血红蛋白携氧-释氧动力学研究

本文研究了鸡、家兔、鲤鱼、蟾蜍4种实验动物血红蛋白(hemoglobin,Hb)携氧-释氧动力学过程,初步建立Hb携氧-释氧动力学研究方法,并探讨Hb携氧-释氧动力学过程与动物生存环境之间的关系.结果显示:4种动物Hb携氧动力学曲线均呈"S"形曲线特征,与传统的Hb氧解离曲线(oxygen dissociation curve,ODC)相似;同时不同动物Hb携氧-释氧动力学曲线也有各自特点,如鸡Hb释氧时间长达(1 411±6)S;在Hb携氧.释氧曲线I阶段,鲤鱼上升斜率远大于家兔等.提示Hb携氧-释氧动力学曲线可反映不同动物Hb携氧效率的差异.与传统ODC参数P50相对应,由动力学曲线可得到Hb携氧动力学参数T50°T50是Hb达到50%氧饱和度所需时间,可直观反映Hb携氧效率的差异.4种实验动物Hb均有较稳定的T50,从大到小依次为:鸡、家兔、鲤鱼和蟾蜍.对Hb携氧动力学曲线与ODC综合分析,可得到Hb携氧效能参数E50,表示Hb达到50%氧饱和度所用时间与环境氧分压之间的关系,即E50(50% Sat,Xeo2,yr).E50有可能成为全面评价Hb携氧效能的综合指标.     

Tackling the challenges of interdisciplinary bioscience

The ultimate goal for biology is to become a science that formulates our understanding of subcellular, cellular and multicellular systems in terms of quantitative, holistic models that are underpinned by the rigorous principles of the physical sciences and mathematics. This can only be achieved through interdisciplinary research that draws heavily on the expertise and technologies of the physical sciences, engineering, computation and mathematics. Here, I discuss the benefits and challenges (both intellectual and practical) of interdisciplinary bioscience.     

Mathematical struggles and ensuring success: post-compulsory mathematics as preparation for undergraduate bioscience

This article reports on data from a large-scale study investigating students’ mathematical transitions to higher education. Three hundred and seventy-one undergraduate bioscientists were surveyed in order to investigate their perceptions and experiences of studying post-compulsory mathematics, as preparation for the mathematics elements of their degree. A-level Mathematics was well-received, with more than three-quarters of participants describing the qualification as good preparation for their degree. Participants particularly valued the statistics units at A-level. However, A-level Further Mathematics was perceived to be less useful preparation, although participants reported that it was enjoyable and challenging. Ongoing qualification reform, as well as the proliferation of post-compulsory mathematics options, means that universities and schools would do well to consider how best to maximise bioscience students’ mathematical preparation prior to beginning their undergraduate studies.     

1.25 A resolution crystal structures of human haemoglobin in the oxy, deoxy and carbonmonoxy forms

The most recent refinement of the crystallographic structure of oxyhaemoglobin (oxyHb) was completed in 1983, and differences between this real-space refined model and later R state models have been interpreted as evidence of crystallisation artefacts, or numerous sub-states. We have refined models of deoxy, oxy and carbonmonoxy Hb to 1.25 A resolution each, and compare them with other Hb structures. It is shown that the older structures reflect the software used in refinement, and many differences with newer structures are unlikely to be physiologically relevant. The improved accuracy of our models clarifies the disagreement between NMR and X-ray studies of oxyHb, the NMR experiments suggesting a hydrogen bond to exist between the distal histidine and oxygen ligand of both the alpha and beta-subunits. The high-resolution crystal structure also reveals a hydrogen bond in both subunit types, but with subtly different geometry which may explain the very different behaviour when this residue is mutated to glycine in alpha or beta globin. We also propose a new set of relatively fixed residues to act as a frame of reference; this set contains a similar number of atoms to the well-known "BGH" frame yet shows a much smaller rmsd value between R and T state models of HbA.     

