Precision determination and Adair scheme analysis of oxygen equilibrium curves of concentrated hemoglobin solution. A strict examination of Adair constant evaluation methods

To examine the validity of the recent finding by Gill et al. (S.J. Gill, E. Di Cera, M.L. Doyle, G.A. Bishop and C.H. Robert, Biochemistry 26 (1987) 3995) that the third overall Adair constant (A3) for human hemoglobin tetramers (Hb A) is too small to be determined and therefore that the contribution of the triply ligated species in the oxygenation process is negligibly small, highly accurate oxygen equilibrium curves for concentrated pure Hb A solutions were determined with an automatic oxygenation apparatus and analyzed by a least-squares curve-fitting method with various options. The present results indicate that an appropriate choice of weighting for data points is the key to the correct evaluation of the Adair constants and the present experimental data cannot accommodate the Adair scheme with A3 = 0, giving distinctly positive values for A3. Several criteria for correct determination of the Adair constants are presented.     

Cobalt hemoglobin: pH dependence of Adair constants and the Bohr effects.

Precise oxygen equilibrium curves have been obtained for cobalt hemoglobin at pH values from 5.5 to 8.2. The Hill plots are symmetric having asymptotes with slopes of unity. At pH 7.0, cobalt hemoglobin has p0.5 = 116 toor (15.45 kPa), pm = 117 torr (15.58 kPa) and a Hill coefficient of n = 1.72. The values of n decrease slightly with either decrease or increase of pH; the protein is almost non-cooperative at pH greater than 8.2. The Adair constants have been calculated with a non-linear least-squares program. From deltalnpm/deltapH a maximum of 2.5 Bohr protons was calculated at physiological pH values. The majority of alkaline Bohr protons are released after binding of the first and the third oxygen with maxima at pH 7.6 and 7.3, respectively. The acid Bohr effect was also observed with the majority of the protons taken up following the first and third oxygen bound. Smaller alkaline Bohr effects were obtained by differential titration and at higher pH than that calculated from oxygen equilibria. The discrepancy can be largely attributed to the binding of salt components to cobalt hemoglobin.     

Analysis of oxygen equilibria of the giant hemoglobin from the earthworm Eisenia foetida using the Adair model

Oxygen equilibrium curves of the giant hemoglobin from the earthworm Eisenia foetida were determined at various concentrations of cations. Using the Adair model of 12 oxygenation steps, we succeeded in fitting the data better than the simple concerted model (MWC model). Analysis of the Adair constants (K1 to K12) indicated that the increase in oxygen affinity occurs in the last six steps (K7 to K12) of the oxygen binding and that it is enhanced by increase in Ca2+ concentration. The Hill coefficient (nmax) at pH 7.5 attained a maximum value of 9.76 at 20 mM CaCl2. In the presence of physiological levels of Ca2+ (5 mM), the Bohr effect was similar to that seen in vertebrates. The data were consistent with the release of two Bohr protons being accompanied by the oxygen-linked binding of one Ca2+. Mg2+ and Na+ exerted a similar effect on the hemoglobin, though to a lesser extent. The stoichiometry of Ca2+ binding of the hemoglobin revealed the presence of two classes of binding sites, of which the affinities are high (Ka = 8.8 x 10(3) +/- 103 M-1) and low. The number of high affinity sites per heme was found to be 0.3, comparable to the number of oxygen-linked Ca2+ binding sites.     

Consequences of neglecting the hemoglobin concentration on the determination of Adair binding constants

It is commonly believed that the tetrameric Adair constants for oxygen binding to human hemoglobin can be evaluated from a single oxygenation experiment at 'high' hemoglobin concentration without considering the consequence of the presence of alpha beta dimers. We present examples which demonstrate that this is a very dangerous assumption. Without a knowledge of the complete oxygenation-linked dimer-tetramer association reaction (alpha beta Xi----(alpha beta)2Xj), it is impossible to predict a priori how high of a hemoglobin concentration would be required to make this assumption. Furthermore, without a knowledge of the complete oxygenation-linked dimer-tetramer association reaction, it is impossible to predict a priori the direction and magnitude of the systematic errors which are induced by making this assumption.     

Spot hemoglobin. Studies on the Root effect hemoglobin of a marine teleost.

