Oxygen Equilibrium Curves Of Hemoglobin Research Articles

Ex-Vivo FT-4202 Treatment Improves Hemoglobin Oxygen Affinity and Membrane Health in Red Blood Cells of Patients with Hemoglobin SS and Hemoglobin SC Disease Irrespective of Prior Hydroxyurea Use

The two most common genotypes causing sickle cell disease (SCD) are homozygous bsbs producing hemoglobin S (HbSS), and compound heterozygous bsbc producing HbS and HbC (HbSC). HbS polymerizes in both, when deoxygenated. Patients with HbSC have distinct hematologic characteristics such as higher Hb levels but lower fetal hemoglobin (HbF); increased corpuscular Hb concentration, due to pronounced red blood cell (RBC) K+ loss and dehydration, contributes to HbS polymerization. While hydroxyurea (HU) as a HbF-inducing agent clearly improves HbSS phenotype, targeting of other pathogenetic mechanisms in SCD may offer additional benefit in both HbSS and HbSC patients. The RBC metabolism depends on anaerobic glycolysis for ATP production. Key RBC functions are intricately linked to metabolism: hemoglobin-oxygen (Hb-O2) affinity is modulated by 2,3-diphosphoglycerate (2,3-DPG), RBC membrane health depends on adequate synthesis of ATP, and heme-iron is maintained in the functional reduced state by the synthesis of antioxidants, such as GSH, supplying NADH to methemoglobin reductase. Erythrocyte pyruvate kinase (PKR) performs a rate limiting step in RBC glycolysis, converting phosphoenolpyruvate to pyruvate and generating ATP. Insufficient PKR activity due to any of a variety of causes, including excessive energy demand, can alter the abundance of metabolic products and intermediates, including ATP and 2,3-DPG, adversely affecting RBC function. We assessed baseline 2,3-DPG and ATP levels in whole blood of patients with HbSS (N=18) by LC/MS analysis. As well-reported, hemoglobin (Hb) levels were significantly decreased in patients with HbSS compared to healthy volunteers (9.68 ± 0.32 g/dL vs 14.88 ± 0.27 g/dL, p<0.0001). After normalizing to the decreased Hb levels, 2,3-DPG levels were significantly increased (4842.78 ± 182.98 vs 4327.73 ± 126.60 µg/g Hb, p<0.05) and ATP levels were significantly decreased (1621.27 ± 133.02 vs 1995.37 ± 60.80 µg/g Hb, p<0.05) in whole blood specimens of patients with HbSS compared to healthy volunteers. FT-4202 is a potent, selective, and orally bioavailable allosteric activator of PKR which is in Phase 1 clinical trial for the treatment of SCD (NCT03815695). We have previously shown that FT-4202 decreases 2,3-DPG within 24 hr of single dose and increases ATP after 14 days of multiple dosing in healthy volunteers (Blood, 2019, 134, Supplement_1:616). Here, we hypothesized that, by reducing 2,3-DPG and increasing ATP, FT-4202 may improve oxygen affinity and membrane health in both HbSS and HbSC RBCs irrespective of hydroxyurea use and HbF expression. Whole blood samples in K2EDTA were collected from donors with confirmed HbSS or HbSC disease in accordance with an approved IRB protocol in Cincinnati Children's Hospital. % HbF and % F-cells were measured for each sample. Packed RBCs were washed, resuspended to 20% v/v (20% hematocrit), and then incubated for 4 hr at 37oC with 20 μM FT-4202 or equivalent volume of vehicle control DMSO. P50 representing the partial pressure of oxygen at which Hb is 50% saturated with oxygen, was determined using non-linear regression analysis of hemoglobin-oxygen equilibrium curves obtained on a HEMOX Analyzer (TCS Scientific). RBC deformability as a function of oxygen pressure, or Oxygenscan, was measured using Lorrca® (RR Mechatronics) (Rab et al., 2019, Am J Hematol). Point of Sickling (PoS) was calculated as the pO2 at which RBC deformability is decreased by 5% of maximum value during deoxygenation. The P50 and PoS values in FT-4202 versus DMSO-treated HbSC or HbSS RBCs from patients treated with or without HU were analyzed by paired Wilcoxon test. FT-4202 treatment significantly decreased P50 in both HU-treated and not HU-treated HbSS RBCs, as well as in HbSC RBCs, as detailed in Table below showing mean ± se (*p<0.05, **p<0.002, ***p<0.0005 for FT-4202 versus vehicle). The decrease in P50 was significantly higher in not HU- versus HU-treated HbSS RBCs (###p<0.001 by two-way ANOVA) likely due to the increased HbF with baseline decreased P50 in HU-treated RBCs (23.6 ± 2.3 %HbF) versus non-HU treated RBCs (10 ± 1.9 %HbF). PoS was decreased after FT-4202 treatment both in HbSS and HbSC RBCs (p<0.05). These data suggest that increasing flux through the glycolytic pathway by FT-4202 treatment may offer dual beneficial effects of increased Hb-O2 affinity and membrane flexibility in HbSC as well as in HbSS RBCs irrespective of HU use. Figure Disclosures Fulzele: FORMA Therapeutics, Inc: Current Employment, Other: Shareholder of Forma Therapeutics. Guichard:FORMA Therapeutics, Inc: Current Employment, Other: Shareholder of Forma Therapeutics; AstraZeneca: Other: Shareholder. Kalfa:Agios Pharmaceuticals, Inc: Consultancy, Research Funding; Forma Therapeutics, Inc: Research Funding.

