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

74 Pages·2016·1.1 MB·English

by  Bradley Allen Williams

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Bradley Allen Williams
Wright State University
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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
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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.

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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
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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 ..............................................................................................

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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

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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

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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 good times while in school.
I would like to share a quote I stumbled upon a couple years ago. “Behind
every success one experiences in life, there are always individuals in the
background who contributed in some way to said success.” I do not know who
said this or where I read the quote to give credit. I would now like to shine light on
those in the background who helped contribute to my success at Wright State
University.
Foremost I would like to express my gratitude to my co-advisors Drs.
David Cool & Peter Lauf. Their patience, guidance, hard work, motivation, and
wealth of knowledge really helped me grow as a person and more so as a
scientist. Without this support I do not believe I would have graduated early.
Furthermore I would like to thank my committee member Dr. Norma
Adragna who helped me with challenging questions during my presentations
preceding my defense. Experiencing more challenging questions helped me
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