Yeast for Mathematicians: A Ferment of Discovery and Model Competition to Describe Data

In addition to the memorization, algorithmic skills and vocabulary which are the default focus in many mathematics classrooms, professional mathematicians are expected to creatively apply known techniques, construct new mathematical approaches and communicate with and about mathematics. We propose that students can learn these professional, higher-level skills through Laboratory Experiences in Mathematical Biology which put students in the role of mathematics researcher creating mathematics to describe and understand biological data. Here we introduce a laboratory experience centered on yeast (Saccharomyces cerevisiae) growing in a small capped flask with a jar to collect carbon dioxide created during yeast growth and respiration. The lab requires no specialized equipment and can easily be run in the context of a college math class. Students collect data and develop mathematical models to explain the data. To help place instructors in the role of mentor/collaborator (as opposed to jury/judge), we facilitate the lab using model competition judged via Bayesian Information Criterion. This article includes details about the class activity conducted, student examples and pedagogical strategies for success.     

Modulation of hemoglobin dynamics by an allosteric effector

Hemoglobin (Hb) is an extensively studied paradigm of proteins that alter their function in response to allosteric effectors. Models of its action have been used as prototypes for structure‐function relationships in many proteins, and models for the molecular basis of its function have been deeply studied and extensively argued. Recent reports suggest that dynamics may play an important role in its function. Relatively little is known about the slow, correlated motions of hemoglobin subunits in various structural states because experimental and computational strategies for their characterization are challenging. Allosteric effectors such as inositol hexaphosphate (IHP) bind to both deoxy‐Hb and HbCO, albeit at different sites, leading to a lowered oxygen affinity. The manner in which these effectors impact oxygen binding is unclear and may involve changes in structure, dynamics or both. Here we use neutron spin echo measurements accompanied by wide‐angle X‐ray scattering to show that binding of IHP to HbCO results in an increase in the rate of coordinated motions of Hb subunits relative to one another with little if any change in large scale structure. This increase of large‐scale dynamics seems to be coupled with a decrease in the average magnitude of higher frequency modes of individual residues. These observations indicate that enhanced dynamic motions contribute to the functional changes induced by IHP and suggest that they may be responsible for the lowered oxygen affinity triggered by these effectors.     

Blood oxygen transport in rats under hypothermia combined with modification of the L-Arginine-NO pathway.

Nitric oxide (NO) has high affinity to heme and by interaction with oxyhemoglobin (HbO2) is converted into nitrate to form methemoglobin (MetHb) as a side product. In combining with deoxy-Hb NO yields a stable molecule of nitrosyl-hemoglobin (HbFe(II)NO) that can further be converted into nitrate and hemoglobin (Hb). In addition, Hb was shown to transport NO in a form of S-nitrosohemoglobin (SNO-Hb). These features of the Hb and NO interaction are important for blood oxygen transport including hemoglobin-oxygen affinity (HOA). The present investigation was aimed to study the blood oxygen transport indices (pO2, pCO2, pH, HOA, etc.) in rats under hypothermia combined with a modification of L-arginine-NO pathway. To modify the L-arginine-NO pathway, rats were administered with N(G)-nitro-L-arginine methyl ester (L-NAME), L-arginine, or sodium nitroprusside (SNP) intravenously before cooling. A substantial impairment of oxygen delivery and development of hypoxia, with an important contribution of HOA into the latter accompanied the deep hypothermia in rats. All the experimental groups developed metabolic acidosis, less pronounced in rats treated with L-arginine only. In the experiments with a modification of the L-arginine-NO pathway, an enhanced cold resistance, attenuated oxygen deficiency, and a weaker oxyhemoglobin dissociation curve (ODC) shift leftwards were observed only after the administration of L-arginine. Neither SNP nor L-NAME had not any protective effects. L-Arginine lowered the value of standard P50 (pO2, corresponding to 50% Hb saturation with oxygen at 37 degrees C, pH 7.4, and pCO2 = 40 mmHg). The actual P50 (at actual pH, pCO2 and temperature) decreased by approximately 15 mmHg and was significantly higher than that under hypothermia without the drug treatment (21.03 +/- 0.35 vs 17.45 +/- 0.60 mmHg). NO also can contribute to this system through different mechanisms (HOA modification, vascular tone regulation, peroxynitrite formation, and effects).     

