Journal article
Solubility of sickle hemoglobin measured by a kinetic micromethod
Biophysical journal, v 70(5), pp 2442-2447
May 1996
PMID: 9172771
Featured in Collection : UN Sustainable Development Goals @ Drexel
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useful for studies of site-directed mutants, and we present results obtained on one such HbS in which Leu 88 beta has been replaced by Ala.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.
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Details
- Title
- Solubility of sickle hemoglobin measured by a kinetic micromethod
- Creators
- D Liao - Department of Physics and Atmospheric Science, Drexel University, Philadelphia, Pennsylvania 19104, USAJ J Martin de Llano - Department of Physics and Atmospheric Science, Drexel University, Philadelphia, Pennsylvania 19104, USAJ P Himanen - Department of Physics and Atmospheric Science, Drexel University, Philadelphia, Pennsylvania 19104, USAJ M Manning - Department of Physics and Atmospheric Science, Drexel University, Philadelphia, Pennsylvania 19104, USAF A Ferrone - Department of Physics and Atmospheric Science, Drexel University, Philadelphia, Pennsylvania 19104, USA
- Publication Details
- Biophysical journal, v 70(5), pp 2442-2447
- Publisher
- Elsevier
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Physics
- Web of Science ID
- WOS:A1996UH72500043
- Scopus ID
- 2-s2.0-0029864048
- Other Identifier
- 991014877956804721
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- Collaboration types
- Domestic collaboration
- Web of Science research areas
- Biophysics