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- Sickle Hemoglobin Polymer Melting in High Concentration Phos...Sickle Hemoglobin Polymer Melting in High Concentration Phosphate Buffer
Biophysical Journal, Volume 76, Issue 4, 1 April 1999, Pages 2216-2222
Joseph G. Louderback, Samir K. Ballas and Daniel B. Kim-Shapiro
AbstractSickle cell hemoglobin (HbS) prepared in argon-saturated 1.8M phosphate buffer was rapidly mixed with carbon monoxide (CO)-saturated buffer. The binding of CO to the sickle hemoglobin and the simultaneous melting of the hemoglobin polymers were monitored by transmission spectroscopy (optical absorption and turbidity). Changes in the absorption profile were interpreted as resulting from CO binding to deoxy-HbS while reduced scattering (turbidity) was attributed to melting (depolymerization) of the HbS polymer phase. Analysis of the data provides insight into the mechanism and kinetics of sickle hemoglobin polymer melting. Conversion of normal deoxygenated, adult hemoglobin (HbA) in high concentration phosphate buffer to the HbA-CO adduct was characterized by an average rate of 83s. Under the same conditions, conversion of deoxy-HbS in the polymer phase to the HbS-CO adduct in the solution phase is characterized by an average rate of 5.8s via an intermediate species that grows in with a 36s rate. Spectral analysis of the intermediate species suggests that a significant amount of CO may bind to the polymer phase before the polymer melts.
Abstract | Full Text | PDF (124 kb) - Metastable Mesoscopic Clusters in Solutions of Sickle-Cell H...Metastable Mesoscopic Clusters in Solutions of Sickle-Cell Hemoglobin
Biophysical Journal, Volume 92, Issue 1, 1 January 2007, Pages 267-277
Weichun Pan, Oleg Galkin, Luis Filobelo, Ronald L. Nagel and Peter G. Vekilov
AbstractSickle cell hemoglobin (HbS) is a mutant, whose polymerization while in deoxy state in the venous circulation underlies the debilitating sickle cell anemia. It has been suggested that the nucleation of the HbS polymers occurs within clusters of dense liquid, existing in HbS solutions. We use dynamic light scattering with solutions of deoxy-HbS, and, for comparison, of oxy-HbS and oxy-normal adult hemoglobin, HbA. We show that solutions of all three Hb variants contain clusters of dense liquid, several hundred nanometers in size, which are metastable with respect to the Hb solutions. The clusters form within a few seconds after solution preparation and their sizes and numbers remain relatively steady for up to 3h. The lower bound of the cluster lifetime is 15ms. The clusters exist in broad temperature and Hb concentration ranges, and occupy 10–10 of the solution volume. The results on the cluster properties can serve as test data for a potential future microscopic theory of cluster stability and kinetics. More importantly, if the clusters are a part of the nucleation mechanism of HbS polymers, the rate of HbS polymerization can be controlled by varying the cluster properties.
Abstract | Full Text | PDF (800 kb) - Liquid-Liquid Phase Separation in Hemoglobins: Distinct Aggr...Liquid-Liquid Phase Separation in Hemoglobins: Distinct Aggregation Mechanisms of the β6 Mutants
Biophysical Journal, Volume 86, Issue 3, 1 March 2004, Pages 1702-1712
Qiuying Chen, Peter G. Vekilov, Ronald L. Nagel and Rhoda Elison Hirsch
AbstractReversible liquid-liquid (L-L) phase separation in the form of high concentration hemoglobin (Hb) solution droplets is favored in an equilibrium with a low-concentration Hb solution when induced by inositol-hexaphosphate in the presence of polyethylene glycol 4000 at pH 6.35 HEPES (50mM). The L-L phase separation of Hb serves as a model to elucidate intermolecular interactions that may give rise to accelerated nucleation kinetics of liganded HbC (6 Lys) compared to HbS (6 Val) and HbA (6 Glu). Under conditions of low pH (pH 6.35) in the presence of inositol-hexaphosphate, COHb assumes an altered R-state. The phase lines for the three Hb variants in concentration and temperature coordinates indicate that liganded HbC exhibits a stronger net intermolecular attraction with a longer range than liganded HbS and HbA. Over time, L-L phase separation gives rise to amorphous aggregation and subsequent formation of crystals of different kinetics and habits, unique to the individual Hb. The composite of R- and T-like solution aggregation behavior indicates that this is a conformationally driven event. These results indicate that specific contact sites, thermodynamics, and kinetics all play a role in L-L phase separation and differ for the 6 mutant hemoglobins compared to HbA. In addition, the dense liquid droplet interface or aggregate interface noticeably participates in crystal nucleation.
Abstract | Full Text | PDF (365 kb) - …more
Copyright © 1996 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 70, Issue 5, 2442-2447, 1 May 1996
doi:10.1016/S0006-3495(96)79815-6
Research Article
Solubility of sickle hemoglobin measured by a kinetic micromethod
D. Liao, J.J. Martin de Llano, J.P. Himanen, J.M. Manning and F.A. Ferrone
Abstract
We have developed a photolytic method to determine the concentration of reactive hemes in a solution in the presence of a trace amount of CO. By measurement of the bimolecular rate of CO binding, and by calibration of the rate constant under equivalent conditions, the concentration of the reactive hemes can be determined. In a solution of sickle hemoglobin, the molecules in the gel contribute negligibly to the recombination rate, allowing the concentration of the molecules in the solution phase to be determined. To optimize signal to noise, modulated excitation methods were employed, although the method could also be used with pulse techniques and suitable signal averaging. Because the optical method employs a microspectrophotometer, only a few microliters of concentrated Hb solution is required to reproduce the entire temperature dependence of the solubility previously determined by centrifugation using milliliter quantities of solutions of the same concentration. This should be especially useful for studies of site-directed mutants, and we present results obtained on one such HbS in which Leu 88 beta has been replaced by Ala. The free energy difference in the polymerization of the Leu 88 beta double mutant is consistent with known differences in the amino acid hydrophobicities. The calibration required for these experiments also provides an excellent determination of the activation energy for binding the first CO to deoxy Hb.
