BIOPHYSICAL STUDIES AIMED AT POTENTIAL THERAPIES OF HEMOGLOBIN ASSOCIATED ...

129 Pages·2015·4.18 MB·English

BIOPHYSICAL STUDIES AIMED AT POTENTIAL THERAPIES OF HEMOGLOBIN
ASSOCIATED DISEASES

BY

ANDREA BELANGER

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

in Partial Fulfillment of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

Physics

May, 2015

Winston-Salem, North Carolina

Approved By:

Daniel B. Kim-Shapiro, Ph.D., Advisor

S. Bruce King, Ph.D., Chair

Samuel S. Cho, Ph.D.

William Kerr, Ph.D.

Jed Macosko, Ph.D.
ACKNOWLEDGMENTS
I would like to thank Dr. Daniel Kim-Shapiro for his mentorship and guidance
over the last five years. I am sincerely grateful for his patience and encouragement as I
delved into the unknown – biology. Additionally, I would like to thank all of the other
faculty members in the department who were always available to help with anything and
everything. I would like to thank my lab mates, particularly Chris Keggi for doing more
good than harm (most of the time) as an undergraduate researcher; Andreas Perlegas for
helping me make sure my experiments ran smoothly and for being a top-notch
phlebotomist; and Swati Basu, Nadeem Wajih, and Christine Helms for answering all of
my questions and being great role models for me in the lab. Thank you to my committee
members Dr. Daniel Kim-Shapiro, Dr. Jed Macosko, Dr. Sam Cho, Dr. William Kerr,
and Dr. Bruce King for your time and to the Physics department and the National
Institute of Health for the financial support.
Additionally, I would like to thank Stephen Baker and Justin Sigley for being my
surrogate brothers who, for the most part, were supportive and encouraging. Tony
Castricone, thank you for helping me maintain my sanity through the tougher times and
for all the much needed Steak-n-Shake breaks.
Lastly, I would like to thank my family for molding me into the person I am today.
Without all of you I wouldn’t be here. To my sisters, Amy and Katie, for making me
tough and setting the bar high, so I had to live up to the Belanger name all through school.
Thanks to my dad, André, for making me get my hands dirty and raising me to work hard,
and to my mom, Dorie, for always making me challenge myself and never letting me take
the easy road.
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TABLE OF CONTENTS
LIST OF FIGURES………………………………………………………………………iv
LIST OF TABLES……………………………………………………………………….vi
LIST OF ABBREVIATIONS………………………………………………………...…vii
ABSTRACT…………………………………………………………………………...…ix
Chapter
I. INTRODUCTION………………………………………………………...1
II. EFFECT OF A2B RECEPTOR BLOCKER ON SICKLING AND
OXYGEN AFFINITY IN sRBC…………………………………………10
III. EFFECTS OF NITRIC OXIDE AND ITS COGENERS ON SICKLE
RED BLOOD CELL DEFORMABILITY………………………………16
Accepted by Transfusion March 2015
IV. FIVE-COORDINATE H64Q NEUROGLOBIN AS A LIGAND-TRAP
ANTIDOTE FOR CARBON MONOXIDE POISONING………………37
Submitted to Nature Medicine, February 2015
V. MECHANISMS OF HUMAN ERYTHROCYTIC BIOACTIVATION OF
NITRITE…………………………………………………………………61
Published in the Journal of Biological Chemistry, December 2014
VI. CONCLUSION…………………………………………………………..93
REFERENCES……………………………………………………..……………………99
APPENDIX………………………………………………………………..……………108
SCHOLASTIC VITAE…………………………………………………………………117