The Spot, Leiostomus xanthrus, has a single tetrameric hemoglobin. Structural studies indicate the presence of alpha- and beta-like chains with COOH-terminal sequences of --Arg and --TYR-His, respectively, the same as is found in human hemoglobin. Spot hemoglobin possesses a Root effect: a heterotropic control mechanism like the Bohr effect but with more extreme pH dependence in the equilibria and kinetics of O2 and CO binding. The Root effect seems to be a molecular adaptation, in that pH- and anion-sensitive hemoglobins may help fish achieve neutral buoyancy by facilitating O2 delivery to the swim bladder. Changes in the kinetics of both "on" and "off" processes contribute to the greatly decreased ligand affinity of Spot hemoglobin at low pH. The time course ofligand combination at low pH is biphasic and wavelength dependent, suggesting a differential effect of pH on the alpha- and beta-like chains. The change in the shape of the ligand-binding curve with pH may be interpreted in terms of a proton-dependent transition between low (T) and high (R) affinity conformations. However, this may not be the only mechanism, since differential pH effects on the two types of chains may also contribute to the observed pH dependence.     

Effect of differences in optical properties of intermediate oxygenated species of hemoglobin A0 on Adair constant determination

Careful evaluation of the so-called isosbestic properties of oxygenated and deoxygenated hemoglobin spectra demonstrates that the spectral changes are not strictly linear with respect to the degree of saturation. In order to quantify the extent of nonlinearity, optical measurements of O2 binding to human hemoglobin were made at different wavelengths in the Soret region approaching the presumed isosbestic point. The results indicate that the extinction coefficient of intermediate oxygenated hemoglobin is 1% less than that of the fully oxygenated hemoglobin, with a resulting 3% (+/- 0.15%) nonlinearity effect on measurements taken at the peak of the oxygenated hemoglobin spectrum (414 nm). The lack of isosbestic conditions allows one to investigate the functional properties of the oxygenated intermediates directly. The small difference in the absorbance of different oxygenated species has practically no influence on the determination of Adair constants at wavelengths removed from the critical isosbestic region.     

Evolution of allosteric models for hemoglobin

We compare various allosteric models that have been proposed to explain cooperative oxygen binding to hemoglobin, including the two-state allosteric model of Monod, Wyman, and Changeux (MWC), the Cooperon model of Brunori, the model of Szabo and Karplus (SK) based on the stereochemical mechanism of Perutz, the generalization of the SK model by Lee and Karplus (SKL), and the Tertiary Two-State (TTS) model of Henry, Bettati, Hofrichter and Eaton. The preponderance of experimental evidence favors the TTS model which postulates an equilibrium between high (r)- and low (t)-affinity tertiary conformations that are present in both the T and R quaternary structures. Cooperative oxygenation in this model arises from the shift of T to R, as in MWC, but with a significant population of both r and t conformations in the liganded T and in the unliganded R quaternary structures. The TTS model may be considered a combination of the SK and SKL models, and these models provide a framework for a structural interpretation of the TTS parameters. The most compelling evidence in favor of the TTS model is the nanosecond - millisecond carbon monoxide (CO) rebinding kinetics in photodissociation experiments on hemoglobin encapsulated in silica gels. The polymeric network of the gel prevents any tertiary or quaternary conformational changes on the sub-second time scale, thereby permitting the subunit conformations prior to CO photodissociation to be determined from their ligand rebinding kinetics. These experiments show that a large fraction of liganded subunits in the T quaternary structure have the same functional conformation as liganded subunits in the R quaternary structure, an experimental finding inconsistent with the MWC, Cooperon, SK, and SKL models, but readily explained by the TTS model as rebinding to r subunits in T. We propose an additional experiment to test another key prediction of the TTS model, namely that a fraction of subunits in the unliganded R quaternary structure has the same functional conformation (t) as unliganded subunits in the T quaternary structure.     