The nicotinamide adenine dinucleotides as allosteric effectors of human hemoglobin

The oxygen binding properties of human hemoglobin are appreciably altered by the nicotinamide dinucleotides NADH, NADP+, and NADPH. These cofactors are important in the control of many metabolic pathways and in providing reductive potential for a number of enzymatic reactions, including in vivo reduction of methemoglobin. Specific binding of these cofactors to hemoglobin and their potential for acting as allosteric modifiers of hemoglobin function have not been previously recognized. Detailed oxygen binding studies utilizing a thin-layer method suggest that the nicotinamide dinucleotides bind with high affinity to the deoxyhemoglobin tetramer at the beta chain anion-binding site and stabilize the low affinity "T-state" conformation. Stripped Hb A in 0.05 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, pH 6.5, at 20 degrees C is half-saturated at a pO2 of 1.6 mm Hg. In the presence of 0.5 mM NADH, NADP+, or NADPH, the P50 is raised to 3.8, 7.1, and 12.5 mm Hg, respectively. The Bohr factor for stripped Hb A in 0.05 M HEPES buffer is sensitive to these effectors and is raised from 0.25 to about 0.65 by the addition of NADPH. The data suggest that routine use of these effectors in studies of human hemoglobin variants or the allosteric mechanism of Hb A be considered carefully. The relatively low intraerythrocytic levels of the nicotinamide dinucleotides in relation to hemoglobin dictate that these cofactors cannot significantly affect in vivo oxygen delivery. However, the converse is theoretically possible. The binding of the cofactors to hemoglobin and the preferential binding of their reduced forms may affect cofactor-dependent metabolic processes in red blood cells.     

Component D of chicken hemoglobin and the hemoglobin of the embryonic Tammar wallaby (Macropus eugenii) self-associate upon deoxygenation: Effect on oxygen binding

Extensive measurements of oxygen binding by some vertebrate hemoglobins (Hbs) have suggested an unusually high degree of cooperativity with reported Hill coefficients, n(H), greater than 4.0. We have reexamined this possibility of "super-cooperativity" with chicken Hb components A (alpha(A) (2)beta(2)) and D (alpha(D) (2)beta(2)). Prior studies have shown that component D but not A self-associates to dimers of tetramers upon deoxygenation. This self-association is reflected in the oxygen equilibrium of Hb D which shows a maximal n(H), greater than 4.0 at approximately 4 mM heme concentration. In contrast, component A has maximal n(H) value below 3. The value of the maximal n(H) for Hb D increases linearly with the fraction of octamer present in the deoxy Hb. We anticipate that deoxygenation-dependent self-association will be shown to be a general property of Hb D from birds and reptiles. Neither oxygen equilibria nor sedimentation measurements show any evidence that components A and D interact to form a complex when deoxygenated. We have also reexamined the oxygen equilibria of Hbs of an embryonic marsupial, the wallaby. The equilibria in red cells have been reported to have Hill coefficients as high as 5-6. Although our oxygen equilibrium measurements of solutions of unfractionated wallaby Hb at a concentration of approximately 1 mM show no n(H) values greater than approximately 3.0, sedimentation velocity measurements provide clear evidence for deoxygenation-dependent self-association.     

Hemoglobin Deer Lodge (beta 2 His replaced by Arg). Consequences of altering the 2,3-diphosphoglycerate binding site.