Hemoglobin–Oxygen Equilibrium Curves Measured during Enzymatic Oxygen Consumption

A rapid, new method to measure hemoglobin–oxygen equilibrium curves is described using the protocatechuic acid/protocatechuic acid 3,4-dioxygenase system [C. Bull and D. P. Ballou (1981)J. Biol. Chem.256, 12673–12680] to deoxygenate hemoglobin solutions enzymatically. The reaction is followed by simultaneous measurements of hemoglobin spectra using a diode array spectrophotometer and oxygen tensions using a polarographic O2microelectrode. Multicomponent analysis allows the determination of fractions of oxyhemoglobin, deoxyhemoglobin, and high-spin and low-spin methemoglobins in each spectrum collected as the reaction proceeds. Fractional saturation as a function of oxygen partial pressure is calculated as the ratio of oxyhemoglobin to oxy- plus deoxyhemoglobin. Several advantages are offered by this method: (i) Hemoglobin–O2binding curves are obtained rapidly and reproducibly; (ii) the speed of the reaction limits methemoglobin formation by autooxidation; (iii) there is no gas–liquid interface, eliminating protein denaturation at the surface; and (iv) direct calculations of fractional saturation are made using spectral analysis, thus avoiding the assumption of a linear transition between deoxy- and oxyhemoglobin.

26] Adair fitting to oxygen equilibrium curves of hemoglobin

This chapter focuses on the Adair fitting to oxygen equilibrium curves of hemoglobin (Hb). The extensive studies on the evaluation of the Adair constants indicate that the dominant factors that influence the reliability of the Adair constant evaluation reside in both the accuracy of the experimental data and the use of the least-squares method. The oxygen equilibrium curve (OEC), which is suitable for least-squares curve fitting, is composed of as many as 30 to 100 data points. To analyze a number of OECs it is useful to construct an on-line system that allows acquisition of the O2 equilibrium data in real time, that is, simultaneously with the measurement and analysis immediately after the measurement. The information contained in the OEC is extracted by fitting a particular function to it. The curve-fitting procedure, which usually employs the least-squares method, yields the best-fit values of the set of the parameters involved in the function. A variety of molecular properties of Hb is derived from the physical meanings of the parameters.

Fitting abnormal oxygen equilibrium curves of hemoglobin

A growing number of oxygen equilibrium curves for hemoglobin (Hb) mutants, post-translational modifications, or the binding of potent new effectors of Hb cannot be fitted adequately with the two-state model. Examples are curves showing double maxima in the derivative of the Hill plot, or slopes of less than unity. We present such examples of modified hemoglobins and strong effectors in this study and calculate at which substate level the two-state model differs from the data. Analysis of hemoglobin oxygen equilibrium curves is reconsidered using the two-state model extended to allow variation of the individual substate probabilities. In this way the effect on the equilibrium due to perturbations in energy of each substate can be studied as a diagnostic tool.

