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Locating Chelerythrine, an Alkaloid, Within a Cytosolic Environment by Maldi-Tof Mass Spectrometry

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Locating Chelerythrine, an Alkaloid, Within a Cytosolic Environment by Maldi-Tof Mass Spectrometry Wright State University CORE Scholar Browse all Theses and Dissertations Theses and Dissertations 2013 Locating Chelerythrine, an Alkaloid, within a Cytosolic Environment by Maldi-Tof Mass Spectrometry Bradley Allen Williams Wright State University Follow this and additional works at: https://corescholar.libraries.wright.edu/etd_all Part of the Pharmacology, Toxicology and Environmental Health Commons Repository Citation Williams, Bradley Allen, "Locating Chelerythrine, an Alkaloid, within a Cytosolic Environment by Maldi-Tof Mass Spectrometry" (2013). Browse all Theses and Dissertations. 1167. https://corescholar.libraries.wright.edu/etd_all/1167 This Thesis is brought to you for free and open access by the Theses and Dissertations at CORE Scholar. It has been accepted for inclusion in Browse all Theses and Dissertations by an authorized administrator of CORE Scholar. For more information, please contact [email protected]. LOCATING CHELERYTHRINE, AN ALKALOID, WITHIN A CYTOSOLIC ENVIRONMENT VIA MALDI-TOF MASS SPECTROMETRY A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science By BRADLEY ALLEN WILLIAMS BS., Youngstown State University, 2010 2013 Wright State University WRIGHT STATE UNIVERSITY GRADUATE SCHOOL December 11, 2013 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Bradley Allen Williams ENTITLED Locating Chelerythrine, an Alkaloid within a Cytosolic Environment via MALDI-TOF Mass Spectrometry BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science ______________________________ Peter K. Lauf, MD., Thesis Director ___________________________ Norma C. Adragna, Ph.D., Interim Chair Pharmacology & Toxicology Committee on Final Examination __________________________________ Peter K. Lauf, MD. __________________________________ David R. Cool, Ph.D., Co-Director __________________________________ Norma C. Adragna, Ph.D. __________________________________ R. William Ayres, Ph.D. Interim Dean, Graduate School ABSTRACT Williams, Bradley Allen. M.S.., Department of Pharmacology & Toxicology, Wright State University, 2013. Locating Chelerythrine, an Alkaloid, Within a Cytosolic Environment via MALDI-TOF Mass Spectrometry. The quaternary benzo-phenanthridine alkaloid (QBA) chelerythrine (CET) is a well-known inhibitor of PKC and cancer growth (Herbert et al., 1990). In human lens epithelial cells (HLECs), CET almost completely inhibits the Na+/K+ pump ATPase (NKA) without changes in phosphorylation (Lauf et al. 2013). Protein alignment studies revealed B-lymphocyte type 2 protein (Bcl-2) BH1- like motifs within the α1 subunit of the NKA to which CET, a drug mimicking Bcl-2 protein BH3-like motifs, might bind thus disrupting NKA function (Lauf et al, 2013). It is unknown by which mechanism CET crosses the plasma membrane to reach its intracellular targets, especially the NKA. In solution, CET exists in two forms, as a positively charged and as uncharged monomer or pseudo-base. The hypothesis is that CET crosses the plasma membrane as monomer and, in the slightly acidic cytosol binds as the positively charged drug to its putative sites on the NKA and pro-survival BcL-2 proteins. The objective of this work is to track the alkaloid CET via MALDI-TOF Mass Spectrometry from the external media bathing HLECs into the cytosol and plasma membrane, if possible into the NKA. A prerequisite of this project is to gain insight into how CET behaves in the biological solutions used, how it might cross the plasma membrane normally impermeable to charged drugs, and how the lower pH within the cytosol affects the ratio of the charged to iii uncharged species of CET. Once evidence is brought forth that CET reaches the plasma membrane, future studies will address by mass spectrometry, the interactions of CET with the NKA during its canonical operation, and perhaps isolate the CET binding site (s) within the NKA. iv TABLE OF CONTENTS Page I. INTRODUCTION .................................................................................. 1 Background of Chelerythrine ........................................................... 1 Potential Applications of Chelerythrine ............................................ 2 Toxicity of Sanguinarines ................................................................ 2 Modern Day Uses of Chelerythrine & Significance .......................... 3 Cell Membrane ................................................................................ 4 Cell Permeability.............................................................................. 5 Human Lens Epithelial Cells ............................................................ 6 Hypothesis ....................................................................................... 6 II. MATERIALS AND METHODS .............................................................. 8 MALDI-TOF Mass Spectrometry ..................................................... 8 Mass Spectrometry Sample Preparation ......................................... 9 Predicting the Presence of the Monomer ...................................... 10 Establishing a Calibration Curve ................................................... 10 Tracking Chelerythrine in the Cytosolic Domain ............................ 10 Tandem Mass Spectrometry ......................................................... 11 Ion Trap Mass Spectrometry ........................................................ 11 Solvent Dependent Serial Dilutions .............................................. 12 v Chelerythrine Dependency on pH ................................................. 15 Modification of the Structure of Chelerythrine ............................... 15 Chelerythrine in Cytosolic Conditions ........................................... 15 III. RESULTS ........................................................................................... 17 Chelerythrine Choice of Matrices .............................................. …17 Chelerythrine Calibration Curves ................................................... 18 Chelerythrine Determination in Biological Media ........................... 19 Factors Modifying CET Detection by MALDI TOF: pH ................... 20 Figures ..................................................................................... 25-39 IV. DISCUSSION ..................................................................................... 42 Chelerythrine in the Cell ................................................................ 43 The Role of pH on Chelerythrine ................................................... 46 Ion Trapping .................................................................................. 46 Quantitation of Chelerythrine by MALDI TOF MS .......................... 47 Implications for Chelerythrine in Medicine ..................................... 49 Conclusions ................................................................................... 51 V. BIBLIOGRAPHY ................................................................................. 53 VI. APPENDIX .............................................................................................. vi LIST OF FIGURES Figure Page 1. Chemical Structure of Chelerythrine ...................................................... 25 2. Mass Spectrum of Chelerythrine ........................................................... 26 3. Serial Dilutions of Chelerythrine ............................................................ 27 4. Initial Calibration Curve ......................................................................... 28 5. Accepted Calibration Curve of Chelerythrine ......................................... 29 6. Cellular Location of Chelerythrine.......................................................... 30 7. Effect of NaOH on Chelerythrine ........................................................... 31 8. HCl Effects on Chelerythrine ................................................................. 32 9. Effects of pH on Chelerythrine ............................................................... 33 10. Effect of pH on the Trimer ..................................................................... 34 11. Chelerythrine Oxidative Demethylation ................................................ 35 12. Tandem Mass Spectrum ...................................................................... 36 13. Ion Trap Tandem Mass Spectrum ........................................................ 37 14. Chelerythrine in DMEM Media .............................................................. 38 15. Fingerprint Analysis .............................................................................. 39 16. Chelerythrine vs Cholesterol .................................................................. 48 vii LIST OF TABLES Table Page 1. Solvent Dependable Serial Dilutions ..................................................... 13 2. Charged Monomer to Pseudobase ........................................................ 14 3. Tandem Mass Spectroscopy Fragmentation ......................................... 40 4. Cellular Concentrations of Chelerythrine .............................................. 41 viii Acknowledgements First, I would like to thank God, and my family back home in Youngstown, Ohio. Without my family (Kathryn McFrazier, Mother; and Daryl Williams, Brother) and divine guidance I would not have made it to where I am today. They have always been there for me, through the ups and downs. This helped me during the good times and not so
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