Hemoglobin Deer Lodge is an abnormal human hemoglobin with arginine substituted for histidine at the beta 2 position. X-ray crystallography of normal human hemoglobin has shown that the beta 2 residue is normally part of the binding site for 2,3-diphosphoglycerate. The substitution of arginine for histidine at beta 2 affects both the kinetics and equilibria of ligand binding. When stripped of anions, Hb Deer Lodge has an increased oxygen affinity and a decreased degree of cooperativity relative to Hb A. The alkaline Bohr effect is slightly increased and there are marked increases in oxygen affinity below pH 6 and above pH 8. In the presence of 2,3-diphosphoglycerate the cooperativity in increases to nromal and the pH dependence of oxygen binding is reduced. This contrasts with the enhanced Bohr effect seen for Hb A in the presence of organic phosphates. Due to enhanced anion binding at high pH, Hb Deer Lodge has a slightly lower oxygen affinity than Hb A at pH 9 in the presence of 2,3-diphosphoglycerate or inositol hexaphosphate. Kinetic studies at neutral pH in the absence of organic phosphates revealed biphasicity in the rate of oxygen dissociation from Hb Deer Lodge, while approximately linear time courses were observed for Hb A. The fast phase of the oxygen dissociation kinetics shows great pH sensitivity, and organic phosphates increase the rate and percentage of the fast phase without greatly affecting the slow phase. The two phases are not resolvable at high pH. CO combination kinetics are much like those of Hb A except that "fast" and "slow" phases were apparent at wavelengths near the deoxy-CO isobestic point. We suggest that functional differences between the alpha and beta chains are enhanced in Hb Deer Lodge. After flash photolysis of the CO derivative, the percentage of quickly reacting material was slightly greater for Hb Deer Lodge than for Hb A. This may imply a somewhat greater tendency to dissociate into high affinity subunits. The substitution of arginine for histidine at beta 2 thus results in a macromolecule whose ligand-binding properties are significantly altered, the primary differences being expressed at high pH where Hb Deer Lodge binds anions more strongly than Hb A. The properties of Hb Deer Lodge are compared to those of other hemoglobin variants with substitutions at residues involved in binding of 2,3-diphosphoglycerate.     

A note on Gutierrez's kinetics model for oxygen delivery to tissue.

The problems associated with Gutierrez model (1986, Resp. Physiol. 63, 79-96) on O2 delivery to tissue are discussed. He has used a dimensionally incorrect function for the oxygen dissociation curve (ODC). The dimensionally correct function for the ODC has been used in the analysis and the correct results are given for normoxic, hypoxic and anaemic conditions.     

Molecular modelling of Trematomus newnesi Hb 1: insights for a lowered oxygen affinity and lack of root effect

Three-dimensional structural models of the hemoglobin (Hb 1) of the Antarctic fish Trematomus newnesi were built by homology modelling, using as template the X-ray crystallographic structures of Trematomus (previously named Pagothenia) bernacchii Hb 1, both in R and T state. The Hbs of these two fishes, although showing remarkably different oxygen binding properties, differ only by 4 residues in the alpha chain (142 aa) and 10 residues in the beta chain (146 aa). T. newnesi Hb1 R-state model, essentially performed as a quality control of the adopted modelling procedure, showed a good correspondence with the crystallographic one. Modelling of T. newnesi Hb1 in the T state was performed taking into account that the proton uptake by aspartate residues, proposed to be responsible for half of the Root effect in T. bernacchii Hb 1 (showing sharp pH dependent oxygen affinity and T-state overstabilization at low pH, i. e. Bohr and Root effect), does not occur in T. newnesi Hb1 (having nearly pH-independent lower oxygen affinity). Comparison with the template structure (submitted to the same minimization procedure) indicates that, in T. newnesi Hb1 T-state model, the substitution of Ile for Thr in 41 C6, in central position of the switch region, induces at the alpha(1)beta(2) interface structural modifications able to hamper the protonation. Similar modifications are also found in T. bernacchii Hb 1 modelled in the T state with the single substitution Thr-->Ile in 41alpha. These models also suggest that the lower oxygen affinity observed in T. newnesi Hb1 is related to structural differences at the alpha(1)beta(2) interface leading to a more stable low-affinity T state. Proteins 2000;39:155-165.     

Virtual cooperativity in myoglobin oxygen saturation curve in skeletal muscle <Emphasis Type="Italic">in vivo</Emphasis>

Background

Myoglobin (Mb) is the simplest monomeric hemoprotein and its physicochemical properties including reversible oxygen (O₂)binding in aqueous solution are well known. Unexpectedly, however, its physiological role in intact muscle has not yet been established in spite of the fact that the role of the more complex tetrameric hemoprotein, hemoglobin (Hb), in red cells is well established. Here, I report my new findings on an overlooked property of skeletal Mb.

Methods

I directly observed the oxygenation of Mb in perfused rat skeletal muscle under various states of tissue respiration. A computer-controlled rapid scanning spectrophotometer was used to measure the oxygenation of Mb in the transmission mode. The light beam was focused on the thigh (quadriceps) through a 5-mm-diameter light guide. The transmitted light was conducted to the spectrophotometer through another 5-mm-diameter light guide. Visible difference spectra in the range of 500–650 nm were recorded when O₂ uptake in the hindlimb muscle reached a constant value after every stepwise change in the O₂ concentration of the buffer.

