Functional and structural analyses on abnormal hemoglobins with impaired oxygen binding properties—To elucidate the allosteric mechanism of hemoglobin

The altered oxygen binding curves for various abnormal hemoglobins were analyzed according to a two-state allosteric model. Of three allosteric parameters computed for abnormal hemoglobins, K _R was nearly constant, but K _T and L varied with the correlation of log c=?0.4 log L, where c is K _R/K _T. This correlation indicates that the abnormal allosteric oxygen binding of hemoglobin is due to altered molecular properties of the deoxy-T state but not that of the deoxy-R state. To clarify the molecular basis of this idea, resonance Raman spectra in the low-frequency region of abnormal hemoglobins were measured under different solvent conditions. Varied frequencies of iron-histidine stretching Raman lines was found to correlate with varied oxygen affinities (K _T) of deoxy-T states. The strength of the iron-histidine bond of deoxy-T states was changed, depending upon the magnitude of the strain imposed on hemes by globin, and this bond presumably comprises an important part of the regulation mechanisms for hemoglobin oxygen binding and structure changes.     

A three-state MWC analysis of oxygenation in tench (Tinca tinca) hemoglobin

Summary Oxygen equilibria in tench hemoglobin were analysed according to a three-state MWC model. In addition to theT andR states of the traditionally used two-state model, the three-state model introduces an additional state, theS state, when organic phosphates bind to theT-structure hemoglobin. Under conditions covering natural red cell pH values and nucleoside triphosphate-hemoglobin ratios, it was possible to closely fit experimental data to the three-state equation with constant values of the association constantsK _R ,K _T , andK _S , and with only the allosteric constantsL andM varying with effector conditions. Thus, in contrast to a twostate analysis of oxygen equilibria, the three-state analysis was consistent with the basic assumption of the MWC model, that heterotropic ligands only affect allosteric constants and not association constants. The temperature-dependence of the three-state parameter values showed that in the presence of nucleoside triphosphate the dominance of theS state over theT state was most pronounced at low temperatures. Furthermore, the numerical values of the enthalpy and entropy change of oxygenation were lower in theS state than in theT andR states, and the enthalpy and entropy change for the allostericSR transition were much larger than for theTR transition.Abbreviations Hb hemoglobin - Y fractional O₂ saturation - ATP adenosine triphosphate     

Tendency for oxidation of annelid hemoglobin at alkaline pH and dissociated states probed by redox titration

The redox titration of extracellular hemoglobin of Glossoscolex paulistus (Annelidea) was investigated in different pH conditions and after dissociation induced by pressure. Oxidation increased with increasing pH, as shown by the reduced amount of ferricyanide necessary for the oxidation of hemoglobin. This behavior was the opposite of that of vertebrate hemoglobins. The potential of half oxidation (E_{1 / 2}) changed from − 65.3 to + 146.8 mV when the pH increased from 4.50 to 8.75. The functional properties indicated a reduction in the log P₅₀ from 1.28 to 0.28 in this pH range. The dissociation at alkaline pH or induced by high pressure, confirmed by HPLC gel filtration, suggested that disassembly of the hemoglobin could be involved in the increased potential for oxidation. These results suggest that the high stability and prolonged lifetime common to invertebrate hemoglobins is related to their low tendency to oxidize at acidic pH, in contrast to vertebrate hemoglobins.     

Characterization by polyacrylamide gel electrophoresis of hemoglobins in rabbit embryo, fetus, and adult

The hemoglobin species of rabbit embryo, fetus, and adult were characterized by quantitative polyacrylamide gel electrophoresis (PAGE). Special statistical methods, including joint confidence envelopes for the slope (K_R) and intercept (Y₀) of the Ferguson plot and analysis of covariance were utilized to identify the hemoglobin species. Five embryonic rabbit hemoglobins could be distinguished. Their relative proportions varied with developmental stage. No specific fetal hemoglobin was detected. The two previously known adult hemoglobins were characterized, prepared by isoclectric focusing on polyacrylamide gel (IFPA) and shown to differ by two amino acid substitutions in the β-chain.A general strategy for testing molecular identity by PAGE is outlined.     

Oxygen equilibrium characteristics of hemoglobins of baboons, Theropithecus gelada,Papio hamadryas and Papio anubis

Hemoglobins of three baboons, Theropithecus gelada, Papio hamadryas- and Papio anubis, were purified and their oxygen equilibrium characteristics were studied. (a) Oxygen affinity, as expressed by P₅₀, oxygen partial pressure for 50% oxygen binding, was in the order of gelada hemoglobin > anubis hemoglobin > hamadryas hemoglobin although the differences were small. (b) The presence of 2,3-diphosphoglycerate reduced their oxygen affinity in a similar manner. The effect on baboon hemoglobins was greater than that on human and Japanese monkey hemoglobins. (c) The intensity of the Bohr effect, as expressed by $\text{?Δlog}\text{P}{}_{50}\text{ΔpH}$, at pH 7·4 agreed well with each other and the value was 0·62 in the presence of 2 mm diphosphoglycerate and 0·52 in its absence. These results indicate that phenotypic adaptation (acclimatory) may play an important role in the adaptation of gelada baboon to high altitudes.     

