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Oxygen-Linked Hydrogen Ion Binding of Human Hemoglobin. Effects of Carbon Dioxide and 2,3-Diphosphoglycerate I. Studies on Erythrolysate
Abstract
Acid-base titrations were performed on oxygenated and deoxygenated erythrolysates at constant PCO2. The Haldane coefficient, - (δHb-bound H+)/(δHb-bound O2), was determined as a function of pH and PCO2 for various concentrations of 2,3-diphosphoglycerate (DPG). At PCO2=0 DPG causes a decrease or increase in the coefficient for pH < 7.0 or pH > 7.0, respectively. The equilibrium constants Kz (acid-base) and Kc (carbamate) of the terminal -NH2 of the β-chains of deoxy-hemoglobin were evaluated. DPG decreases both constants, especially Kz.
... This includes incorporation of modeling of the red blood cells, available only in the newest Stewart type models [59], and representation of Bohr-Haldane effects, i.e. the oxygen binding to haemoglobin or the competitive binding of oxygen, hydrogen ions and carbon dioxide on haemoglobin. The Bohr-Haldane effects have not previously been included in any of the models formulated using the Stewart approach, but have been experimentally deterimined and mathematically described by Siggaard-Andersen and colleagues [60,61]. ...
... The parameters describing heamoglobin buffering in fully oxygenated blood (pKz o , pKc o ) were estimated from only three data points with measured values of pH p and PCO 2 , and a value of the normal buffer base in erythrocyte (BB e ). The parameters describing haemoglobin buffering in fully deoxygenated blood (pKz d , pKc d ) were estimated from only two data points, the Haldane coefficient at pH e = 7.2 in the absence of CO 2 and the Haldane coefficient at pH e = 7.2, at PCO 2 = 5.33 kPa [60,61]. ...
... These simulations accurately described: the addition or removal of CO 2 or strong acid to plasma; the addition or removal of CO 2 , strong acid, or haemoglobin to blood; and the effects of deoxygenating erythrocyte or blood at a wide range of values of pH and PCO 2 . In addition the model was shown to be able to simulate data [60,61] describing values of the Haldane coefficient and Base Excess coefficient over a wide range of values of pH and PCO 2 , as exemplified in Fig. 8B. This evaluation illustrates the generality of the model in that substantial functionality can be validated in a model which includes relatively few parameters, identified from very little data. ...
This dissertation has addressed the broad hypothesis as to whether building mathematical models is useful as a tool for translating physiological knowledge into clinical practice. In doing so it describes work on the INtelligent VENTilator project (INVENT), the goal of which is to build, evaluate and integrate into clinical practice, a model-based decision support system for control of mechanical ventilation. The dissertation describes the mathematical models included in INVENT, i.e. a model of pulmonary gas exchange focusing on oxygen transport, and a model of the acid–base status of blood, interstitial fluid and tissues. These models have been validated, and applied in two other systems: ALPE, a system for measuring pulmonary gas exchange and ARTY, a system for arterialisation of the acid–base and oxygen status of peripheral venous blood.
... The curve is shifted to the right (i.e. lower saturation for a given PO2) by higher PCO2, greater acidity (lower pH), higher temperature, and higher concentration of 2,3-DPG [1][2][3][4][5][6][7] . The factors that shift the ODC to the right are directly relevant to the conditions that prevail in metabolizing tissues, as they facilitate the unloading of oxygen from hemoglobin. ...
Abnormal hemoglobins can have major consequences for tissue delivery of oxygen. Correct diagnosis of hemoglobinopathies with altered oxygen affinity requires a determination of hemoglobin oxygen dissociation curve, which relates the hemoglobin oxygen saturation to the partial pressure of oxygen in the blood. Determination of the oxygen dissociation curve of human hemoglobin is typically carried out under conditions in which hemoglobin is in equilibrium with O2 at each partial pressure. However, in the human body due to the fast transit of red blood cells through tissues hemoglobin oxygen exchanges occur under nonequilibrium conditions. We describe the determination of non-equilibrium oxygen dissociation curve and show that under these conditions the true nature of hemoglobin cooperativity is revealed as emerging solely from the consecutive binding of oxygen to each one of the four subunits of hemoglobin until the entire tetramer is saturated. We call this form of cooperativity the sequential cooperativity of hemoglobin and define the simplest model that includes it as the minimalist model of hemoglobin. A single instantiation of this model accounts for ~70% of hemoglobin cooperativity under non-equilibrium conditions. The total cooperativity of hemoglobin can be viewed more correctly as the summation of two instantiations of the minimalist model (each one corresponding to a tetramer of low and high affinity for O2, respectively) in equilibrium with each other, as in the Monod-Wyman-Changeux model of hemoglobin. In addition to offering new insights on the nature of hemoglobin reaction with oxygen, the methodology described here for the determination of hemoglobin non-equilibrium oxygen dissociation curve provides a simple, fast, low-cost alternative to complex spectrophotometric methods, which is expected to be particularly valuable in regions where hemoglobinopathies are a significant public health problem, but where highly specialized laboratories capable of determining a traditional oxygen dissociation curve are not easily accessible.
... Effect is the mutual interaction of oxygen binding and hydrogen ion binding of oxyhemoglobin 49 . This mutual interaction allows a decrease in pH near tissue capillaries to increase delivery of O 2 at the tissue level 48,50 . ...
Performing uncertainty quantification (UQ) and sensitivity analysis (SA) is vital when developing a patient‐specific physiological model because it can quantify model output uncertainty and estimate the effect of each of the model’s input parameters on the mathematical model. By providing this information, UQ and SA act as diagnostic tools to evaluate model fidelity and compare model characteristics with expert knowledge and real world observation. Computational efficiency is an important part of UQ and SA methods and thus optimization is an active area of research. In this work, we investigate a new efficient sampling method for least‐squares polynomial approximation, weighted approximate Fekete points (WAFP). We analyze the performance of this method by demonstrating its utility in stochastic analysis of a cardiovascular model that estimates changes in oxyhemoglobin saturation response. Polynomial chaos (PC) expansion using WAFP produced results similar to the more standard Monte Carlo in quantifying uncertainty and identifying the most influential model inputs (including input interactions) when modeling oxyhemoglobin saturation, PC expansion using WAFP was far more efficient. These findings show the usefulness of using WAFP based PC expansion to quantify uncertainty and analyze sensitivity of a oxyhemoglobin dissociation response model. Applying these techniques could help analyze the fidelity of other relevant models in preparation for clinical application.