Results

The O₂ dissociation curve (ODC) of Mb, when the effluent buffer O₂ pressure was used as the abscissa, was of a sigmoid shape under normal and increased respiratory conditions whereas it was of rectangular hyperbolic shape under a suppressed respiratory condition. The dissociation curve was shifted toward the right and became more sigmoid with an increase in tissue respiration activity. These observations indicate that an increase in O₂ demand in tissues makes the O₂ saturation of Mb more sensitive to O₂ pressure change in the capillaries and enhances the Mb-mediated O₂ transfer from Hb to cytochrome oxidase (Cyt. aa₃), especially under heavy O₂ demands.

Conclusion

The virtual cooperativity and O₂ demand-dependent shifts of the ODC may provide a basis for explaining why Mb has been preserved as monomer during molecular evolution.

     

Comparison of non-linear and linear models for estimating haemoglobin adduct stability

According to the kinetic theory for the build-up and elimination of haemoglobin (Hb) adducts, unstable Hb adducts are simultaneously eliminated by zero-order Hb turnover and first-order chemical instability. Thus, the elimination of unstable Hb adducts is non-linear with respect to time. Nonetheless, many studies of Hb adduct stability have characterized the elimination of Hb adducts using linear zero-order or linear first-order models. This paper demonstrates the use of non-linear regression to estimate the first-order rate constant of Hb adduct instability (k) using data on the elimination of Hb adducts in rats dosed with benzene or ortho -toluidine. Results obtained using non-linear regression models are compared with results from the more commonly employed zero- and first-order linear models. It is shown that exposure estimates based on measured levels of unstable Hb adducts can be severely biased if zero-order turnover is assumed. Furthermore, based on published data, estimates of k are subject to estimated relative biases in the range of -4% to 96% when first-order linear models are used to characterize Hb adduct instability.     

Fisherian and Wrightian perspectives in evolutionary genetics and model-mediated imposition of theoretical assumptions

I investigate how theoretical assumptions, pertinent to different perspectives and operative during the modeling process, are central in determining how nature is actually taken to be. I explore two different models by Michael Turelli and Steve Frank of the evolution of parasite-mediated cytoplasmic incompatility, guided, respectively, by Fisherian and Wrightian perspectives. Since the two models can be shown to be commensurable both with respect to mathematics and data, I argue that the differences between them in the (1) mathematical presentation of the models, (2) explanations, and (3) objectified ontologies stem neither from differences in mathematical method nor the employed data, but from differences in the theoretical assumptions, especially regarding ontology, already present in the respective perspectives. I use my "set up, mathematically manipulate, explain, and objectify" (SMEO) account of the modeling process to track the model-mediated imposition of theoretical assumptions. I conclude with a discussion of the general implications of my analysis of these models for the controversy between Fisherian and Wrightian perspectives.     

Hb Santa Clara (beta 97His-->Asn), a human haemoglobin variant: functional characterization and structure modelling

This study examines the functional and structural effects of amino acid substitution at alpha(1)beta(2) interface of Hb Santa Clara (beta 97His-->Asn). We have characterized the variation by a combination of electrospray ionisation mass spectrometry and DNA sequence analysis followed by oxygen-binding experiments. Functional studies outlined an increased oxygen affinity, reduced effect of organic phosphates and a reduced Bohr effect with respect to HbA. In view of the primary role of this interface in the cooperative quaternary transition from the T to R conformational state, a theoretical three-dimensional model of Hb Santa Clara was generated. Structural investigations suggest that replacement of Asn for His beta 97 results in a significant stabilization of the high affinity R-state of the haemoglobin molecule with respect to the low affinity T-state. The role of beta FG4 position has been further examined by computational models of known beta FG4 variants, namely Hb Malm? (beta 97His-->Gln), Hb Wood (beta 97His-->Leu), Hb Nagoya (beta 97His-->Pro) and Hb Moriguchi (beta 97His-->Tyr). These findings demonstrate that, among the various residues at the alpha(1)beta(2) (and alpha(2)beta(1)) intersubunit interface, His beta FG4 contributes significantly to the quaternary constraints that are responsible for the low oxygen affinity of human deoxyhaemoglobin.     