Regulation of reactivated contraction in teleost retinal cone models by calcium and cyclic adenosine monophosphate

We have been using lysed cell models of teleost retinal cones to examine the mechanism of contraction in nonmuscle cells. We have previously reported that dark-adapted retinas can be lysed with the detergent Brij-58 to obtain cone motile models that undergo Ca++- and adenosine triphosphate (ATP)-dependent reactivated contraction. In this report we further dissect the roles of ATP and Ca++ in activation of contraction and force production by (a) characterizing the Ca++ and nucleotide requirements in more detail, (b) by analyzing the effects of inosine triphosphate (ITP) and the ATP analog ATP gamma S and (c) by testing effects of cyclic adenosine monophosphate (cAMP) on reactivated cone contraction. Exposing lysed cone models to differing free Ca++ concentrations produced reactivated contraction at rates proportional to the free Ca++ concentration between 3.16 X 10(-8) and 10(-6) M. A role for calmodulin (CaM) in this Ca++ regulation was suggested by the inhibition of reactivated contraction by the calmodulin inhibitors trifluoperazine and calmidazolium ( R24571 ). The results of analysis of nucleotide requirements in lysed cone models were consistent with those of smooth muscle studies suggesting a role for myosin phosphorylation in Ca++ regulation of contraction. ATP gamma S and ITP are particularly interesting in that ATP gamma S, on the one hand, can be used by kinases to phosphorylate proteins (e.g., myosin light chains) but resists cleavage by phosphatases or adenosine triphosphatases (ATPases), e.g., myosin ATPase. ITP, on the other hand, can be used by myosin ATPase but does not support Ca++/calmodulin mediated phosphorylation of myosin light chains by myosin light chain kinase. Thus, these nucleotides provide an opportunity to distinguish between the kinase and myosin ATPase requirements for ATP. When individual nucleotides were tested with cone motile models, the nucleotide requirement was highly specific for ATP; not only ITP and ATP gamma S, but also guanosine triphosphate, cytosine triphosphate, adenylyl-imidodiphosphate (AMPPNP) failed to support reactivated contraction when substituted for ATP throughout the incubation. However, if lysed cones were initially incubated with ATP gamma S and then subsequently incubated with ITP, the cones contracted to an extent that was comparable to that observed with ATP. As observed in skinned smooth muscle, adding cAMP to contraction medium strongly inhibited contraction in lysed cone models.     

Regulation of reactivated elongation in lysed cell models of teleost retinal cones by cAMP and calcium

Teleost retinal cones elongate in the dark and contract in the light. In isolated retinas of the green sunfish Lepomis cyanellus, cone myoids undergo microtubule-dependent elongation from 5 to 45 micron. We have previously shown that cone contraction can be reactivated in motile models of cones lysed with Brij-58. Reactivated contraction is both actin and ATP dependent, activated by calcium, and inhibited by cAMP. We report here that we have obtained reactivated cone elongation in lysed models prepared by the same procedures. Reactivated elongation is ATP dependent, activated by cAMP, and inhibited by calcium. The rate of reactivated elongation is proportional to the cAMP concentration between 10 microM and 0.5 mM, but is constant between 10 microM and 1.0 mM Mg-ATP. No elongation occurs if cAMP or Mg-ATP concentration is less than or equal to 5 microM. Mg-ATP is required for both cAMP-dependent and cAMP-independent processes, suggesting that Mg-ATP is required both for a regulatory process entailing cAMP-dependent phosphorylation and for a force-producing process. Free calcium concentrations greater than or equal to 10(-7) reduce the elongation rate by 78% or more, completely inhibiting elongation at 10(-5) M. This inhibition is not due to competition from calcium-activated contraction. Cytochalasin D blocks reactivated contraction, but does not abolish calcium inhibition of reactivated elongation. Thus calcium directly affects the elongation mechanism. Calcium inhibition is calmodulin dependent. The calmodulin inhibitor trifluoperazine abolishes calcium inhibition of elongation. Furthermore, calcium blocks elongation only if present during the lysis step; subsequent calcium addition has no effect. However, if calcium plus exogenous calmodulin are subsequently added, elongation is again inhibited. Thus calcium inhibition appears to require a soluble calmodulin which is lost shortly after lysis.     

Mathematical models of the oxygen-binding function of intact human hemoglobin and hemoglobin modified by UV-radiation in the presence of carbon oxide

The influence of carbon oxide and UV-radiation in doses of 151-453 J/m2 on the physiological properties of human oxyhemoglobin has been studied. Mathematical models of the oxygen-binding function of intact and modified hemoprotein have been developed. It has been found that saturation of human hemoglobin with oxygen obeys the logistic dependence. In the presence of carboxyhemoglobin, the oxygenation parameters change and saturation curves are described by the equations of degree dependence. It has been shown that UV light had the stimulating influence on the functional properties of human hemoglobin modified by carbon oxide if the concentration of carboxyform of the hemoprotein in solution was no higher than 10 per cent. The disturbance of the oxygen-binding ability of hemoglobin by the action of higher concentrations of carboxyhemoglobin was irreversible and was not corrected by UV-radiation.     