Improved polyacrylamide gel electrophoresis with different amino acids as the trailing constituent

Using a polyacrylamide disc gel electrophoretic system similar to that described by J. T. Clarke (1964, Ann. N. Y. Acad. Sci.121, 428–436), we have achieved an improved separation of hemoglobins from Rana catesbeiana tadpoles by substituting one of several amino acids in the place of glycine in the electrode chamber buffer. The relative migrations (R_f) and degree of separation of these similar hemoglobins are proportional to the pK′ of the α-amino group of the amino acid used in the buffer. Specifically, for these proteins, log (R_f × 100) was found to be directly proportional to the pK′₂ of the amino acid divided by the volume conductivity (specific conductance) of the electrode chamber buffer. For example, improved separation of these hemoglobins in short electrophoretic times can be achieved, at low cost, by using dl-alanine instead of glycine in the buffer. Improved separation of other proteins which migrate at basic pH might be achieved by a similar approach.     

Analysis of teleost hemoglobin by Adair and Monod-Wyman-Changeux models

Summary The allosteric effects of the erythrocytic nucleoside triphosphates (NTP) and of proton concentrations were investigated by precise measurement of Hb–O₂ equilibria of tench hemoglobin (including extreme, high and low saturation ranges) and analysed in terms of the MWC two state model and the Adair four step oxygenation theory.At low concentrations (NTP/Hb ratio=1.0, and pH>7.3) ATP, GTP and protons decrease Hb–O₂ affinity by increasing the allosteric constantL and reducingK _T, the association constant¹ of the deoxy, tense state of the Hb, without significantly affecting that (K _R) of the oxy state, increasing the free energy of cooperativity (G). High concentrations of these effectors, however, also reduceK _R. The greater sensitivity of the half-saturation O₂ tension (P ₅₀) of the Hb to GTP than to ATP at the same concentration, correlates with greater effects of GTP on bothK _T andK _R. The pH and NTP dependence of the four Adair association constants and the calculated fractional populations of Hb molecules in different stages of oxygenation show that the autochthonous NTP effectors and protons stabilize the T structure and postpone the TR transition basic to cooperativity in fish Hb.The possible implications of the findings for aquatic respiration are discussed.Abbreviations ATP adenosine triphosphate - DPG 2,3-diphosphoglycerate (glycerate-2,3-bisphosphate) - GTP guanosine triphosphate - IHP inositol hexaphosphate - NTP nucleoside triphosphates In this paperK _T andK _R are defined as theassociation equilibrium constants instead of dissociation constants (as originally defined by Monod et al. 1965) to facilitate comparison with the Adair constants     

Interaction of allosteric effectors (ATP,CO2, H+) modulating oxygen affinity of the hemoglobin in the carp,Cyprinus carpio,in vitro

Summary The interaction of allosteric effectors (CO₂, ATP, H⁺) with respect to the oxygen affinity of carp hemoglobin was analyzed by determining oxygen binding curves spectrophotometrically in dilute solutions of stripped hemoglobin at 20°C. The pH range studied was 6.8–8.2.P _CO2 was 0, 10 and 70 mmHg (0, 1.33 and 9.3 kPa). ATP/Hb₄ was 0, 8 and 24. In the presence of either CO₂ or ATP, the effects of the cofactors onP ₅₀ were as expected over the whole pH range. In contrast to other published data, each cofactor also had a significant effect onP ₅₀ in the presence of the other cofactor. Evidence was obtained that oxylabile carbamate is formed by carp hemoglobin and that the formation of carbamate persists at a lower level in the presence of ATP. The results support the view that the binding of ATP to carp hemoglobin requires only one terminal amino group, leaving the other N-terminal of the -chain free to react with CO₂.     

Thyroid Hormone-Induced Alterations in Membrane Structure-Function Relationships: Studies on Kinetic Properties of Rat Kidney Microsomal Na+,K+-ATPase and Lipid/Phospholipid Profiles

The effects of thyroidectomy (Tx) and subsequent treatment with 3,5,3′-triiodothyronine (T₃) or combined replacement therapy (T_R) with T₃ and thyroxine (T₄) on the substrate and temperature kinetics properties of Na⁺,K⁺-ATPase and lipid/phospholipid makeup of rat kidney microsomes were examined. Enzyme activity was somewhat high in the hypothyroid (Tx) animals and increased significantly following T₃ treatment, while T_R treatment caused a decrease. In the Tx and T₃ groups enzyme activity resolved in two kinetic components, while in the T_R group the enzyme showed allosteric behavior up to 0.5 mm ATP concentration. The K _m and V _max values of both the components decreased in Tx animals without affecting the catalytic efficiency. T₃ treatment caused a significant increase in the V _max of both the components, with a significant increase in the catalytic efficiency, while the K _m values were not upregulated. The T_R regimen lowered the K _m and V _max of component II but improved the catalytic efficiency. Thyroid status-dependent changes were also noted in the temperature kinetics of the enzyme. Regression analysis revealed that changes in the substrate and temperature kinetics parameters correlated with specific phospholipid components.     

Single amino acid substitution in important hemoglobinopathies does not disturb molecular function and biological process

Hemoglobin is an important protein found in the red cells of many animals. In humans, the hemoglobin is mainly distributed in the red blood cell. Single amino acid substitution is the main pathogenesis of most hemoglobin disorders. Here, the author used a new gene ontology technology to predict the molecular function and biological process of four important hemoglobin disorders with single substitution. The four studied important abnormal hemoglobins (Hb) with single substitution included Hb S, Hb E, Hb C, and Hb J-Baltimore. Using the GoFigure server, the molecular function and biological process in normal and abnormal hemoglobins was predicted. Compared with normal hemoglobin, all studied abnormal hemoglobins had the same function and biological process. This indicated that the overall function of oxygen transportation is not disturbed in the studied hemoglobin disorders. Clinical findings of oxygen depletion in abnormal hemoglobin should therefore be due to the other processes rather than genomics, proteomics, and expression levels.