Posttranscriptional regulation of ornithine decarboxylase activity

We have used a Chinese hamster ovary cell line (DF3) that overproduces ornithine decarboxylase (ODC) to examine various parameters in the cell cycle-dependent regulation of this enzyme. Under a variety of conditions, alterations in the activity of ODC were accompanied by parallel changes in the levels of the protein, as measured by immunologically cross-reactive material (CRM). While putrescine has been known to suppress the induction of ODC, we have found that in DF3 cells 10(-4)M ornithine completely suppresses ODC activity. We also show that the levels of ODC mRNA are not modulated when the levels of ODC activity and CRM change drastically. The data can be interpreted in terms of models involving either an effect of putrescine on the translation of ODC mRNA, or on the activity of a relatively specific protease with ODC as its target.     

Optimal hemoglobin concentration and high altitude: a theoretical approach for Andean men at rest.

The beneficial role of erythrocytosis for O2 transport has been questioned by evidence from bloodletting and hemodilution research as well as by studies suggesting the existence of an "optimal" hematocrit (Hct) or hemoglobin concentration ([Hb]) value. To assess to what extent erythrocytosis is beneficial in Andean men at high altitude, we examined and discussed optimal [Hb] using a mathematical approach by modeling the mixed (mean) venous Po2 (Pv(O2)) and arterial O2 content, considering for both the relation between [Hb] and arterial Po2. Relations of [Hb] to other physiological variables such as cardiac output and convective arterial O2 transport were also discussed, revealing the importance of Pv(O2) in this model. Our theoretical analysis suggests that increasing [Hb] allows increase and maintenance of Pv(O2) with only moderate declines in arterial Po2 as a consequence of moderate increases in altitude, reaching its maximum at the optimal [Hb] of 14.7 g/dl. Our analysis also shows that [Hb] corresponding to high arterial O2 content and O2 transport values is apparently not quite advantageous for improvement of oxygenation. Furthermore, chronic mountain sickness is discussed as an insightful example of the effects of excessive erythrocytosis at high altitude.     

Comparison of non-linear and linear models for estimating haemoglobin adduct stability

According to the kinetic theory for the build-up and elimination of haemoglobin (Hb) adducts, unstable Hb adducts are simultaneously eliminated by zero-order Hb turnover and first-order chemical instability. Thus, the elimination of unstable Hb adducts is non-linear with respect to time. Nonetheless, many studies of Hb adduct stability have characterized the elimination of Hb adducts using linear zero-order or linear first-order models. This paper demonstrates the use of non-linear regression to estimate the first-order rate constant of Hb adduct instability (k) using data on the elimination of Hb adducts in rats dosed with benzene or ortho -toluidine. Results obtained using non-linear regression models are compared with results from the more commonly employed zero- and first-order linear models. It is shown that exposure estimates based on measured levels of unstable Hb adducts can be severely biased if zero-order turnover is assumed. Furthermore, based on published data, estimates of k are subject to estimated relative biases in the range of -4% to 96% when first-order linear models are used to characterize Hb adduct instability.     

Simplified models for gas exchange in the human lungs

This paper presents a hierarchy of models with increasing complexity for gas exchange in the human lungs. The models span from a single compartment, inflexible lung to a single compartment, flexible lung with pulmonary gas exchange. It is shown how the models are related to well-known models in the literature. A long-term purpose of this work is to study nonlinear phenomena seen in the cardio-respiratory system (for example, synchronization between ventilation rate and heart rate, and Cheyne-Stokes respiration). The models developed in this paper can be regarded as the controlled system (plant) and provide a mathematical framework to link between "molecular-level", and "systems-level" models. It is shown how changes in molecular level affect the alveolar partial pressure. Two assumptions that have previously been made are re-examined: (1) the hidden assumption that the air flow through the mouth is equal to the rate of volume change in the lungs, and, (2) the assumption that the process of oxygen binding to hemoglobin is near equilibrium. Conditions under which these assumptions are valid are studied. All the parameters in the models, except two, are physiologically realistic. Numerical results are consistent with published experimental observations.