The Effect of Temperature on the Oxygen-linked Ionizations of Hemoglobin

Abstract On the assumption that only two of the acid groups per heme are affected by oxygenation, the difference in electrical charge, ΔX, between oxygenated and deoxygenated hemoglobin (O2Hb and Hb) at the same pH (as determined from the difference in their titration curves) is given by the equation. [see PDF for equation] where h = hydrogen ion concentration and Ko, K'o, Kr, and K'r are the ionization constants of these "Bohr groups" in O2Hb and Hb, respectively. The above equation has been found to fit (within the limits of experimental error) the data on the "difference" titration curves for bovine, human, and horse O2Hb and Hb over the pH range 5.0 to 9.3 at 25–37°; "best" (i.e. least mean square) values of the ionization constants have been estimated statistically by means of an automatic computer program, written in Fortran language. Thus, for human hemoglobin at 25°, pKr = 7.84 (±0.006), pKo = 6.84 (±0.009), pK'r = 5.13 (±0.04), and pK'o = 5.60 (±0.03). These figures differ by up to 0.7 pK unit from those obtained by other recent workers, who did not, however, analyze their data statistically. Values for the corresponding heats of ionization, Qo, Q'o, Qr and Q'r, have been calculated from the effect of temperature on the respective ionization constants. The most accurate results have been obtained in the case of human hemoglobin, wherein it is found that (Qr - Qo) is 5,000 (±800) cal. The actual value of Qr is of the order of 11,000 cal and thus lies outside the usual range for the heat of ionization of imidazole and its derivatives. These conclusions are based upon the experimental data not only of the present paper but also of the recent paper (11) of Antonini, Wyman, Brunori, Fronticelli, Bucci, and Rossi-Fanelli, the concordance between these two independent sets of results, when statistically analyzed, being very satisfactory. (Q'o - Q'r) has also been found to be of the order of 5000 cal, but this figure is subject to a wide margin of uncertainty, owing to the difficulty in obtaining accurate estimates of pK'o and pK'r from data in the acid pH range, wherein hemoglobin is unstable. Preliminary experiments have shown that at pH below 6.2 "rapid" titration figures (i.e. within 10 msec) differ significantly from the results obtained by the customary slow titration procedure, which takes several minutes. The results of this paper are discussed in light of current views on the chemical and x-ray structure of O2Hb and Hb

The binding of CO2 to human hemoglobin.

CO2-dissociation curves of concentrated human deoxy- and carbonmonoxyhemoglobin at 37 degrees, pH 7.6 to 7.0, PCO2 equal to 10 to 160 mm Hg, have been obtained by a rapid mixing and ion exchange technique. The CO2-dissociation curves for deoxyhemogloblin can only be fitted by assuming two classes of binding sites for carbon dioxide. The simplest way to account for the experimental data is to assume that the alpha-amino groups of the alpha and beta chains react with carbon dioxide with affinities that differ by at least a factor of 3. No difference in reactivity with CO2 was found among the four terminal alpha-amino groups of carbonmonoxyhemoglobin

Direct Calorimetric Studies on the Heats of Ionization of Oxygenated and Deoxygenated Hemoglobin

Abstract The total heats of ionization, Qo and Qr, of bovine, human, and horse oxygenated and deoxygenated hemoglobin (O2Hb and Hb) have been directly measured by the rapid calorimetric method over the pH range from 5.7 to 9.0, at 12–28°. The most extensive determinations have been those on bovine hemoglobin: above about pH 6.6 the thermal titration curve for Hb lies systematically above that for O2Hb by about 600 cal, this difference presisting practically unchanged up to the most alkaline pH (8.7) studied. The two thermal titration curves cross at approximately pH 6.3, below which the O2Hb curve lies above the Hb curve by an increasing amount (up to 1,000 cal). The fact that Qr remains greater than Qo at pH 8.7, at which the absolute value of Qr is about 11,000 cal, implies that the heme-linked group, which ionizes in this pH range in the case of Hb, must have a heat of ionization, Qr, of around 11,000 cal. This figure, which was confirmed by an approximate method of calculation, lies outside the range usually attributed to the heat of ionization of imidazole or its derivatives. There is some indication, from a comparison of the difference between the two thermal titration curves for human Hb and O2Hb at approximately pH 7.3, that (Qr - Qo) is of the order of 4,000 cal, Qo being the heat of ionization of the corresponding heme-linked group in O2Hb. The results thus support the conclusions reached in the adjoining paper by Rossi-Bernardi and Roughton on the effect of temperature on the oxygen-linked ionizations of hemoglobin. The relation of the present studies to the cognate effects of pH on the heat of oxygenation of hemoglobin is briefly indicated

The intermediate compounds between human hemoglobin and carbon monoxide at equilibrium and during approach to equilibrium.

