Supersaturation in sickle cell hemoglobin solutions.

Abstract

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...

The role of β93 Cys in the inhibition of Hb S fiber formation

“…Solubility is described as the concentration of deoxy Hb S present in the supernatant after fibers are allowed to form and sedimented by centrifugation. The ratio of initial concentration (C 0 ) to the soluble fraction (C s ) is known as the supersaturation ratio and can be expressed as: S ≡ C 0 /C s [54]. The solubility and the supersaturation ratio are a measure of the relative propensity of Hb molecules to form fibers.…”

“…Hb S 1.44 ± 0.14 1.11 ± 0.13 0.14 a S is the supersaturation ratio; S =(γ 0 C 0 )/(γ s C s ) [54]; γ values taken from Ross and Minton [44]. b i ⁎ is the size of the homogeneous nucleus; i ⁎ = −(4RT + δ 1 μ PC ) /RT ln S where δ 1 describes the fraction of intermolecular bonds in the nucleus relative to the infinite polymer, μ PC is the chemical potential contribution to the infinite polymer from the bonding free energy between molecules.…”

Universal Metastability of Sickle Hemoglobin Polymerization

“…A well known way to study such behavior is by sedimentation equilibrium 2; 3; 4 , in which the supernatant concentration reaches a saturating value of the solubility. Careful experiments showed that this concentration is independent of initial concentration, centrifugal force and duration (above a minimum concentration, force and time) 5 . This argued strongly in favor of this final concentration being a thermodynamic parameter, since it depended only on the conditions of the final state, not the path by which the final state was achieved.…”

A transcriptional signature of Alzheimer’s disease is associated with a metastable subproteome at risk for aggregation

“…To understand why some proteins aggregate whereas others remain soluble, we recently observed that many proteins in the proteome are insufficiently soluble relative to their expression levels (24). Such proteins are metastable to aggregation as their concentrations exceed their solubilities, that is, they are supersaturated (24)(25)(26)(27). Upon formation of aggregate seeds by nucleation events, a supersaturated protein will form insoluble deposits until the concentration of its soluble fraction is reduced to match its solubility (24)(25)(26)(27)(28).…”