Electrospray mass spectrometry : an investigation of non- covalent interactions of cytochrome c/crown ether complexes and applied methods of computational chemistry

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dc.contributor.advisor Kruger, Terry L. en_US
dc.contributor.author Sproch, Norman K. en_US
dc.date.accessioned 2011-06-03T19:36:38Z
dc.date.available 2011-06-03T19:36:38Z
dc.date.created 1994 en_US
dc.date.issued 1994
dc.identifier LD2489.Z78 1994 .S68 en_US
dc.identifier.uri http://cardinalscholar.bsu.edu/handle/handle/184918
dc.description.abstract This research is directed at developing the interplay of experimental and computational methods in the area of biochemical mass spectrometry. The experimental method is that of electrospray ionization mass spectrometry (ESI-MS). The computational methods employed are those of semi-empirical quantum mechanics and molecular modeling.The use of Electrospray Mass Spectrometry was developed to investigate whole proteins and the non-covalent complexes that may be formed with small molecules. This method provides the soft ionization needed to accurately determine a noncovalently bound complex's mass with an error of less than 0.1 %. An original design electrospray ionization source (ESI) and a syringe pump have been built to fulfill the goals of the research. The ESI source design has been published in The Journal of the American Society for Mass Spectrometry, (1993, 4, 964-967).In this work the protein selected was cytochrome c and its variants from different species. The small molecules chosen were a broad class of structures known as crown ethers. With the ESI technique the proteins are prepared in an acidic solution that fully protonates the solvent-exposed basic amino acid residues. This provides the protein with many positive charges which makes the analysis by ESI mass spectrometry possible with a single quadrupole instrument, an Extrel ELQ 400. The mass of the protein is divided by the number of positive charges. The small molecules, the crown ethers, were chosen due to their ability to bind ammonium ion and protonated amino groups. This binding is non-covalent, hydrogen bonds stabilize the complex formation. Because this complex is non-covalent in nature the charge of proteins does not change. To aid in the interpretation of our electrospray mass spectra we have originated a new kind of linear plot for use with ESI data. It was found that in using the ESI technique that ion currents representing non-covalent complexes of cytochrome c and crown ethers could be observed in the mass spectra. The measurements of the total ion counts of peaks in the mass spectra allowed binding constants to be calculated. This had not been reported before in the literature.The accurate weight determination and the characteristic charge distribution in the ESI mass spectrum provides data suitable for computer modeling. The nature of the protein's positive charges in ESI had not been well defined. The experimentally determined binding constants allowed comparison to results from computational chemistry and molecular modeling. This result shows that the binding occurs at specific, protonated amino acid residues. The calculations performed yielding the Heats of Formation (OH f) for protonated amino acids complexed with crown ethers indicates that the OH binding of crowns increases from histidine, to arginine, to lysine. The use of a 3D model of cytochrome c from crystallographic data provided in the Brookhaven Protein Database and the SYBYL molecular modeling program allows a structural correlation to be made between the 3D model of the protein and protein/crown ether complex. The stoichiometric ratios of bound crown ether to protein determined from experiment, along with the computational results, have been used to rationalize a protein molecular model that allows predictions to be made about the potential for binding of other small molecules.
dc.description.sponsorship Department of Chemistry
dc.format.extent xvi, 194 leaves : ill. ; 28 cm. en_US
dc.source Virtual Press en_US
dc.subject.lcsh Mass spectrometry. en_US
dc.subject.lcsh Biochemistry -- Technique. en_US
dc.subject.lcsh Ions -- Spectra. en_US
dc.title Electrospray mass spectrometry : an investigation of non- covalent interactions of cytochrome c/crown ether complexes and applied methods of computational chemistry en_US
dc.description.degree Thesis (M.S.)
dc.identifier.cardcat-url http://liblink.bsu.edu/catkey/917033 en_US

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  • Master's Theses [5370]
    Master's theses submitted to the Graduate School by Ball State University master's degree candidates in partial fulfillment of degree requirements.

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