The procedure of Perrella et al. (Perrella, M., Benazzi, L., Cremonesi, L., Vesely, S., Viggiano, G., and Rossi-Bernardi, L. (1983) J. Biol. Chem. 258, 4511-4517) for trapping the intermediate compounds between human hemoglobin and carbon monoxide was validated by quantitatively determining during the approach to equilibrium all the species present in a solution containing large amounts of intermediates. An accurate estimate of the intermediate compounds at 50% carbon monoxide saturation in 0.1 M KCl, pH 7, at 22 degrees C, allowed the calculation, according to Adair's scheme, of the four equilibrium constants. At 50% ligand saturation, the pool of intermediate species was about 12% of the total. A slightly greater concentration of tri-liganded than mono-liganded species was found. Carbon monoxide bound to beta chains in slightly greater excess with respect to alpha chains in both the mono- and tri-liganded species. The symmetrical bi-liganded intermediates, alpha 2 beta CO2 and alpha 2CO beta 2, were absent. The nature of the bi-liganded intermediate found to be present in detectable amounts by our technique has yet to be clarified: it could be either the asymmetrical species (alpha beta) (alpha CO beta CO) and (alpha beta CO) (alpha CO beta) or both of them. Such a finding on the functional heterogeneity among the four possible bi-liganded intermediates is consistent with hypotheses of the existence of more than two quaternary structures in the course of ligand binding to hemoglobin

Isolation of intermediate valence hybrids between ferrous and methemoglobin at subzero temperatures.

Quenching a hemoglobin solution partially saturated with carbon monoxide into a hydro-organic solvent containing ferricyanide will produce under suitable conditions a population of partially oxidized and CO-bound hemoglobin molecules. Since each Fe3+ heme carries one extra charge, it should be possible, in theory, to resolve the spectrum of intermediate compounds between hemoglobin and carbon monoxide, which was originally present in solution. In this study we report: 1) the development of a simple and rapid method to quench aqueous hemoglobin solutions into a hydro-organic solvent at subzero temperatures; 2) the determination of suitable experimental conditions to isolate valence hybrids between carbonmonoxy- and methemoglobin by isoelectric focusing at temperatures as low as -25 degrees C; and 3) the identification and isolation of all valence hybrids of different charge between carbonmonoxy- and methemoglobin

Role of the α-Amino Groups of the α and β Chains of Human Hemoglobin in Oxygen-linked Binding of Carbon Dioxide

Abstract Human hemoglobin has been reacted with potassium cyanate and purified to yield three species, α2cβ2c,α2cβ2, and α2β2c, where superscript c denotes specific reaction of cyanate with the α-amino group of the particular chain. The effect of carbon dioxide on the oxygen affinity of these species in the presence and in the absence of 2,3-diphosphoglycerate has been measured. Carbon dioxide has no effect on the oxygen affinity of α2cβ2c, confirming that the usual lowering of the oxygen affinity of carbon dioxide in normal hemoglobin is mediated by the α-amino groups of the α and β chains. The lowering of the oxygen affinity of α2β2c by carbon dioxide was not affected by the presence or absence of 2,3-diphosphoglycerate, showing that 2,3-diphosphoglycerate does not interfere with the oxygen-linked binding of carbon dioxide at the α chain α-amino group. In α2cβ2 there was a much larger effect of carbon dioxide on the oxygen affinity in the absence of 2,3-diphosphoglycerate than in α2β2c; however, on addition of 2,3-diphosphoglycerate the effect of carbon dioxide on the oxygen affinity of α2cβ2 was much smaller and similar to that occurring in α2β2c. This shows that there is a large difference in the carbon dioxide binding constants of the β chain α-amino group in the oxy and deoxy forms of human hemoglobin, and that 2,3-diphosphoglycerate suppresses this difference, probably by binding strongly to the β chain α-amino group of deoxyhemoglobin and displacing any bound carbon dioxide

Isolation of intermediate compounds between hemoglobin and carbon monoxide.

