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Studies of Reversible Inhibition, Irreversible Inhibition and Activation of Alkaline Phosphatase by Capillary Electrophoresis

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Studies of Reversible Inhibition, Irreversible Inhibition and Activation of Alkaline Phosphatase by Capillary Electrophoresis University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Masters Theses Graduate School 5-2004 Studies of Reversible Inhibition, Irreversible Inhibition and Activation of Alkaline Phosphatase by Capillary Electrophoresis Angela Randall Whisnant University of Tennessee, Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_gradthes Part of the Chemistry Commons Recommended Citation Whisnant, Angela Randall, "Studies of Reversible Inhibition, Irreversible Inhibition and Activation of Alkaline Phosphatase by Capillary Electrophoresis. " Master's Thesis, University of Tennessee, 2004. https://trace.tennessee.edu/utk_gradthes/4828 This Thesis is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Masters Theses by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a thesis written by Angela Randall Whisnant entitled "Studies of Reversible Inhibition, Irreversible Inhibition and Activation of Alkaline Phosphatase by Capillary Electrophoresis." I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Master of Science, with a major in Chemistry. S. Douglass Gilman, Major Professor We have read this thesis and recommend its acceptance: James Q. Chambers, David C. Baker Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a thesis written by Angela Whisnant entitled "Studies of Reversible Inhibition, Irreversible Inhibition and Activation of Alkaline Phosphatase by Capillary Electrophoresis". I have examined the final paper copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of the Master of Science, with a major in chemistry. ouglass Gilman, Major Professor We have read this thesis and recommend its acceptance: Acceptance for the Council: Vice Char.cellar Graduate Studi Studies of Reversible Inhibition, Irreversible !Inhibition and Activation of Alkaline Phosphatase by Capillary Electrophoresis A Thesis presented for the Master of Science Degree The University of Tennessee, Knoxville Angela Randall Whisnant May 2004 Acknowledgments I wish to thank all those who supported me while I worked on this degree. I must first thank my husband, Jody, who supports me in whatever I choose to pursue. I also thank my little girl, Caitlin, for being the sunshine of my day and cheering me up when I so very often have needed it. I also thank my father, mother, and sister for the love and support they give me. I also thank Dr. Gilman for the ideas, guidance and encouragement that he gave me to get this project started from the very beginning to the point of being successful. I also must thank my fellow group members, especially Jason Pittman, for ideas and help given when needed. I also thank Carol Moulton for her encouragement and friendship over the years that we worked together; she will be greatly missed. iii Abstract Reversible inhibition, irreversible inhibition, and activation of calf intestinal alkaline phosphatase (EC 3.1.3.1) have been studied by capillary electrophoresis. The capillary electrophoretic enzyme-inhibitor assays were performed by electrophoretically mixing zones of inhibitor and enzyme in a substrate-filled capillary. Enzyme inhibition was indicated by a decrease in product formation detected in the capillary by laser-induced fluorescence. Reversible enzyme inhibitors could be quantified by Michaelis-Menten treatment of the electrophoretic data. Reversible, competitive inhibition of alkaline phosphatase by sodium vanadate and sodium arsenate has been examined. The calculated K; value for the capillary electrophoretic enzyme-inhibitor assays was 2.1 µM for sodium vanadate and 21 µM for sodium arsenate. The limit of detection for sodium vanadate was 3 µM, and the limit of detection for sodium arsenate was 10 µM. Reversible, non-competitive inhibition of alkaline phosphatase by theophylline has been studied. The calculated K; value for the capillary electrophoretic enzyme-inhibitor assays for theophylline was 102 µM, and the limit of detection was 3 µM. Irreversible inhibition of alkaline phosphatase by EDTA at concentrations of 1.0 mM or higher has been observed. Activation of alkaline phosphatase has also been observed for EDTA at con�ntrations from 20 to 400 µM. V Table of Contents Chapter 1: Introduction to Enzyme Assays .................................................. 1 1.1 Enzyme Assays .......................................................................1 1.2 Enzyme Inhibition............ ......................................................... 5 1.3 On-line Enzyme Assays....................................... ..................... 9 1.4 On-line Enzyme-Inhibition Assays ............................................. 12 Chapter 2: Capillary Electrophoretic Analysis of Alkaline Phosphatase Inhibition by Theophylline ............................................................................... 17 2.1 Introduction ........................................................................... 17 2.2 Experimental. ........................................................................18 2.2.1 Reagents .................................................................. 18 2.2.2 CE-LIF Instrumentation and Experimental Conditions... ...... 18 2.2.3 Microplate Experiments.................................. .............. 20 2.3 Results and Discussion................................. .......................... 21 2.3.1 On-column Capillary Electrophoretic Assay of Alkaline Phosphatase.......................................... ....................... 21 2.3.2 Capillary Electrophoretic Enzyme-Inhibitor Assay of Alkaline Phosphatase................................................ ................. 25 2.3.3 Theophylline Quantitation ............................................. 27 Chapter 3: Capillary Electrophoretic Analysis of Alkaline Phosphatase Inhibition of Irreversible and Competitive, Reversible Inhibitors .............................. 33 3.1 Introduction........................................................ ................... 33 3.2 Experimental................................................. ........................34 vii 3.2.1 Reagents .................................................................. 34 3.2.2 Experimental Conditions.............................................. 35 3.3 Results and Discussion ........................................................... 35 3.3.1 Alkaline Phosphatase Inhibition by Competitive, Reversible Inhibitors............................................................... ....... 35 3.3.2 Alkaline Phosphatase Inhibition by an Irreversible Inhibitor.................................................................. ......43 Chapter 4: Conclusions and Future Work ..................................................51 4.1 Conclusions... ........................................................................51 4.2 Future Studies .......................................................................53 References .........................................................................................55 Appendix ............................................................................................63 Vita .......................... : ......................................................................... 71 viii List of Figures Chapter 1 Figure 1.1: General enzyme-catalyzed reaction scheme... .............................. 2 Figure 1.2: Reaction pathways for reversible inhibitors ................................... 7 Figure 1.3: Description of the two types of EMMA. ...................................... 11 Chapter 2 Figure 2.1 : Electropherogram of an alkaline phosphatase enzyme assay with no inhibitor ..................................................................................... 22 Figure 2.2: Electropherogram without theromstatting........................... ........ 24 Figure 2.3: Electropherogram of an alkaline phosphatase-theophylline enzyme- inhibition assays ......................................................................... 26 Figure 2.4: Plot of Rice versus theophylline concentration ............................. 30 Figure 2.5: Plot of Rlmp versus theophylline concentration .............................31 Chapter 3 Figure 3.1 : Electropherogram of a sodium vanadate--alkaline phosphatase enzyme-inhibitor assay........................................... ...................... 36 Figure 3.2: A plot of Rice versus sodium vanadate concentration ....................38 Figure 3.3: A plot of Rlmp versus the concentration of sodium vanadate...... .....40 Figure 3.4: Electropherogram of a sodium arsenate-alkaline phosphatase enzyme-inhibitor assay ................................................................. 41 ix Figure 3.5: Electropherogram of an EDTA-alkaline phosphatase enzyme- inhibitor assay............................................................................ 44 Figure 3.6: Activation of alkaline phosphatase by 0.030 mM EDTA ................ .45 Figure 3.7: A plot of fractional activity versus concentration.........................
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