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... pH's large influence on oxyhemoglobin dissociation, called the Bohr Effect, plays a major physiological role in transporting oxygen. The Bohr Effect is the mutual interaction of oxygen binding and hydrogen ion binding of oxyhemoglobin [54]. This mutual interaction allows a decrease in pH near tissue capillaries to increase delivery of O2 at the tissue level [18,48]. ...
Performing uncertainty quantification (UQ) and sensitivity analysis (SA) is vital when developing a patient-specific physiological model because it can quantify model output uncertainty and estimate the effect of each of the model's input parameters on the mathematical model. By providing this information, UQ and SA act as diagnostic tools to evaluate model fidelity and compare model characteristics with expert knowledge and real world observation. Computational efficiency is an important part of UQ and SA methods and thus optimization is an active area of research. In this work, we investigate a new efficient sampling method for least-squares polynomial approximation, weighted approximate Fekete points (WAFP). We analyze the performance of this method by demonstrating its utility in stochastic analysis of a cardiovascular model that estimates changes in oxyhemoglobin saturation response. Polynomial chaos (PC) expansion using WAFP produced results similar to the more standard Monte Carlo in quantifying uncertainty and identifying the most influential model inputs (including input interactions) when modeling oxyhemoglobin saturation, PC expansion using WAFP was far more efficient. These findings show the usefulness of using WAFP based PC expansion to quantify uncertainty and analyze sensitivity of a oxyhemoglobin dissociation response model. Applying these techniques could help analyze the fidelity of other relevant models in preparation for clinical application.
... where [H + ] = 10 −pH in RBCs. Given the pH of plasma, the pH of RBCs can be obtained using the simple relationship established by Siggaard-Andersen and colleagues (Siggaard-Andersen 1971;Siggaard-Andersen and Salling 1971): pH rbc = 0.795pH pl + 1.357, giving the Gibbs-Donnan ratio for electrochemical equilibrium of H + and HCO 3 − across the RBC membrane as a function of pH pl : R rbc = 10 −(pHpl − pHrbc) = 10 −(0.205pHpl − 1.357) (see Table 1). In our previous S HbO 2 and S HbCO 2 models (Dash and Bassingthwaighte 2010), R rbc was considered as a constant: R rbc = 0.69, a value corresponding to pH pl = 7.4 and pH rbc = 7.24 (see Table 1). ...
Equations for blood oxyhemoglobin (HbO2) and carbaminohemoglobin (HbCO2) dissociation curves that incorporate nonlinear biochemical interactions of oxygen and carbon dioxide with hemoglobin (Hb), covering a wide range of physiological conditions, are crucial for a number of practical applications. These include the development of physiologically-based computational models of alveolar-blood and blood-tissue O2-CO2 transport, exchange, and metabolism, and the analysis of clinical and in vitro data.
To this end, we have revisited, simplified, and extended our previous models of blood HbO2 and HbCO2 dissociation curves (Dash and Bassingthwaighte, Ann Biomed Eng 38:1683-1701, 2010), validated wherever possible by available experimental data, so that the models now accurately fit the low HbO2 saturation ([Formula: see text]) range over a wide range of values of [Formula: see text], pH, 2,3-DPG, and temperature. Our new equations incorporate a novel [Formula: see text]-dependent variable cooperativity hypothesis for the binding of O2 to Hb, and a new equation for P 50 of O2 that provides accurate shifts in the HbO2 and HbCO2 dissociation curves over a wide range of physiological conditions. The accuracy and efficiency of these equations in computing [Formula: see text] and [Formula: see text] from the [Formula: see text] and [Formula: see text] levels using simple iterative numerical schemes that give rapid convergence is a significant advantage over alternative [Formula: see text] and [Formula: see text] models.
The new [Formula: see text] and [Formula: see text] models have significant computational modeling implications as they provide high accuracy under non-physiological conditions, such as ischemia and reperfusion, extremes in gas concentrations, high altitudes, and extreme temperatures.
... (10) (Obrázok 3, F) zohľadnením merania nezávislého nad Bohrovými protónmi od Atha a Ackers (11), ktorý zmerali enthalpiu odviazania kyslíka ako 59 kJ/mol. [12] pri okysličovaní (A), od Bauera a Schrödera [7] ako strata CO2 pri titrovaní v otvorenom systéme (B), od Siggaard-Andersena [8] ako korekcie kyslosti pri plnom odkysličení (C), od Naereau ako zmena sO2 pri zmene kyslosti (D), od Matthewa a spol. [9] ako zmena naviazaného CO2 pri titrovaní v uzatvorenom systéme (E) and Reevesa [10] pri okysličovaní za rozdielnej teploty(F). ...
Tento príspevok nadväzuje na náš model viazania krvných plynov a protónov na hemoglobín publikovaný v roku 2015 v impaktovanom časopise " Scandi-navian Journal of Clinical and Laboratory Investigation " v článku pod názvom " Adair-based hemoglobin equilibrium with oxygen, carbon dioxide and hydrogen ion aktivity ". Ukazuje, ako je možné tento model implementovať v Mode-lice (http://www.modelica.org) za pomoci knižnice Physiolibrary (http://www. physiolibrary.org). Nakoniec ukazuje možnosť využiť tento implementovaný model hemoglobínu pre simuláciu rôznych stavov a experimentov oxygeniná-cie, carboxylácie a/alebo titrácie kyselinami či zásadami.
... H max to a higher pH. In human Hb the allosteric effector DPG likewise increases AzHmax and shifts this maximum value to a higher pH (Siggaard-Andersen, 1971;De Bruin et aI., 1973). However, these effects do not seem to be as pronounced as those here observed in teoch Hb, at naturally occurring organic P/Hb ratios. ...
The free open-source library called Physiolibrary (https://github.com/MarekMatejak/Physiolibrary) in version 3.0 recast components from physiological domains such as hydraulic (cardiovascular), thermal, osmotic and chemical into Modelica Standard Library (MSL) concept of Fluid/Media. Components are expanded to include gases transports, acid-base, electrolytes, nutrients delivery and endocrines by simple selecting pre-packaged media. They can be connected directly (the same medium) or across membranes (different media), allowing small physiological models to be easily coupled within more quantitative ones with minimal effort.
The free open-source Physiolibrary version 3.0 (https://github.com/MarekMatejak/Physiolibrary) has transformed components from physiological domains such as hydraulic (cardiovascular), thermal, osmotic, and chemical into the Modelica Standard Library (MSL) concept of Fluid/Media and Chemical library. Components are extended to include gas transports, acids-bases, electrolytes, nutrient delivery, and endocrines by simply selecting pre-made media. They can be connected directly (same medium) or across membranes (different media), allowing small physiological models to be integrated into more quantitative models with minimal effort.
