Thermodynamic studies of polymerization of deoxygenated sickle cell hemoglobin.

Magdoff-Fairchild, Beatrice; Poillon, William N.; Li, Ting-I.; Bertles, John F.

doi:10.1073/pnas.73.4.990

Cited by 84 publications

(32 citation statements)

References 24 publications

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“…over which c8 and delay times can both be measured on the same sample. The independence of c, from ct has also been reported by Magdoff-Fairchild et al (10). Within the experimental errors of our technique, the pellet concentration is also nearly constant at 48-55 g/dl at pH 7.05-7.1.…”

Section: Methodssupporting

confidence: 67%

Supersaturation in sickle cell hemoglobin solutions.

Hofrichter¹,

Ross²,

Eaton³

1976

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

197

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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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Section: Methodssupporting

confidence: 67%

Supersaturation in sickle cell hemoglobin solutions.

Hofrichter¹,

Ross²,

Eaton³

1976

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

197

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“…For hemoglobin S gelation, on the other hand, calorimetric and van't Hoff results are in agreement (20,21).…”

Section: Discussionsupporting

confidence: 73%

Calorimetric studies of the in vitro polymerization of brain tubulin.

Sutherland¹,

Sturtevant²

1976

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

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The enthalpy change for chain propagation in the polymerization of bovine tubulin has been studied directly by stopped-flow microcalorimetry at 170 and 250, and found to be 0 + 1 kcal per mol of 6S tubulin dimer at both temperatures.Substantial heat evolution with a half-time of decay of approximately 1 hr was observed when tubulin was injected into the calorimeter. This heat was shown to result from contamination of the tubulin by small amounts of some material from the crude brain homogenate from which the tubulin was prepared, and to be totally unconnected with microtubule assembly. Model In the following discussion, it should be remembered that thermodynamics strictly applies only to reversible processes between well defined states. Microtubule assembly is only quasi-reversible (3) and may involve nucleotide hydrolysis (4). Moreover, the intermediate species and even the polymers may vary with the biological system or from one preparation to another. We therefore attempted to keep our experimental procedure as nearly constant as possible. Of course, the same restrictions apply as well to van't Hoff measurements but for these measurements the interpretation is complicated by the existence of states intermediate between the polymerizing unit and the final polymer, as will be discussed below.Although the measurement discussed herein applies primarily to the chain lengthening process and not to the nucleation process, the amount of tubulin in the form of nuclei is likely to comprise at most only a small percentage of the total mass of tubulin in any microtubule preparation. Therefore, the overall enthalpy change involved in the formation of microtubules reflects primarily this enthalpy and not the enthalpy of nucleation. MATERIALS AND METHODSTubulin, twice polymerized and depolymerized, was prepared by the method of Shelanski et al. (5) The GTP was purchased from Sigma (Type II-S) and fl,ymethyleneguanosine triphosphate (GMPPCP) was from P-L Biochemicals. Protein concentrations were determined by a modified Lowry procedure (6), using a bovine serum albumin standard.Flow Calorimetry. Our most reliable thermochemical data were obtained by means of flow calorimetry. The instrument employed is a modification of the Beckman model 190 batch calorimeter, and was previously described (7). The flow tubing is platinum of 1 mm internal diameter, and a total volume of 1 ml in thermal contact with the measuring thermopile. The volume of delivery tubing from each syringe to the mixer is 0.58 ml; part of this tubing is Teflon in good thermal contact with the constant temperature water bath surrounding the calorimeter submarine, and part of it is platinum in good thermal contact with the massive aluminum heat sink of the calorimeter.Various experimental formats were tried with this instrument, of which the only successful one may be described as a 3565 Abbreviations: DSC, differential scanning calorimetry; GMPPCP, f3, 'y-methyleneguanosine triphosphate.

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“…The measurement, usually referred to as C SAT , is regarded as the criterion standard. 1,2 Relatively large amounts of hemoglobin are required, the final pH is low (approximately 6.8), and the measurement is made under fully deoxygenated conditions in the presence of dithionite. Intracellular components that either do or may affect polymerization are generally not present because they have been removed by dialysis.…”

Section: Introductionmentioning

confidence: 99%

Direct intracellular measurement of deoxygenated hemoglobin S solubility

Fabry

Desrosiers

Suzuka

2001

Blood

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The solubility of deoxygenated hemoglobin S (HbS), which is the concentration of fully deoxygenated HbS in equilibrium with polymer (C SAT ), is a factor that determines in vivo polymer formation. However, measurement of C SAT is usually performed under conditions that are far from physiological. In solution studies of HbS by Benesch et al, it was demonstrated that p50, the point at which hemoglobin is half-saturated with oxygen, is proportional to the amount of polymer formed and that it may be used to measure C SAT . This method has been extended to measure C SAT

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Thermodynamic studies of polymerization of deoxygenated sickle cell hemoglobin.

Cited by 84 publications

References 24 publications

Supersaturation in sickle cell hemoglobin solutions.

Supersaturation in sickle cell hemoglobin solutions.

Calorimetric studies of the in vitro polymerization of brain tubulin.

Direct intracellular measurement of deoxygenated hemoglobin S solubility

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