values increasing from 32.2 +/- 1.8 torr for control erythrocytes to 86 +/- 60 torr (pH 7.40; PCO2 40 torr at 37 degrees C; 1 torr = 1.333 X 10(2) Pa). The shape of the dissociation curve was still sigmoidal, although the Hill coefficient was decreased. The life span of InsP6-loaded erythrocytes equaled that of control erythrocytes. The long-term physiological effects of the InsP6-loaded erythrocytes on piglets were increased O2 release and reduced cardiac output. The reduced O2 affinity of the InsP6-loaded erythrocytes was still effective 20 days after transfusion in awake piglets. The electrolyte concentration appeared stable over the 5-day observation period except for a transient, but significant, hyperkalemia immediately after transfusion. The reductions in the O2 affinity of Hb reported here are large compared with previously reported values. Introduction of InsP6 into viable erythrocytes improves tissue oxygenation when, for any reason, normal blood flow is impaired.

A high resolution NMR study of localized dynamic and structural perturbations in human hemoglobin modified with thiol reagents.

The hydrogen exchange kinetics of the N delta H proton in His F8 of iodoacetamide- and N-ethylmaleimide-treated human deoxyhemoglobins were studied using a NMR method. Comparison with unmodified hemoglobin shows that the reagents, covalently bound to Cys beta 93, significantly increase (about one order of magnitude) the exchange kinetics in beta chains only. This effect was partially reversed by the strong allosteric effector inositol hexaphosphate. Study of the high resolution 400-MHz NMR spectra of modified oxy- and deoxy-hemoglobins permitted localization of the extent of chemically induced structural perturbations. The resonances corresponding to hydrogen bonds specific to the deoxy conformation are not changed, in accord with the preserved cooperativity. Under the experimental conditions (0.1 M bis-Tris, 10 mM Cl-, pH 7.2), the salt bridge at the C terminus of the beta chain in the deoxy state (His beta 146-Asp beta 94) is perturbed by both modifications. The His beta 146 appears to be rendered more immobilized by the reagents in the oxy conformation. From the resonances corresponding to heme pocket protons of oxyhemoglobin it is deduced that the perturbations do not extend over the distal side of the heme pocket but are limited to the FG, F, and HC segments of the beta chain.

Structural and functional studies of hemoglobin Suresnes (arg 141 alpha 2 replaced by His beta 2). Consequences of disrupting an oxygen-linked anion-binding site.

Hb Suresnes is a human hemoglobin variant in which histidine replaces arginine at the COOH terminus of the alpha chains. The COOH-terminal arginines of the alpha chains play a major role in normal human hemoglobin (HbA), both by electrostatic interactions which constrain deoxygenated hemoglobin in a low affinity quaternary conformation and by involvement in oxygen-linked anion binding knoiwn to give rise to a large part of the alkaline Bohr effect. An x-ray crystallographic analysis of deoxyHb Suresnes reveals the loss of the normal intersubunit salt bridge to lysine 127 alpha and a decrease in the occupancy of inorganic anions at the alpha chain aniom-binding site. Relative to normal HbA, Hb Suresnes has a high affinity for oxygen, a reduced cooperativity in oxygen biding, and greatly reduced pH and chloride sensitivity. similar changes are found in carboxypeptidase B-digested hemoglobin Ao where the COOH-terminal arginine is removed by enzymatic digestion. Both equilibrium and kinetic manifestations of the destabilization of the normal low affinity configuration are observed. Inositol hexaphosphate restores cooperativity and pH sensitivity but, even in the presence of this strong allosteric effector, the low and high affinity conformations of Hb Suresnes appear to differ from those of HbA. In both the unliganded and liganded states, apreciable subunit dissociation of Hb Suresnes is apparent, which suggests that the substitution disrupts bonds which normally stabilize the tetramer. Structural changes at both the quaternary and tertiary level appear to contribute to alterations in the high and low affinity conformations of ths abnormal hemoglobin. Hb Suresnes thus illustrtaes the consequences of the loss of an important oxygen-linked anion-binding site.