For a century, the influence of the Bohr effect on the utilization of blood-borne oxygen has been deemed secondary to its influence on the uptake of carbon dioxide by the blood. Here, we show that the opposite is the case. Using a simple two-ligand, two-state formulation, we modeled the simultaneous oxygen and proton binding to hemoglobin, as well as the resulting acid-base changes of the surrounding solution. Blocking of the Bohr effect in this model system results in a dramatic increase in the oxygen affinity, as expressed by the oxygen partial pressure at half saturation, the P 50 . It also becomes clear that the P 50 and the Bohr factor (a measure of the size of the Bohr effect) are not independent but directly related. Thus, everything else being equal, varying the number of Bohr groups from 0 to 8 per tetramer results in an increase in the Bohr factor from 0 to −0.9 and an increase in P 50 from 6 to 46 mmHg at a constant Pco 2 of 40 mmHg. Therefore, changes in hemoglobin structure that lead to changes in the Bohr factor will inevitably also change hemoglobin oxygen affinity.
NEW & NOTEWORTHY Using a mathematical model, we show that the Bohr effect has a more profound effect on gas exchange than is evident when comparing oxygen equilibrium curves measured in the laboratory at different constant values of Pco 2 or pH. Protons preloaded on the Bohr groups, as well as the protons taken up during oxygen unloading, dramatically decrease oxygen affinity of the physiological oxygen equilibrium curve. Therefore, the Bohr effect is instrumental in setting the oxygen affinity.
In birds and mammals hemoglobin (Hb) plays a central role in the delivery of the oxygen that drives aerobic metabolism and in the removal of the carbon dioxide and the hydrogen ions that would otherwise disrupt aerobic metabolism. In large measure, Hb is important because it reversibly binds O2. One important feature of O2 binding is an increased blood-O2 capacity; essentially all of the O2 supplied to tissues in birds and mammals is carried as HbO2. A second important feature of O2 binding is the affinity of Hb for O2 which insures O2 loading in lungs and sets the pressures for O2 release in tissues. A third important feature of O2 binding is cooperativity. O2 binding shows positive cooperativity, linkage of O2 to deoxy Hb increases the affinity of Hb for subsequent O2. Finally, in addition to O2, Hb reversibly binds CO2 and H+ and in so doing transports a significant portion of these metabolic products from their sites of production to lungs and other sites of turnover in the body.
As has been known for over a century, oxygen binding onto hemoglobin is influenced by the activity of hydrogen ions (H+), as well as the concentration of carbon dioxide (CO2). As is also known, the binding of both CO2 and H+ on terminal valine-1 residues is competitive. One-parametric situations of these hemoglobin equilibria at specific levels of H+, O2 or CO2 are also well described. However, we think interpolating or extrapolating this knowledge into an ‘empirical’ function of three independent variables has not yet been completely satisfactory. We present a model that integrates three orthogonal views of hemoglobin oxygenation, titration, and carbamination at different temperatures. The model is based only on chemical principles, Adair’s oxygenation steps and Van’t Hoff equation of temperature dependences. Our model fits the measurements of the Haldane coefficient and CO2 hemoglobin saturation. It also fits the oxygen dissociation curve influenced by simultaneous changes in H+, CO2 and O2, which makes it a strong candidate for integration into more complex models of blood acid-base with gas transport, where any combination of mentioned substances can appear.
The Bohr coefficient, b, and the Haldane coefficient, h, were measured simultaneously in erythrolysates in the presence and absence of CO2and 2,3-diphosphoglycerate. We found b—h=—0.04 (—0.023 to —0.054) although b—h=—0 according to the linkage equation. The cause of the observed difference seems to be that his measured as the mean value for oxygen saturation fractions (zHbO2) between 0 and 1, while brefers to a zHbO2of 0.54. Apparently b(and h) varies with zHbO2and reaches a numerical maximum at an intermediate zHbO2value.
After intense exercise muscle may give off hydrogen ions independently of lactate, perhaps by a mechanism involving sodium ions. To examine this possibility further five healthy young men cycled for 2 min to exhaustion. Blood was drawn from catheters in the femoral artery and vein during exercise and at 1-h intervals after exercise. The blood samples were analysed for pH, blood gases, lactate, haemoglobin, and plasma proteins and electrolytes. Base deficit was calculated directly without using common approximations. The leg blood flow was also measured, thus allowing calculations of the leg’s exchange of metabolites. The arterial blood lactate concentration rose to 14.2 ± 1.0 mmol L–1, the plasma pH fell to 7.18 ± 0.02, and the base deficit rose 22% more than the blood lactate concentration did. The femoral-venous minus arterial differences peaked at 1.8 ± 0.2 mmol L–1 (lactate), –0.24 ± 0.01 (pH), and 4.5 ± 0.4 mmol L–1 (base deficit), and –2.5 ± 0.7 mmol L–1 (plasma sodium concentration corrected for volume changes). Thus, near the end of the exercise and for the first 10 min of the recovery period the leg gave off more hydrogen ions than lactate ions to the blood, and sodium left plasma in proportion to the extra hydrogen ions appearing. The leg’s integrated excess release of hydrogen ions of 0.88 ± 0.45 mmol kg–1 body mass was 67% of the integrated lactate release. Base deficit calculated by the traditional approximate equations underestimated the true value, but the error was less than 10%. We conclude that intense exercise and lactic acidosis may lead to a muscle release of hydrogen ions independent of lactate release, possibly by a Na+,H+ exchange. Hydrogen ions were largely buffered in the red blood cells.
Acid-base titrations were performed on oxygenated and deoxygenated blood at constant PCO2. The BE-coefficient, - (δBlood-base excess)/(δHb-bound O2), is 0.3 mol/mol for normal blood, varying between 0 and 0.4 with variations in pH, PCO2, and 2,3-diphosphoglycerate concentration. The ratio between the BE-coefficient for a given plasma pH and the Haldane coefficient (-δHb-bound H+)/(δHb-bound O2)) for the corresponding erythrocyte pH is found to be 0.77. Comparison of the two coefficients indicates that (Ery-pH)=0.796 (Plasma-pH) + 1.356.
The four different ways of improvement of oxygen supply to tissues in different phases of the burn syndrome are: 1. increase in cardiac output, 2. increase in haemoglobin level, 3. increase in arterial oxygen saturation and 4. decrease in oxygen affinity of haemoglobin.The observed decrease in oxygen affinity of haemoglobin due to increased synthesis of 2,3-DPG in the erythrocytes (‘the oxygen affinity reserve’) is of great importance for the maintenance of a constant and sufficient tissue oxygen tension in patients with severe burns during several weeks after the trauma.
