Hemoglobin Interaction: Modification of Solid Phase Composition in the Sickling Phenomenon

Bertles, John F.; Rabinowitz, Rosanne; Döbler, Johanna

doi:10.1126/science.169.3943.375

Cited by 127 publications

(36 citation statements)

References 18 publications

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“…Hybrid formation can be prevented by mixing the hemoglobins in the absence of oxygen. However, experiments to determine the extent of copolymerization have led to conflicting results, especially for Hb F. Measurement of the concentration of Hb F in the pellet after centrifugation of the gel led Bertles et al (15) to the conclusion that Hb F does not copolymerize with Hb S, in agreement with Behe and Englander (16), who analyzed the supernate. Goldberg et al (17), on the other hand, found evidence of copolymerization with Hb F under both hybridizing and nonhybridizing conditions.…”

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confidence: 80%

Solubilization of hemoglobin S by other hemoglobins.

Edalji¹,

Benesch²,

Kwong³

1980

Proc. Natl. Acad. Sci. U.S.A.

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The polymerization of mixtures of Hb S with hemoglobins A, A2, and F has been investigated by analysis of the proportions of S and non-S hemoglobin both in the supernate and-in the pellet after centrifugation. In all cases the non-S hemoglobin was incorporated into the polymer even in the absence of hybrids in the order A > A2 > F. The solubility of Hb S is substantially increased by the other hemoglobins, especially by Hb F. which would account for its antisickling effect. It appears that the excluded volume effect of the other hemoglobin on Hb S is largely counterbalanced by the solubilizing effect arising from the interaction between the two hemoglobins in solution. The ability of hybrid hemoglobins to gel was demonstrated directly with tetramers in which afts dimers were covalently linked to aflA, a5A2, and ayF dimers.The so-called "sickle-sparing" effect of other hemoglobins on hemoglobin S (Hb S) was described more than 20 years ago by Singer and Singer (1) and by Allison (2), who found that some hemoglobins such as Hb A and Hb C greatly decrease the concentration of Hb S necessary for gelling, whereas Hb F had a much smaller effect. It was assumed by these authors and in much of the subsequent work with other hemoglobins (3-6) that the "sickle-sparing" hemoglobins act by substituting for Hb S in the polymer, i.e., by copolymerizing with it. By contrast, it was concluded that Hb F and, more recently, Hb A2 (7-9) do not copolymerize with Hb S.However, it has now been realized that another factor plays a major role in the gelling of Hb S in the presence of other hemoglobins (10-12). In these very concentrated and nonideal solutions the presence of any other hemoglobin [and, indeed, any The purpose of the work reported here was therefore to investigate the participation of Hb F and Hb A2 in the polymerization of Hb S by direct analysis, not only of the supernate, but also of the gel. In addition, hemoglobins in which an af3s dimer was covalently crosslinked to either an acy or an a6 dimer were used to test the ability of these mixed tetramers to polymerize. MATERIALS AND METHODSThe hemoglobins were isolated from hemolysates, prepared as described (19) from normal blood, sickle trait blood, and cord blood. After dialysis against 0.1 M glycine at pH 7.6, the hemolysates were fractionated on DEAE-cellulose, Whatman DE52 (20), under the conditions shown in Table 1. Sectional columns (Kontes Glass) were used and the desired hemoglobin was removed from the appropriate segment without elution of the column (21).Crosslinked (XL) hemoglobins were prepared by using the reagent 2-nor-2-formylpyridoxal 5'-phosphate as described (22,23). For the preparation of crosslinked mixed tetramers, the reaction was performed on equimolar amounts of Hb S and the other hemoglobin that had been mixed under oxygen to allow for hybridization. After the coupling reaction, which takes place under anaerobic conditions, the hemoglobins were converted to the cyanmet form by oxidation with 1.2 equivalents of potassium ferricyanide...

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confidence: 80%

Solubilization of hemoglobin S by other hemoglobins.

Edalji¹,

Benesch²,

Kwong³

1980

Proc. Natl. Acad. Sci. U.S.A.

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“…Once gelation was complete, as judged from the turbidity progress curve, the equilibrium properties of the resulting gel were characterized using a centrifugation technique similar to the one initially described by Bertles et al (9). After the polymers had been sedimented, the concentration of hemoglobin S in the supernatant (c8), the concentration of hemoglobin S in the pellet (cp), and the volume fraction of the pellet (vp) were measured.…”

Section: Methodsmentioning

confidence: 99%

Supersaturation in sickle cell hemoglobin solutions.

Hofrichter¹,

Ross²,

Eaton³

1976

Proc. Natl. Acad. Sci. U.S.A.

198

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The kinetic inhibition of the gelation of hemoglobin S is compared to the change in hemoglobin S solubility, when the so-ubility is altered by carbn monoxide, pH, or urea. By means of a new technique, the delay time and the extent of gelation are measured on the same sample. The delay time, td, is found to be proportional to a hig power (30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) of the hemoglobin S solubility. Together with the previously reported concentration dependence, this result demonstrates that the rate is proportional to a high power of the supersaturation, S, defined as the ratio of the total hemoglobin S concentration to the equilibrium solubility. The results obey the supersaturation equation td-' = ySFI, where 'y is an empirical constant (about 10-7 sec') and n is about 35. The supersaturation equation can successfully account for observations on the kinetics of cell sickling and is therefore used to estimate the increase in the delay time for sickling necessary to produce significant clinical benefit to patients with sickle cell disease. In previous papers we reported initial results on the kinetic and thermodynamic behavior of deoxyhemoglobin S gelation (1,2). When gelation was induced by temperature jumps, there was a delay period during which no observable polymerization took place (1, 3, 4). The delay time (td) was found to depend inversely on an extraordinarily high power (about 30) of the deoxyhemoglobin S concentration (ct) (1), and to be extremely sensitive to changes in temperature (1, 3). We also found that over a limited temperature range the delay time was proportional to a very high power of the equilibrium solubility (1, 2), where the solubility (c,) was taken as the concentration of deoxyhemoglobin S in the supernatant after sedimentation of the polymers by high-speed ultracentrifugation. The concentration and temperature dependence of the delay time could then be summarized to first order by a single empirical equation: 1/td = TS; S -Ce/c. [l] where y is an experimental constant. By analogy to crystallization or condensation processes, the ratio of the total initial concentration, ct, to the solubility, c5, was called the supersaturation ratio, S. The form of this "supersaturation equation" could be justified theoretically on the basis of our proposed nucleation-controlled polymerization mechanism (1).Using the supersaturation equation, we carried out a series of calculations on the influence of physiological variables on the delay time of intracellular gelation in vvo. The results of these calculations led to the kinetic hypothesis that the rate of intracellular gelation relative to the capillary transit time of the red cell is a major determinant of clinical severity in sickle cell disease (1,5). A crucial assumption in these calculations was that the supersaturation equation would hold approximately, no matter how the solubility was changed. Experimental data, however, on the influence of physiological variables, such as oxygen concentration and pH, on the delay t...

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“…The maximum value of the square of the multiple correlation coefficient, which measures the goodness-of-fit of the analysis, was found to be <3%. Thus, no laboratory parameter was found FIGURE 1 The frequency distribution of the percentage of FIGURE 2 The frequency distribution of MCV what lower than our mean percent Hb-F of 12.3±7.0, the methodologies for Hb-F determination were different. In general, alkaline denaturation methods as used in Jamaica and Turkey give lower readings particularly at higher levels (above 20%) of Hb-F so that these apparent differences may not be significant.…”

Section: Introductionmentioning

confidence: 99%