Prey capture by Luciocephalus pulcher: implications for models of jaw protrusion in teleost fishes

Synopsis Luciocephalus pulcher possesses one of the most protrusible jaws known among teleosts, the premaxillae extending anteriorly a distance of 33% of the head length during feeding. Jaw bone movement during feeding proceeds according to a stereotypical pattern and resembles that of other teleosts except for extreme cranial elevation and premaxillary protrusion. Anatomical specializations associated with cranial elevation include: a highly modified first vertebra with a separate neural spine, articular fossae on the posterior aspect, greatly enlarged zygapophyses on the second vertebra with complex articular condyles, and highly pinnate multi-layered epaxial musculature with multiple tendinous insertions on the skull. Luciocephalus, despite the extreme jaw protrusion, does not use suction during prey capture: rather, the prey is captured by a rapid lunge (peak velocity of about 150 cm per sec) and is surrounded by the open mouth. Previous hypotheses of the function of upper jaw protrusion are reviewed in relation to jaw movements inLuciocephalus. Protrusion is not obligatorily linked with suction feeding; behavioral aspects of the feeding process limit the possible range of biological roles of a given morphological specialization, and make prediction of role from structure risky.     

PH dependence of the Adair constants of human hemoglobin. Nonuniform contribution of successive oxygen bindings to the alkaline Bohr effect.

In order to solve the problem of an apparent discrepancy between the pH variance of oxygen equilibrium curve and the linear relation between the number of released Bohr protons and the degree of ligation, precise oxygen equilibrium curves of human hemoglobin were determined at a number of pH values from 6.5 to 8.8. From the equilibrium data individual steps (Adair constants), ki (i equals 1, 2, 3, 4), were obtained and the number of Bohr protons (deltaHi+) released on the ith stage of oxygenation was estimated. The pH dependence of k4 was very small, while the other ks strongly depended on pH over the pH range examined. As a consequence, the contribution of each step of oxygen binding to the alkaline Bohr effect nonuniform: deltaH4 was very small compared with deltaH1+, deltaH2+, and deltaH3+. In spite of this, calcuation has shown that the fractional number of released protons is essentially proportional to fractional oxygen saturation because of cooperative effects in hemoglobin. Thus, the present study indicates that the linear relationship between the fractional number of released protons and the degree of ligation, as obtained from titration experiments, is not necessarily incompatible with the pH variance of the shape of the oxygen equilibrium curve. The nonuniform pH depencence of the Adair constants implies that the two-state allosteric model of Monod, J., Wyman, J., and Changeus, J.P. (1965) J. Mol. Biol. 12, 88-118 is not adequate to describe the heterotropic effect caused by protons.     

Dynamics of the Adair model for ligand-receptor binding

The dynamics of the Adair model for ligand-receptor binding involving the case with interacting receptors is investigated. Using the methods of formal reaction kinetics, the existence, uniqueness, and asymptotic stability of equilibria within the model are demonstrated. The approximate solutions to the Adair model are found.     

Hill Coefficients of a Polymodal Monod-Wyman-Changeux Model for Ion Channel Gating

Allosteric transitions of ion channels can be driven by multiple sources of free energies. One class of model for describing such transitions is the multistimulus Monod-Wyman-Changeux model, in which each stimulus interacts with a specific sensor on the protein and activation of the sensor is allosterically coupled to conformational changes of the protein. In general, when a protein is stressed by multiple stimuli, one stimulus can influence the response to another, which can result in both a shift of the midpoint of the dose-response curve and a change of the slope of the curve. Here I show that, for a Monod-Wyman-Changeux model with independent sensors, the different dose-response curves of open probability for one stimulus have the same slope at the same agonist concentration. In the other words, the slope of the dose-response curve for one stimulus is an intrinsic property of the sensors for that stimulus; it is independent of other stimuli or their sensor properties. As the dose-response curve for many receptors can be fit to a Boltzmann or Hill equation, this property provides a practical, usable test for applicability of such models.     