The sections in this article are:
The effect of the concentration of red cell 2,3-diphosphoglycerate (2,3-DPG, 0.5–21
D\textpH\texti /D\textpH\texte = 1 + log\user2 r\textH\text + = 1 + \textpH\texti - \textpH\texte .\Delta {\text{pH}}_{\text{i}} /\Delta {\text{pH}}_{\text{e}} = 1 + \log \user2{ }r_{{\text{H}}^{\text{ + }} } = 1 + {\text{pH}}_{\text{i}} - {\text{pH}}_{\text{e}} .
The validity of this equation appears to be restricted to conditions where the Donnan ratior
H
+ is altered between 0.3 and 1 either by changes of the red cell concentration of buffering anions such as 2,3-DPG or by changes of the extracellular pH.As determined in suspensions of red cells with intact membranes, the 2,3-DPG- and pH-induced changes of pHi/pHe lead to proportional changes in the buffering power of the non-bicarbonate buffers of erythrocytes. Due to this effect the buffering power of suspensions of cells containing 5 times the normal concentration of the buffer 2,3-DPG islower than that of cells with normal 2,3-DPG content (at extracellular pH values above 7). These findings demonstrate that the action of intracellular nonbicarbonate buffers in blood is effectively modulated by the physico-chemical properties of the red cell membrane.
Evidence suggests that strong ions can exist reversibly bound to proteins in a pH-dependent manner and that they can be recruited into the biological solution, modulating its strong ion difference in a process that opposes the acid base disturbances imposed on the system. These recruitable strong ions represent the solution's 'strong ion reserve'. The physiologic [corrected] role of these protein-bound strong ions [corrected] in the buffering of acid base disorders is discussed.
The first apparent dissociation constant of carbonic acid, pK'1, of plasma and red cells was determined on venous blood of ten healthy, adult, male, human subjects. pH and PCO2 of plasma and red cells were analyzed electrometrically and a micromanometric method was used for the determination of total carbon dioxide content. Erythrocyte carbamino hemoglobin levels were estimated and used for the correction of erythrocyte pK'1. Each blood sample was subjected to the following regimen before centrifugation, 1) As drawn from the antecubital vein, 2) Oxygenated with a 5% CO2, O2 balance gas mixture, and 3) Reduced with a 5% CO2, N2 balance gas mixture. pK'1 of plasma and red cells are presented: (see article). The consistently larger values for red cell pK'1 than the respective plasma data may be attributed to the greater amount of carbamino hemoglobin concentration present in the erythrocytes. A simplified method for the calculation of erythrocyte bicarbonate concentration using the experimentally determined red cell pK'1 value has been formulated. The method involves the use of a regression equation relating plasma and red cell pH, the equivalence of plasma and red cell PCO2, along with the experimentally determined red cell pK'1.
The Haldane coefficient (the amount of the oxygen-linked hydrogen ion binding of hemoglobin) was determined in bovine erythrolysate (Hb concentration equals 13.5 mM) by means of the differential titration method with varying PCO2 from 0 to 74 mm Hg and pH from 6.0 to 8.5 at 37 degrees C. The maximum value of the coefficient was found to be 0.49 mM per mM Hb at PCO2 equals 0 and pH 7.20. With increasing of PCO2, the coefficient became smaller in all ranges of pH studied. The coefficient under the conditions of pH 7.20 and PCO2 equals 45 mm Hg that are normally prevailing in the interior of bovine erythrocytes was 0.31.
A comparative study is reported of the effect of temperature and acidity on the oxygen affinity of Hbs A and S when the Hbs are present in both intact red cells and in aqueous solution in both the presence and absence of 2,3-DPG.The reciprocal temperature plots for the oxygen affinity (expressed in terms of p50) of Hb A and Hb S stripped of organic phosphate and dissolved in phosphate buffer are found to be linear over the whole range of temperatures studied (2–40°C); the plots for cells, are, however, significantly curved. No such finding has been previously reported.The alkaline Bohr effect is found to be temperature dependent for both the Hbs, whether in cells or in phosphate buffer. At any given temperature, the Bohr value for Hb SS cells is more negative than that for Hb AA cells.Another important finding reported is that, whereas Hill plots for solutions of Hbs A and S are found, as expected, to be linear between 20 and 80% oxygenation, those for cells and for solutions containing 2,3-DPG in three- and sixfold molar excess are curved over the whole range of oxygen pressures studied. Also, n50, the Hill plot slope at 50% conversion to oxyhemoglobin, is profoundly temperature dependent in the case of cells though less so for Hb solutions. In agreement with the literature, n50 values in bis-Tris-HCl buffer are found to be dependent on the 2,3-DPG concentration.The above findings indicate that, although the behavior of Hbs in dilute solutions is simple, the situation is much more complex in red cells, where the concentration of the protein is 200–400-fold greater. Intertetramer interactions must become more important within the erythrocyte.
In experiments with graded exercise of 15 men (6 untrained, 3 semitrained, 6 endurance-trained) the trained subjects showed a massive shift to the right of thein vivo O2 dissociation curve (ODC) of femoral venous blood. At a saturation of 20 to 25% (18 mkp/sec)P
O2 was about 9 mm Hg higher for the trained than for the untrained group. The following factors play a role: 1. The 2,3-diphosphoglycerate [2,3-DPG] concentration was increased by 15 to 20% in the trained group which explains about 2 mm Hg of the difference inP
O2. 2. Exercise acidosis in the femoral venous blood depends to a large extent on CO2 in the trained, but on lactic acid in the untrained group. At low saturations the CO2-Bohr effect increases sharply thus having a greater importance in the trained subjects. This factor can explain about 2 mm Hg of the difference. However, influence of chloride and 2,3-DPG on the Bohr effect must be taken into consideration. 3. Since the large ODC-shift to the right of the trained group was not reproducible underin vitro conditions, it is suggested that a rapidly decaying unknown substance accounts for the remaining difference inP
O2.