Comparative least-squares analysis of hemoglobin oxygen equilibrium curves

The oxygen-binding properties of hemoglobin have been studied at 600 microM protein concentration with organic phosphate, and analyzed by a series of different nonlinear least-squares analysis methods to determine whether reports of negligibly small values of the third overall Adair parameter, A3, are consequences of the data or a product of the data analysis. Data from other laboratories were analyzed as well. The single most important factor in creating a measurement that yields a small A3 is the use of equally weighted fitting in the Adair equation, while end-weighted fitting generally yields a larger A3. Endpoint extrapolation is ruled out as a major cause of abnormal A3 values. Monte Carlo simulations of the 600 microM results suggest that, if a small A3 were present, end weighting is at least as sensitive to a small A3 as equal weighting. We conclude that equally weighted fitting of the tetrameric Adair equation is unable to resolve the upper asymptote of the oxygen-binding data, resulting in an unusually small value for A3.

Analysis of hemoglobin oxygen equilibrium curves are unique solutions possible?

As a hemoglobin tetramer is oxygenated, it converts from a form with low ligand affinity to a high-affinity structure. This allosteric transition occurs in partially liganded molecules, typically after two or three ligands are bound. As a result of the co-operative nature of the process, the populations of the partially liganded forms are low. The relative proportions and precise properties of these intermediate substrates are therefore difficult to measure and are subject to controversy. The problem is compounded by compensating effects; for example, over-estimation of the oxygen affinity of triply liganded forms will result in under-estimation of the proportion of these species. Specifically, published values for the oxygen affinity of the triply liganded species vary for identical conditions by a factor of more than 6. In analyses based on the highest affinity values, the triply liganded species virtually disappears. However, this affinity is usually calculated from the last few per cent of the oxygenation curve, and this part of the curve is extremely sensitive to the normalization of the data. We conclude that unique solutions for the ligand affinity and substate populations may be impossible to achieve, and that unusual mechanisms based on particular combinations of parameters, therefore, should be viewed with caution.

Properties of carboxymethylated cross-linked hemoglobin A.

The selective carboxymethylation of the N-terminal amino groups of hemoglobin A with glyoxylic acid and sodium cyanoborohydride has been studied as a function of the state of ligation of hemoglobin. The N-terminal residues have been established as the primary sites of reaction by peptide mapping of the tryptic digest of each chain and subsequent amino acid analysis of the modified peptides. With oxyhemoglobin, the desired derivatives with a carboxymethyl group at the N-terminal of either or both chains amounted to 55% [Di Donato, A., Fantl, W. J., Acharya, A. S., & Manning, J. M. (1983) J. Biol. Chem. 258, 11890-11895]. In the present study it is shown that with deoxyhemoglobin the amount of the desired derivative is increased to 75%. The oxygen equilibrium curve of hemoglobin A carboxymethylated on its four N-terminal residues [0.5 mM as tetramer in 50 mM [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane (Bis-Tris), pH 7.5, 37 degrees C] had a P50 value of 30 mmHg (Hill coefficient n = 2.8, alkaline Bohr value = 0.4) compared to a P50 of 9 mmHg for unmodified hemoglobin under the same conditions (n = 2.5, alkaline Bohr value = 0.5). In carboxymethylated oxyhemoglobin A, cross-linked with the mild agent glycolaldehyde for 3.5 h, there was 85% of Mr 64,000 species and 15% of Mr 128,000 or higher species. For the former, the extent of cross-linking between two subunits was 19%. For the latter, there was 29% of two cross-linked subunits and 13% of three cross-linked subunits. Termination of cross-linking, which may be desirable in some circumstances, can be successfully achieved with isonicotinic acid hydrazide.(ABSTRACT TRUNCATED AT 250 WORDS)

Reverse temperature dependence of tuna hemoglobin oxygenation

At a pH around 7.5 with 0.05 M NaP i, the shape of the oxygen equilibrium curve of hemoglobin from the bluefin tuna ( Thunnus thynnus) is temperature dependent. The affinity at low saturation increases, and that at high saturation decreases on cooling from 20°C to 10°C. The equilibrium curves at the two temperatures therefore cross over. This behavior is physiologically advantageous to a warm-bodied fish. It may be explained in terms of the two-state model by supposing that the allosteric constant L increases markedly on cooling the solutions.