Three-state combinatorial switch models as applied to the binding of oxygen by human hemoglobin

We have generated a series of all 6561 unique, discrete three-state combinatorial switch models to describe the partitioning of the cooperative oxygen-binding free change among the 10 variously ligated forms of human hemoglobin tetramers. These models were inspired by the experimental observation of Smith and Ackers that the cooperative free energy of the intersubunit contact regions of the 10 possible ligated forms of human hemoglobin tetramers can be represented by a particular distribution of three distinct energy levels [Smith, F. R., & Ackers, G. K. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5347-5351]. A statistical thermodynamic formulation accounting for both dimer-tetramer equilibria and ligand binding properties of hemoglobin solutions as a function of oxygen and protein concentrations was utilized to exhaustively test these thermodynamic models. In this series of models each of the 10 ligated forms of the hemoglobin tetramer can exist in one, and only one, of three possible energy levels; i.e., each ligated form was assumed to be associated with a discrete energy state. This series of models includes all possible ways that the 10 ligation states of hemoglobin can be distributed into three distinct cooperative energy levels. The mathematical models, as presented here, do not permit equilibria between energy states to exist for any of the 10 unique ligated forms of hemoglobin tetramers. These models were analyzed by nonlinear least-squares estimation of the free energy parameters characteristic of this statistical thermodynamic development.(ABSTRACT TRUNCATED AT 250 WORDS)     

Iron acquisition by teleost fish

Iron is a vital micronutrient for teleost fish, being an integral component of proteins involved in cellular respiration and oxygen transfer. However, in excess iron is toxic, and fish need to balance uptake to prevent deficiency vs. potential toxicity. This review assesses the current physiological and molecular knowledge of the mechanisms of iron acquisition in the teleost fish. It focuses on freshwater teleost fish when assessing the gill as a possible site for iron acquisition, and includes a summary of geochemical processes that govern aquatic iron bioavailability. It focuses on marine teleost fish for assessing the mechanism of intestinal iron uptake. Physiological evidence indicates that iron preferentially crosses the apical membrane of both the gills and intestine in the ferrous (Fe2+) state. Molecular evidence supports this, demonstrating the presence of homologues in fish to the large Slc 11a family of evolutionary conserved proteins linked to Fe2+ transport. This symporter is probably linked to a reductase, which reduces either ferric (Fe3+) or organic complexed iron to Fe2+ prior to uptake.     

The amino acid sequence and oxygen-binding properties of the single hemoglobin of the cold-adapted Antarctic teleost Gymnodraco acuticeps.

The complete amino acid sequence of the single hemoglobin of the Antarctic teleost Gymnodraco acuticeps has been determined. The alpha chain contains 142 amino acid residues; an acetylated seryl residue is at the amino terminal. The beta chain contains 146 residues. A very high degree of sequence identity has been found with hemoglobins of other Antarctic fishes. Oxygen binding is not modulated by pH and allosteric effectors. The Bohr and Root effects are absent, although specific amino acid residues, considered responsible of most of these functions, are conserved in the sequence, thus posing new questions about the molecular basis of these mechanisms. The low heat of oxygenation may be interpreted as one of the mechanisms involved in the process of cold adaptation.     

The inhibition of hemoglobin C crystallization by hemoglobin F

We have reported that circulating CC erythrocytes containing HbO2 C crystals exhibit little or no Hb F suggesting that Hb F may inhibit the crystallization of Hb C. We report now that Hb F inhibits in vitro crystallization of HbO2 and HbCO C when compared to the effect of Hb A in a wide range of mixture proportions. For example, while HbCO C solutions form tetragonal C crystals within 25 min, no crystals form within 2 h with 30% Hb F, whereas 550 crystals/mm3 form with 30% Hb A. Furthermore, an increase in the percent of Hb A is correlated with a greater number of orthorhombic crystal formation rather than the tetragonal morphology observed with 100% Hb C. We also report that Hb A2 (containing delta chains that exhibit 10 sequence differences with beta chains) and Hb Lepore Boston-Washington (a fusion mutant of delta and beta chains that contains only six of these differences) both inhibit Hb C crystallization. By comparing the sequences of the three inhibitory hemoglobins, we conclude that position Gln-87 in the gamma chains is, at least partially, the cause of the inhibitory effect of Hb F on the crystallization of Hb C.