A theoretical model of the process of respiratory gas exchange between capillary and tissue is described, with special reference to the importance of variations in blood properties. The volume of tissue supplied with oxygen from a single vessel, as a function of the blood flowrate (u), hematocrit (h), and 2,3 diphosphoglycerate concentration (DPG), is calculated from a solution to the set of equations governing species distributions in the blood and tissue. The results, which are presented in the form of crossplots of the three blood parameters (u, h, and DPG) at a constant oxygen supply rate, show the possible significance of in vivo variations in the oxygen affinity of hemoglobin as a compensatory mechanism. Of further physiological interest is the sharp increase in venous erythrocyte pH in response to decreases in hematocrit, once the hematocrit is below a certain level. These results, and those relating DPG to hematocrit at constant O2 supply, are consistent with experimental observations of elevated DPG and pH levels in anemic individualts, and the dependence of erythrocyte DPG concentration upon pH.
In 1969 a model was proposed (BARNIKOL et al.) to explain the effect of the Hb- concentration on the O2- Hb- binding curve (GROTE, 1967). The existence of at least one low molecular substance (called Z) besides H and CO2 was postulated, which is equimolar with Hb (≈ 5 mM) and which is bound preferentially to deoxy- Hb. From a simple stoichiometric consideration it follows, that a substance can only have a measurable influence on the O2- Hb- binding curve, if its molar concentration is not to low compared with the molar Hb- concentration. For example, 2,3- diphosphogly-cerate (= DPG), glutathione and Mg++ have intracellular concentrations of the necessary magnitude. Only a small part of Magnesium is bound to the membrane of the erythrocytes (HARRISON et al.). Principally all these substances are candidates for Z. In 1967 it was shown by BENESCH et al. and by CHANUTIN et al., that ATP and DPG influence the O2-Hb- binding curve as was postulated for Z, and it was shown (BENESCH et al., 1968), that the organic phosphates are bound preferentially to deoxy-Hb. In our speculation on the chemical nature of Z first we prefered Mg++ because of its complex forming properties. It is known for a long time, that Mg++ forms a complex with ATP (MARTELL et al.,1956) and in 1970 it was found, that DPG forms also a complex with Mg++ (COLLIER et al.).
Eighteen anesthetized baboons were studied to determine the effects of passive hyperventilation and phlebotomy on oxygen transport. After 1 hour of hyperventilation a significant increase in the red cell affinity for oxygen occurred in vivo. This was not associated with any significant changes in cardiac output, oxygen consumption, or in lactic acid production. There was a 40% decrease in cerebral blood flow, a 10 mm Hg decrease in the pulmonary artery Po2 level, and a 17 mm Hg decrease in the jugular venous Po2 level. After 1 hour of hyperventilation, the plasma inorganic phosphorus level decreased significantly, the red cell ATP level decreased slightly, and the red cell 2. 3 DPG level increased significantly, indicating that inorganic phosphorus had been removed from the blood during hyperventilation. Passive hyperventilation was maintained, and the baboons were bled 32% of their red cell volume. The blood volume was partially restored with nonbuffered isotonic saline. One hour after the phlebotomy and volume restoration (2 hours of hyperventilation) there were no changes in oxygen consumption, cardiac output, cerebral blood flow, or blood lactate levels, but the pulmonary artery Po2 level was decreased by 15 mm Hg, and the jugular venous Po2 level was decreased by 20 mm Hg. Systemic oxygen consumption was not affected by the significant decrease in pulmonary artery Po2.
To describe the overall gas exchange rates in red blood cells (RBC), a computer program for solving the diffusion equations for O2, CO2, and HCO3-that accompany the chemical reactions of Bohr- and Haldane-effects was developed. Three diffusion equations were solved alternatively and repeatedly in an increment time of 2 ms. After solving the diffusion equations the Po2, O2 saturation (So2), Pco2, pH, and HCO3-content were corrected by using the Henderson-Hasselbalch equation, where the buffer value was newly derived from the CO2 dissociation curve. In computing the Haldane effect, the buffer value was taken to be 44mmol X l(RBC)-1 X pHc-1, so that the change in intracellular dissolved CO2 caused by the So2 change was fully compensated by the subsequent CO2 diffusion. The oxygenation and deoxygenation rate factors of hemoglobin were assumed to be 2.09 X (1-S)2.02 and 0.3s-1 X Torr-1, respectively. The Po2 change due to the Bohr-shift was computed from Hill's equation, in which the K value was given by a function of the intracellular pH. When the parameter values thus far measured were used, the computed Bohr- and Haldane-effects coincided well with the experimental data, supporting the validity of the equations. The overall gas exchange profiles calculated in the pulmonary capillary model showed that the CO2 equilibration time was significantly longer than the oxygenation time.
A transport model for translocation of the protonophore CCCP across the red cell membrane has been established and cellular CCCP binding parameters have been determined. The time course of the CCCP redistribution across the red cell membrane, following a jump in membrane potential induced by valinomycin addition, has been characterized by fitting values of preequilibrium extracellular pH vs. time to the transport model. It is demonstrated, that even in the presence of valinomycin, the CCCP-anion is "well behaved," in that the translocation can be described by simple electrodiffusion. The translocation kinetics conform to an Eyring transport model, with a single activation energy barrier, contrary to translocation across lipid bilayers, that is reported to follow a transport model with a plateau in the activation energy barrier. The CCCP anion permeability across the red cell membrane has been calculated to be close to 2.0 X 10(-4) cm/sec at 37 degrees C with small variations between donors. Thus the permeability of CCCP in the human red cell membrane deviates from that found in black lipid membranes, in which the permeability is found to be a factor of 10 higher.
In man adaptation of oxygen transport to low ambient pO2 comprises a series of physiological responses, including increases in ventilation, cardiac output, and oxygen carrying capacity (hemoglobin concentration). The increase in ventilation is generally considered to be the most responsive and effective of these. A fourth adaptive mechanism which involves changes in oxygen affinity of circulating red cells has been focused on recently (Astrup et al, 1968; Lenfant et al, 1968). Theoretically, a decrease in red cell affinity for oxygen will facilitate oxygenation of the tissues and an increased amount of oxygen will be released from the red cells at a given pO2. However, oxygen uptake by the red cells in the lungs may be impaired. A decrease in red cell affinity for oxygen is beneficial for the total oxygen transport only when the arterial oxygen saturation is greater than 70%.
The effect of changes in effector ligand activity on the oxygen affinity of human whole blood was calculated for protons, carbon dioxide, and 2,3-diphosphoglycerate (DPG) at various concentrations of these ligands. The data were used to obtain, from experimentally obtained oxygen dissociation curves of blood from apparently healthy subjects, the standard oxygen dissociation curve (pH = 7.40, Pco2 = 40 mm Hg, DPG = 5.0 mmol/l, and HbCO = 1.0% of normal human blood. © 1974 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
Oxygen binding curves were determined, at 2 plasma pH values in the range 7.0 to 7.5, each at 2 pCO2 values of 22 and 77 mm Hg, respectively, on human whole blood where the red cell 2,3 diphosphoglycerate (DPG) concentration had been decreased to about 1 mmol/l of packed cells and increased to about 9 mmol/l of packed cells. Numerical values are given for the oxygen saturation dependent ligand interaction coefficients ΔlogpO2/ΔpH, ΔlogpO2/ΔlogpCO2 and ΔlogpO2/ΔlogDPG and their dependence on pH, pCO2 and DPG, respectively.
