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Dissertation
Kinetic micromethod for measuring the solubility of native and mutant sickle hemoglobin
Doctor of Philosophy (Ph.D.), Drexel University
1997
DOI:
https://doi.org/10.17918/00007731
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 hemoglobin 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 the double mutant HbS [beta]L88A, HbS [beta]T87L, HbS [beta]K95I. The free energy difference in the polymerization of the double mutant HbS [beta]L88A 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 hemoglobin. The solubilities of other HbS double mutant HbS [beta]T87L, HbS [beta]K95I were also measured. The solubility of HbS [beta]K95I was found much higher than that of native HbS. Nonlinear effect for thick samples like HbS [beta]K95I gave a reasonable explanation of extraordinary results on calculated initial concentration. The solubility of HbS [beta]T87L was found close to that of native HbS.
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Details
- Title
- Kinetic micromethod for measuring the solubility of native and mutant sickle hemoglobin
- Creators
- Dan Liao
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xii, 146 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- College of Arts and Sciences; Drexel University
- Other Identifier
- 991021889094004721