A human hemoglobin solution partially saturated with carbon monoxide was rapidly quenched at -25 degrees C into a hydro-organic buffer containing ferricyanide. Under the experimental conditions of pH, ionic strength, and buffer composition used in this work, it was found that the deoxy hemes were rapidly transformed into their met form, whereas practically no carbon monoxide-bound hemes were oxidized before the separation of the mixture from the oxidizing agent. As a preliminary step to the analysis of the resulting solution, carbonylhemoglobin solutions partially oxidized with ferricyanide were studied by isoelectric focusing at -25 degrees C under identical conditions. The relative position in the gel of all nine possible valence hybrids was established as follows (going from the anodic to the cathodic side of the gel) alpha CO2 beta CO2, (alpha CO beta +)(alpha CO beta CO) (alpha CO beta CO), (alpha CO2 beta +2), (alpha + beta CO), (alpha + beta +)-(alpha CO beta CO), (alpha + beta +)(alpha CO beta +), (alpha +2 beta CO2), (alpha + beta +)(alpha + beta CO), alpha +2 beta +2. When carbonylhemoglobin and methemoglobin were mixed in equal proportion at -25 degrees C and then analyzed by isoelectric focusing at the same temperature, it was found that the contribution of valence hybrids other than alpha CO2 beta CO2 and alpha +2 beta +2 to the total amount of hemoglobin in the gel was no more than 6%. When carbonylhemoglobin and deoxyhemoglobin were mixed in the same proportion and incubated at 20 degrees C so to allow the redistribution of the carbon monoxide molecules between all possible binding sites to occur, a substantially higher amount of valence hybrids, derived from the oxidation of intermediate compounds of hemoglobin with carbon monoxide, was found. The isoelectric focusing separation indicated the presence in the original solution of intermediate species other than carbonylhemoglobin and deoxyhemoglobin at a concentration of about 10% of the total

The dissociation of carbon monoxide from hemoglobin intermediate.

To investigate the mechanism of allosteric switching in human hemoglobin, we have studied the dissociation of the ligand (CO) from several intermediate ligation states by a stopped-flow kinetic technique that utilizes competitive binding of CO by microperoxidase. The hemoglobin species investigated include Hb(CO)4, the diliganded symmetrical species (alpha beta-CO)2 and (alpha-CO beta)2, and the di- and monoliganded asymmetrical species (alpha-CO beta-CO)(alpha beta), (alpha-CO beta)(alpha beta-CO), (alpha beta-CO) (alpha beta), and (alpha-CO beta)(alpha beta). They were obtained by rapid reduction with dithionite of the corresponding valence intermediates that in turn were obtained by chromatography or by hybridization. The nature and concentration of the intermediates were determined by isoelectric focusing at −25 degrees C. The study was performed at varying hemoglobin concentrations (0.1, 0.02, and 0.001 mM [heme]), pH (6.0, 7.0, 8.0), with and without inositol hexaphosphate. The results indicate that: (a) hemoglobin concentration in the 0.1-0.02 mM range does not significantly affect the kinetic rates; (b) the alpha chains dissociate CO faster than the beta chains; (c) the symmetrical diliganded intermediates show cooperativity with respect to ligand dissociation that disappears in the presence of inositol hexaphosphate; (d) the monoliganded intermediates dissociate CO faster than the diliganded intermediates; (e) the asymmetrical diliganded intermediates are functionally different from the symmetrical species

Innovative observing strategy and orbit determination for Low Earth Orbit Space Debris

We present the results of a large scale simulation, reproducing the behavior of a data center for the build-up and maintenance of a complete catalog of space debris in the upper part of the low Earth orbits region (LEO). The purpose is to determine the performances of a network of advanced optical sensors, through the use of the newest orbit determination algorithms developed by the Department of Mathematics of Pisa (DM). Such a network has been proposed to ESA in the Space Situational Awareness (SSA) framework by Carlo Gavazzi Space SpA (CGS), Istituto Nazionale di Astrofisica (INAF), DM, and Istituto di Scienza e Tecnologie dell'Informazione (ISTI-CNR). The conclusion is that it is possible to use a network of optical sensors to build up a catalog containing more than 98% of the objects with perigee height between 1100 and 2000 km, which would be observable by a reference radar system selected as comparison. It is also possible to maintain such a catalog within the accuracy requirements motivated by collision avoidance, and to detect catastrophic fragmentation events. However, such results depend upon specific assumptions on the sensor and on the software technologies

Hybrid formation for liganded hemoglobins A and C at subzero temperatures

The kinetics of formation of the asymmetric carbon-monoxyhemoglobin hybrid (\u3b1\u3b2)(A)(\u3b1\u3b2)(C) from the parent molecules \u3b12\u3b22(A) and \u3b12\u3b22(C) have been studied by electrophoresis at subzero temperatures (down to -40\ub0C) using as supporting media gels of acrylamide/methylacrylate in dimethyl sulfoxide/water mixtures. It has been found that in these media the rate of hybrid formation is markedly affected by pH and decreases by an order of magnitude between pH 7.3 and 8.3. At pH>10, t=-40\ub0C, the hybrid between \u3b12\u3b22(A) and \u3b12\u3b22(C) is stable for several hours. A rapid thermal quenching of a mixture of \u3b12\u3b22(A) and \u3b12\u3b22(C) prevented hybrid formation during the time required to separate the 2 molecules