Hardly any molecule has attracted the interest of as many scientific disciplines as has haemoglobin. Contributions from physicists, chemists and biologists have combined to bring about a full understanding of the relationship between its structure and function. In particular, the finding that red cell metabolism and haemoglobin function are interdependent has provided important biological insights that have transformed the red cell from a seemingly inconspicuous biological unit into a model demonstrating several fundamental biological principles.
It is the aim of this article to summarise current knowledge about the physiological properties of haemoglobin in transport of oxygen and carbon dioxide in the blood, and to relate this to its molecular structure.
1. The carbamate (HbCO 2 ) concentration in oxygenated and deoxygenated human adult and foetal red blood cells was estimated at a constant pressure of carbon dioxide ( P CO 2 = 40 mm Hg) and various pH values of the serum. The Donnan ratio for chloride and bicarbonate ions was used to calculate the bicarbonate concentration in the red cells. With this figure the carbamate concentration was calculated as follows:
[HbCO 2 ] = [Total CO 2 ] — [HCO ⁻ 3 ] — [dissolved CO 2 ].
2. At a given pH value in the red cell deoxygenated foetal red cells contain more HbCO 2 than deoxygenated adult ones. Upon oxygenation (at constant pH) HbCO 2 drops in both types of erythrocytes to lower values than in deoxygenated cells. The fraction of ‘oxylabile carbamate’ (−ΔHbCO 2 /ΔHbO 2 ) at a red cell pH of 7·2 and a P CO 2 of 40 mm Hg is 0·117 in foetal and 0·081 in adult erythrocytes.
3. From the fraction of moles carbamate formed per Hb monomer (moles CO 2 /mole Hb i ) K ′ c and K ′ z , the apparent carbamate equilibrium constants were calculated which can be used to estimate the carbamate concentration in normal adult and foetal blood.
4. The first apparent dissociation constant of carbonic acid is significantly higher in oxygenated (−log 10 K ′ 1 = pK′ 1 = 6·10) than in deoxygenated (pK′ 1 = 6·12) adult red cells, whereas in foetal red cells the difference is smaller and statistically not significant.
5. For a given set of physiological conditions in arterial and mixed venous blood in respect to oxygen saturation, P CO 2 and pH, the fractional contribution of carbamino compounds of haemoglobin to the amount of carbon dioxide which is exchanged during the respiratory cycle was computed on the basis of the present results and found to be 10·5% in adult and 19% in foetal blood.
Extract: The buffering properties and the Bohr effect were studied in vitro on 10 human fetal and 9 adult whole blood samples. At the beginning of the experiments, the fetal and adult blood samples were similarly acidotic (BE = - 9 mEq/liter).
The level of 2,3-diphosphoglycerate (2,3-DPG) in erythrocytes was found to be increased in patients with cirrhosis of the liver, and as a consequence the oxygen affinity of hemoglobin was decreased. Part of the 2,3-DPG increase in the patients with cirrhosis was caused by a concomitant anemia. At all hemoglobin levels, however, patients with cirrhosis showed an additional 2,3-DPG increase in comparison with patients without cirrhosis. Red cell 2,3-DPG was positively correlated to pH in plasma and in erythrolysate. It is likely that pH changes contribute to the regulation of the red cell 2,3-DPG concentration in cirrhotic patients. In a group of these patients (n=48), the arterial pH and pCO2 did not differ significantly from normal values. Other, as yet unknown, factors must presumably contribute to the increase in red cell 2,3-DPG. No direct effect of NH4+ on red cell 2,3-DPG could be demonstrated in vitro. The plasma concentration of NH4+ in vivo correlated positively to plasma pH. The lowered oxygen affinity does not contribute significantly to the decrease in oxygen saturation of arterial blood often found in patients with hepatic cirrhosis.
Prolonged incubation (5 hr) of canine erythrocyte at pH 7.2, or pH 7.7 and PCO2 3.5 or 35 were used to determine the effect of CO2 on glycolysis. The following was observed: 1) an inverse relationship exists between lactate production and CO2. 2) the inhibitory effect of H+ ion on glucose uptake is decreased by prolonged incubation (5 hr compared to 1 hr). 3) carbon dioxide becomes inhibitory for glucose uptake following prolonged incubation. 4) increasing external bicarbonate decreases erythrocyte 2–3 DPG. 5) iodoacetate inhibition of glycolysis reverses the inverse relationship of lactate (pyruvate) to CO2 suggesting that the CO2 sensitive enzyme lies proximal to 1–3 diphosphoglycerate.
The specific effect of carbon dioxide on blood affinity for oxygen (P50 at pH 7.2) and on the Bohr effect (Δ log P50/ΔpH) has been measured in adult and cord blood in the presence (fresh blood) and the absence (stored blood) of 2,3-diphosphoglycerate (DPG). Studies were performed without C02 and at a P50, of 40 torr at 37 °C. Blood pH was varied with fixed acid (HC1) and base NaOH) from 6.8 to 7:4. Carbon dioxide lowers the Bohr coefficients similarily in cord and adult blood; this effect is 50% inhibited by DPG. In the absence of DPG, the CO 2 specific effect on P50 is lower in cord blood than in adult blood. DPG increases P50 less in cord blood than in adult blood. In cord blood the DPG and CO2 effects are additive. The higher affinity of foetal blood for oxygen may be explained by the lower response of foetal haemoglobin to CO5 and DPG. This is considered as an example of physiological adaptation of foetal erythrocytes to the conditions of intra-uterine life.
The Bohr effect related to whole blood (Be) and erythrocyte pH (Bi) was determined over a wide oxygen saturation range at an almost constant 2,3-diphosphoglycerate concentration. In two different sets of experiments acidification was caused either by fixed acid (lactic acid) or by CO2. Fixed acid-induced Bohr coefficients can be approximated by a second-order function of O2 saturation with a maximum at mid-saturation range. CO2-inducedBe andBi values yield a third-order relationship with highest results at low O2 saturation. The ratioBi/Be with respect to fixed acid-acidification exhibits a positive correlation with oxygenation, whereas the corresponding ratio referring to CO2 does not. For this different behavior a possibly more pronounced diminution of the Haldane effect, i.e. oxygen-linked proton release, in the presence of fixed acid is discussed. The physiological importance of the magnitude of CO2-inducedBi values at low O2 saturation is emphasized.