Hemoglobin Chesapeake (92α, Arginine → Leucine): PRECISE MEASUREMENTS AND ANALYSES OF OXYGEN EQUILIBRIUM

Abstract Precise oxygen equilibrium curves of hemoglobin (Hb) Chesapeake and human adult hemoglobin (Hb A) were determined by the automatic recording method in the presence and absence of NaCl, 2,3-diphosphoglycerate (DPG), and inositol hexaphosphate (IHP). The Adair constants, k1, k2, k3, and k4, and some oxygenation parameters were obtained from the equilibrium data. Maximum slope of the Hill plot for Hb Chesapeake, which was 1.22 under stripped conditions, was profoundly increased on the addition of 2 mm DPG and still more, beyond 2, on the addition of 2 mm IHP. It has been shown that DPG increases the cooperativity of oxygen binding to Hb Chesapeake by reducing k1, k2, and k3 without significant change of k4, as previously observed in Hb A. Within experimental errors, the k4 value for Hb Chesapeake was equal to that for Hb A whether DPG was present or absent. The ki value for Hb Chesapeake did not monotonously increase with i: k3 was significantly smaller than k2 whether DPG and IHP were present or absent. This resulted in slightly biphasic oxygen equilibrium curves of Hb Chesapeake. The biphasic oxygen binding was tentatively interpreted as a result of significantly cooperative interactions between the α1 and β1 (and α2 and β2) subunits and impaired subunit interactions at the α1β2 (and α2-β1) interface.

Thermodynamical analysis of oxygen equilibrium of stripped hemoglobin

Precise oxygen equilibrium curves of hemoglobin stripped of phosphates were determined at pH 7.4 and five different temperatures. The data were thermodynamically analyzed according to Adair's stepwise oxygenation theory and the allosteric model of Monod et al . Heat of oxygenation of Hb(O 2) 3 was significantly larger than that of Hb, indicating that the shape of oxygen equilibrium curve is not invariant with the change of temperature. The results do not support the idea that the cooperative effects are essentially entropic in nature, suggesting that the allosteric transition from the unliganded T-state to the unliganded R-state is an endothermic process, during which the hemoglobin molecule gains entropy.

Similation of biphasic oxygen equilibrium curves of hemoglobin in the presence of small amounts of 2,3-diphosphoglycerate

Oxygen equilibria of hemoglobin in the presence of different concentrations of 2,3-diphosphoglycerate were simulated by using Adair's stepwise oxygenation scheme in which changes of the concentration of free 2,3-diphosphoglycerate during oxygenation were taken into account. The calculated equilibrium curves fitted well to experimental points and also well expressed biphasic features in the presence of less than 1 mole of the phosphate per mole of hemoglobin.

Studies on the function of abnormal hemoglobins I. An improved method for automatic measurement of the oxygen equilibrium curve of hemoglobin

An apparatus with which the continuous oxygen equilibrium curve of hemo globin can be recorded automatically was constructed and its performance was examined. The oxygen pressure in the hemoglobin solution and the degree of saturation of the hemoglobin with oxygen are determined by means of a Clark's oxygen electrode and by spectrophotometry, respectively. The deoxygenation of oxyhemoglobin is attained by introducing nitrogen gas into the reaction cell after about 90 min and the reoxygenation of deoxyhemoglobin by introducing air. The deoxygenation curve coincided well with the successive oxygenation curve of the same sample. The curves measured using monochromatic lights of various wavelengths in the visible range coincided well with each other, in so far as the measurements were done under the same conditions. This method is applicable to hemoglobin solutions of concentrations of 0.01–2%. The curve is reproduced very well if the experiments are carried out under the same conditions and within several days. This reproducibility, however, becomes worse when the experiments are carried out over a longer period. The standard error of the fractional saturation for the curves which have been measured over the past one year depends on the position on the curve and is a maximum, about 2%, near the point of half saturation. This apparatus can furnish us with easy, speedy and accurate means to measure the oxygen equilibrium curve and can facilitate the observation of fine structures of the curve. This method is suitable for studies on the function of abnormal hemoglobins.