• Login
    View Item 
    •   UMB Digital Archive
    • School, Graduate
    • Theses and Dissertations All Schools
    • View Item
    •   UMB Digital Archive
    • School, Graduate
    • Theses and Dissertations All Schools
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Browse

    All of UMB Digital ArchiveCommunitiesPublication DateAuthorsTitlesSubjectsThis CollectionPublication DateAuthorsTitlesSubjects

    My Account

    LoginRegister

    Statistics

    Display statistics

    Luminescent probes for the detection of glutamine and protein hydrodynamics

    • CSV
    • RefMan
    • EndNote
    • BibTex
    • RefWorks
    Find Full text
    Author
    Dattelbaum, Jonathan David
    Advisor
    Lakowicz, Joseph R.
    Date
    2000
    Type
    dissertation
    
    Metadata
    Show full item record
    Abstract
    We have prepared novel fluorescent probes for protein hydrodynamics and for the determination of glutamine in solution. Ru(II) and Re(I) metal ligand complexes (MLCs) have been synthesized with amine- and sulfhydryl-reactive moieties suitable for modifying biomolecules. The spectral properties of MLCs when free in solution and when conjugated to proteins have been studied. We have characterized MLCs which display photoluminescent lifetimes between 60 ns and 3.5 mus and possess useful anisotropy values between 0.1 and 0.32. Time resolved anisotropy measurements were used to demonstrate the potential utility of these complexes in measuring the long rotational correlation times of macromolecules in solution. The usefulness of this class of molecules in fluorescence polarization immunoassay (FPI) was explored using an association reaction between HSA and polyclonal anitbody. A series of water soluble MLCs have been synthesized and characterized which may be used to measure correlation times between 0.1 and 10 mus. We have developed a reagentless optical assay for glutamine based on the Escherichia coli glutamine binding protein (GlnBP). Site-directed mutagenesis was performed to engineer single cysteine mutants which were covalently modified with environmentally sensitive sulfhydryl-reactive probes. The fluorescence emission of acrylodan and 2-(4'(iodoacetamido)anilino)naphthalene-6-sulfonic acid (IAANS) attached to GlnBP mutant S179C was shown to decrease 65 and 35 percent, respectively, upon titration with increasing amounts of glutamine (0 to 6.4 muM; KDapp 160 nM). No significant changes in the fluorescence intensity were observed for the structurally similar amino acids glutamate, asparagine and arginine. Time-resolved intensity decays showed a 2.4-fold decrease in mean lifetime for GlnBP S179C-acrylodan upon the addition of glutamine, indicating the possibility of a lifetime-based assay. Anisotropy decay measurements for GlnBP S179C-acrylodan showed a 13 ns rotational correlation time in the ligand free state; whereas, multiple correlation times were assigned in the glutamine bound conformation. The decrease in fluorescence intensity of S179C-acrylodan was adapted to polarization sensing of glutamine. A generic approach to the design of ratiometric biosensors is also discussed. The engineered GlnBP is a first step towards the development of a nonenzymatic biosensor capable of determining glutamine concentrations in cell cultures.
    Description
    University of Maryland, Baltimore. Biochemistry and Molecular Biology. Ph.D. 2000
    Keyword
    Chemistry, Biochemistry
    Identifier to cite or link to this item
    http://hdl.handle.net/10713/1235
    Collections
    Theses and Dissertations School of Medicine
    Theses and Dissertations All Schools

    entitlement

     
    DSpace software (copyright © 2002 - 2023)  DuraSpace
    Quick Guide | Policies | Contact Us | UMB Health Sciences & Human Services Library
    Open Repository is a service operated by 
    Atmire NV
     

    Export search results

    The export option will allow you to export the current search results of the entered query to a file. Different formats are available for download. To export the items, click on the button corresponding with the preferred download format.

    By default, clicking on the export buttons will result in a download of the allowed maximum amount of items.

    To select a subset of the search results, click "Selective Export" button and make a selection of the items you want to export. The amount of items that can be exported at once is similarly restricted as the full export.

    After making a selection, click one of the export format buttons. The amount of items that will be exported is indicated in the bubble next to export format.