The Bohr coefficient, b, and the Haldane coefficient, h, were measured simultaneously in erythrolysates in the presence and absence of CO2and 2,3-diphosphoglycerate. We found b - h= - 0.04 ( - 0.023 to - 0.054) although b - h= - 0 according to the linkage equation. The cause of the observed difference seems to be that his measured as the mean value for oxygen saturation fractions (zHbO2) between 0 and 1, while brefers to a zHbO2of 0.54. Apparently b(and h) varies with zHbO2and reaches a numerical maximum at an intermediate zHbO2value. © 1972 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
The oxygen affinity of normal human blood was measured at two different pH values and three different pCO2 values over the whole saturation range. From these data, and from data on the interaction of 2,3-diphosphoglycerate with hemoglobin, values were calculated for the proton-linked oxygen affinity at different pCO2 values and for the carbamino-linked oxygen affinity at different pH values. Both the proton-linked and the carbamino-linked oxygen affinity were found to be markedly dependent on the degree of oxygen saturation. The relative contribution of the three ligands, i.e. protons, carbon dioxide and 2,3-diphosphoglycerate, to the classical Bohr effect was estimated and found to be dependant on the oxygen saturation level. The values for the proton-linked oxygen affinity and the carbamino-linked oxygen affinity were found, by applications of Wyman's reciprocal relations and 2,3-diphosphoglycerate binding data, to give good estimates of the oxygen-linked proton binding and the oxygen-linked carbamino binding. The data show that the oxygen-linked carbamino reaction takes place mainly during the binding of the first 2 oxygen molecules with a value of about 0.25 mol per mol of oxygen. The proton release accompanying this reaction was estimated to be 1.3–1.7 mol per mol.
Human erythrocyte fluid is described as a thermodynamical system. As independent variables are chosen: Oxygen saturation, pH, pCO2, and total DPG concentration. The dependent variables are: pO2, titratable base, total CO2, and DPG activity. Four differential equations relating these variables constitute the basis of the description. 10 different partial differential coefficients between the variables are described. All the remaining 310 different partial differential coefficients may be derived in terms of these 10.
Factors affecting oxygen transport to tissue are cardiac output and its distribution, blood oxygen content, and affinity of hemoglobin for oxygen. Hemoglobin-oxygen affinity, in turn, depends upon pH, CO2, and DPG. The physiologic significance of such alterations in oxygen affinity of hemoglobin has been a matter of some interest. In the course of studying the in vivo consequences of increased hemoglobin-oxygen affinity, we recently noted that rats with a 15 mm Hg decrease in PO2 due to DPG depletion were able to perform in a maximal exercise test as well or very nearly as well as rats with normal hemoglobin-oxygen affinity. By contrast, rats with normovolemic anemia showed a marked reduction in work capacity.
Acid-base titrations were performed on oxygenated and deoxygenated blood at constant PCO2. The BE-coefficient, - (δBlood-base excess)/(δHb-bound O2), is 0.3 mol/mol for normal blood, varying between 0 and 0.4 with variations in pH, PCO2, and 2,3-diphosphoglycerate concentration. The ratio between the BE-coefficient for a given plasma pH and the Haldane coefficient (-δHb-bound H+)/(δHb-bound O2)) for the corresponding erythrocyte pH is found to be 0.77. Comparison of the two coefficients indicates that (Ery-pH)=0.796 (Plasma-pH) + 1.356.
The oxygenation of haemoglobin is accompanied by structural changes in the subunits triggered by shifts of the iron atoms relative to the porphyrin and, in the β-subunits, also by the steric effect of oxygen itself. The oxygen-free form is constrained by salt-bridges which are broken by the energy of haem–haem interaction with the release of H+. 2,3-Diphosphoglycerate may add to the constraints by being stereochemically complementary to a site between the β-chains ; this complementarity is lost on oxygenation.
The CO2 content of dialyzed solutions of human haemoglobin has been measured at a PCO2, of 40 mm Hg and varying pH values without and with added 2,3 diphosphoglycerate (2,3 DPG). It has been found that 2,3 DPG reduces the CO2 affinity of deoxygenated haemoglobin. The decreased CO2 content in oxygenated haemoglobin solutions compared with deoxygenated haemoglobin at the same pH and PCO2, could be established only in the absence of 2,3 DPG. Haemoglobin solutions containing 2,3 DPG showed an even higher CO2 affinity of oxygenated haemoglobin compared with deoxygenated haemoglobin.The Bohr effect, when measured as equivalents OH−/ heme released from haemoglobin upon deoxygenation at a constant Pco2, and pH was lower in the absence than in the presence of 2,3 DPG. It is suggested that the binding of 2,3 DPG to deoxygenated haemoglobin interferes with the carbamate formation at the terminal amino groups of the β-chain. The physiological implications of these results for the CO2 transport and exchange are discussed.
The model has been constructed by combining information from the three-dimensional Fourier syntheses of horse oxyhaemoglobin at 5·5 Å and of sperm whale myoglobin at 1·4 Å resolution. Between them, these two sets of experimental data were sufficient to locate the positions of each of the haem groups and amino acid residues in haemoglobin within narrow limits. The nature of the residues was known from the chemical sequence. The accuracy of atomic positions is estimated to be of the order of 1 to 2 Å. The model has not yet been checked by comparison of observed and calculated intensities of X-ray reflexions.The model is sufficiently accurate, however, to show how the different kinds of residues are distributed between the interior and the surface of the four subunits; to locate the residues lying at the interfaces between the subunits and at the contacts between neighbouring molecules in the crystal; to determine the surroundings of the haem groups; and to reveal the existence of an internal cavity which is populated by a variety of polar side-chains and is filled with water.The nature of the intra- and intermolecular forces and the dissociation properties of haemoglobin are discussed in the light of the model. Relations between structure and function are dealt with in subsequent papers.
The change in the sum of the liquid-junction potentials of the junctions ‘test solution|KCl dilution|KCl saturated’ was measured as a function of the concentration of the KCl dilution. The results indicate that the junction potentials calculated by means of the Henderson equation are fairly reliable. For the junction ‘test solution|KCl saturated’ the values are of the same order of magnitude for the standard equimolar NBS phosphate buffer and plasma or erythrolysate, being approximately 2.9, 1.9, and 2.5 mV respec tively, at temperature 310 K.
