Isoelectric focusing with simple buffers: Studies of liver alcohol dehydrogenase and its complexes with ligands.
Item
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Title
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Isoelectric focusing with simple buffers: Studies of liver alcohol dehydrogenase and its complexes with ligands.
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Identifier
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AAI9707101
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identifier
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9707101
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Creator
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Hausfeld, A. David.
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Contributor
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Adviser: J. B. A. Ross
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Date
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1996
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry | Biophysics, General
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Abstract
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A new method for establishing a highly stable pH gradient for isoelectric focusing (IEF) is presented. The pH gradients cover intervals of the order of 0.5 to 1 pH unit; each pH gradient is established with a pair of simple buffering species. Selection of varied buffering pairs allows gradients to cover the pH range 3 to 10. The pH gradients are formed in granulated gel beds supported by a cation-selective membrane over a buffer filled channel. The new IEF method was applied to measure isoelectric points (pI) of alcohol dehydrogenase from horse liver (LADH) and a complex with coenzyme and substrate. The experimental pI values were compared to pI values calculated using the Finite Difference Poisson-Boltzmann (FDPB) equation and structures known from x-ray crystallography as recorded in the Protein Data Bank (Brookhaven). This comparison has afforded an evaluation of theoretical results calculated with dielectric constants fixed alternately at 4 and 20 in the protein interior. It is concluded that an improved theoretical model should allow the dielectric constant to vary in the protein interior. The new IEF method was also employed to search for multiple isoelectric points for LADH or its complexes as evidence of a pH-dependent conformational change; this conformational change has been hypothesized to explain the decrease in coenzyme association rates with increasing pH. Two isoelectric points have been found for the LADH/salicylate complex.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