1.1) The Bohr effect expressed as has been measured in dialysed haemoglobin solutions with and without added 2,3 DPG in the absende or in the presence of CO2.2.2) In the absence of CO2 and 2,3 DPG the Bohr factor was 0.47 and increased insignificantly to 0.53 after addition of 2,3 DPG. At a pCO2 of 40 mm Hg it decreased to 0.34 and rose to 0.48 after addition of 2,3 DPG.
THE alkaline Bohr effect is the uptake of protons at a pH greater than 6 when oxygen is removed from haemoglobin1. In horse haemoglobin the alpha-amino groups of the alpha-chain contribute about one-quarter of the alkaline Bohr effect2. Recently Perutz et al.3 proposed that histidine 146beta contributes about half the alkaline Bohr effect. Their evidence was based on the interpretation of a difference Fourier map of deoxyhaemoglobin after reaction with N-ethylmaleimide4. This haemoglobin has lost half its alkaline Bohr effect. We have prepared des-(His 146beta) human haemoglobin: it exhibits haem-haem interaction and lacks half the alkaline Bohr effect, and thus fully confirms the ideas of Perutz et al.3.
Blocking of the α-amino groups by cyanate inhibits the uptake of
CO2 by haemoglobin. It also inhibits the influence which
changes in pCO2 at constant pH normally have on the oxygen
affinity and on the Bohr effect. Blocking the α-amino groups of
the α chain reduces the alkaline Bohr effect by 25 per cent.
The imidazole groups of the C-terminal histidines of the beta-chains, together with the alpha-amino groups of the alpha-chains, are responsible for most of the Bohr effect. In oxyhaemoglobin these groups are free, while in deoxyhaemoglobin their pKs are raised, probably by linkage to carboxyl groups.
The interaction of 2,3-diphosphoglycerate with hemoglobin is markedly influenced by environmental factors. The strength and extent of binding is inversely proportional to pH, as would be expected for electrostatic interaction with a polyanion. The effect of 2,3-diphosphoglycerate in lowering the oxygen affinity of hemoglobin can be regarded as a special salt effect, since neutral salts act similarly, albeit in concentrations 1000 times greater than 2,3-diphosphoglycerate. This molecule is thus capable of regulating oxygen affinity at the millimolar concentrations in which it occurs in the erythrocyte without disturbing the osmotic equilibrium. Both oxygen and 2,3-diphosphoglycerate binding by hemoglobin are exothermic. Since oxygenation involves displacement of 2,3-diphosphoglycerate, the temperature dependence of the oxygenation reaction is lowered in the presence of the phosphate cofactor, permitting correct oxygen release over a wider range of temperature. The reaction of 2,3-diphosphoglycerate with deoxyhemoglobin is accompanied by a decrease in entropy, which, together with other evidence, bears out the previously proposed model for the interaction of 2,3-diphosphoglycerate with hemoglobin.
The oxygen saturation of capillary blood (approximately 25 μpl) is determined spectrophotometrically on hemolyzed packed red cells at 600 and 505 nm. The blood is centrifuged and the packed red cells hemolyzed by successive freezing and thawing. For a 0.1 mm cuvette an ordinary blood counting chamber is found convenient.
This chapter discusses linked functions and reciprocal effects in hemoglobin. Because the components are defined thermodynamically in terms of independently variable chemical entities and, therefore, a given component may exist in any number of different forms in equilibrium with one another, these considerations show that not only do the linkage relations apply irrespective of whether the macromolecules undergo chemical change or polymerization, but also irrespective of whether the ligands themselves associate and dissociate, possibly as macromolecules. From a formal point of view the distinction between ligand and macromolecule breaks down. This principle may be useful in the application of linkage relations to antibody–antigen systems and to enzyme systems when both enzyme and substrate are proteins. Another more general, and perhaps more interesting, question is whether the dissociation process is also dependent on oxygenation, i.e., whether the protons involved are oxygen-linked. There is now strong evidence that the dissociation at acidic pH is, indeed, dependent on oxygenation.
1. The difference of pH (ΔpH) between human deoxygenated haemoglobin (Hb) and oxygenated haemoglobin (O 2 Hb) solutions when equilibrated with physiological pressures of carbon dioxide is (experimentally) much less than previously supposed.
2. This smaller ΔpH is in contradiction to Wyman's (1948) theoretical calculations, wherein no allowance was made for the specific effect of carbamino‐compounds on the amount of base neutralized by haemoglobin. Other previous authorities have also neglected this factor, which when properly allowed for restores the role of carbamino‐compounds in CO 2 transport practically to that previously estimated by Ferguson & Roughton (1934 a, b ).
3. At a given pH and P CO 2 , more CO 2 is bound by Hb solutions than by O 2 Hb, the difference increasing with pH. This result provides further, and seemingly decisive, evidence that the bound‐CO 2 in the blood other than HCO 3 — (i.e. x ‐bound CO 2 ) is oxygen‐linked.
4. According to a modified form of the Henderson—Hasselbalch equation for haemoglobin solutions [Formula: see text]. The value of pK 1 ′ is the same in Hb as in O 2 Hb solution and from the data in 3 is found to have the value 6·15 at 37° C.
5. The difference between the titration curves of O 2 Hb and Hb (Δ X̄ ), at a given pH had been hitherto supposed to be the same in presence of CO 2 as in its absence. Our experiments show, however, that Δ X̄ is less in presence of CO 2 and at pH > 7·5 may even change sign. This paradoxical effect is also explicable, at any rate semi‐quantitatively, by the effect of carbamino compounds on the buffer power, according to the theory put forward in the paper.
6. The results show that the buffer power (d B /dpH) of haemoglobin solution under physiological conditions is 20‐30% greater than previously estimated, and this also is in line with the new theory.
7. In graphs of total CO 2 versus P CO 2 in haemoglobin solutions (or blood) it has been customary to suppose that points on straight lines radiating from the origin are points of equal pH. Our data, however, show that the iso‐pH lines drawn through the experimental points in the pH range, 7·2‐7·4 do not, when produced as straight lines, pass through the origin, but intercept the P CO 2 axis significantly to the right thereof.
8. Calculations indicate that most of the x ‐bound CO 2 in haemoglobin solutions at pH 7·2‐7·4 and at 37° C can be accounted for by carbaminobound CO 2 without the need of postulating the existence of appreciable amounts of yet other forms, i.e. y ‐bound CO 2 , in this range.
Estimation of 2,3-DPG in whole blood
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