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LIST OF FIGURES
CHAPTER I
1. Schematic of ektacytometer……………………………………………………….4
2. Osmoscan of healthy RBC………………………………………………………...4
3. Schematic of photolysis…………………………………………………………...7
4. Hemoglobin basis spectra…………………………………………………………9
CHAPTER II
1. Oxygen saturation curves………………………………………………………...13
2. Effect of adenosine on sRBC p50…………………………….………………....13
CHAPTER III
1. Osmotic deformability curves for healthy and sickle RBCs…………………….27
2. Effect of NO donors and NOS-affecting agents on RBC deformability………...28
3. Effect of SNP and calcium on RBC deformability………………………………28
4. Effects of SNP on deformability of sRBCs……………………………………...29
5. Effects of nitrite on deformability and hydration of sRBCs…………………….30
6. Effect of SNP on ion transport…………………………………………………..31
7. Effects of other nitrosating and oxidizing agents on RBCs……………………..32
CHAPTER IV
1. Structure of wild-type and H64Q Ngb unliganded and with bound CO………..40
2. Kinetics of association and dissociation of CO with Hb and Ngb………………43
3. CO transfer from Hb to Ngb……………………………………………………..46
4. CO transfer from RBC-encapsulated Hb to Ngb………………………………..48
5. In vivo CO transfer from RBC-encapsulated Hb to Ngb………………………..51
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CHAPTER V
1. Effect of XOR on nitrite reduction by Hb……………………………………….75
2. Effect of Allopurinol on HbNO yield detected by EPR…………………………80
3. Role of XOR in nitrite reductase activity………………………………………..81
4. Nitrite inhibition of platelet activation…………………………………………...82
5. Effect of DZ on erythrocytic nitrite reduction…………………………………...85
6. Evidence of Hb as the primary RBC nitrite reductase…………………………...87
7. Effect of CO on RBC nitrite reductase…………………………………………..89
APPENDIX
1. Standard absorbance reference spectra…………………………………………104
2. CO transfer from pure Hb to Ngb under atmospheric conditions………………105
3. Dissociation of CO from Hb in presence of excess Ngb……………………….106
4. Example measurement of p50 of Ngb………………………………………….107

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LIST OF TABLES

CHAPTER II
1. Solubility calculations……………………………………………………………15
CHAPTER III
1. Preparation of sRBC samples…………………………………………………....23
CHAPTER V
1. Nitrite bioactivation by RBCs……………………………………………………78

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LIST OF ABBREVIATIONS
2,3-DPG – 2,3-Disphosphoglycerate
CA – carbonic anhydrase
CO – carbon monoxide
DEA-NONOate – Diethylamine NONOate
DI Max – maximum deformability
DZ – dorzolamide
eNOS – endothelial nitric oxide synthase
EPR – electron paramagnetic resonance
Hb – hemoglobin
HbS – sickle hemoglobin
L-NAME – L-NG-nitroarginine methyl ester
MCHC – mean cell hemoglobin concentration
NECA – 5’-N-ethylcarboxamidoadenosine
Ngb – neuroglobin
NO – nitric oxide
Osm Max – osmolality at maximum deformability
PAO – phenylarsine oxide
PBS – phosphate buffered saline
PEG-ADA – polyethylene glycol-adenosine deaminase
PDI – protein disulfide isomerase
PRP – platelet rich plasma
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RBC – red blood cell
SAD – S-Antilles-D Punjab (Antilles (beta 23Ile) and D Punjab (beta 121Gln) mutations
to promote sickling
SCD – sickle cell disease
SNP – sodium nitroprusside
UV – ultra-violet
Vis – visible
XOR – xanthine oxidoreductase

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ABSTRACT

Andrea Belanger
BIOPHYSICAL STUDIES OF POTENTIAL THERAPIES OF HEMOGLOBIN-
ASSOCIATED DISEASES
Dissertation under the direction of
Daniel B. Kim-Shapiro, Professor of Physics

Hemoglobin (Hb), the most abundant protein found in the red blood cell (RBC),
carries gases like oxygen and carbon dioxide throughout the body. However, various
conditions, both genetic and environmental, can impede its ability to perform this (and
other) necessary functions. We studied some of the most imperative properties of Hb and
the RBC as they pertain to sickle cell disease (SCD): oxygen affinity and RBC
deformability. A Hemox Analyzer, a dual-wavelength spectrophotometer, was used to
measure oxygen affinity in sRBC treated with a potential drug intended to reduce sickling
and improve oxygen affinity. Deformability, the ability of the cells to elongate when
exposed to shear, was measured as a function of osmotic pressure in a refurbished
Technicon Ektacytometer. This characteristic of the cells is extremely important,
especially in SCD, as rigid cells 1) are unable to traverse the narrow capillaries and 2) are
more likely to get filtered out by the spleen prematurely, leading to severe anemia. The
effects of nitric oxide (NO) and its cogeners were rigorously tested and were shown to
improve deformability under specific conditions.
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In addition to genetic abnormalities of Hb, alterations to the gaseous homeostasis
of the body, such as the presence of carbon monoxide (CO), can lead to diminished Hb
function and subsequent injury or even death. We used laser-assisted flash photolysis to
measure the binding affinities of CO to Hb and a genetically modified neuroglobin (Ngb)
that was designed to have a superphysiologically high affinity for ligands such as CO.
We found that this mutated Ngb had a binding affinity for CO almost 500 times greater
than that of Hb for CO, demonstrating its promising potential as an antidote for CO
poisoning.
It is well understood that NO and nitrite play a major role in the modulation of
blood flow and proper platelet function. Several proteins, including deoxygenated Hb, are
capable of reducing nitrite to NO, however the mechanism by which this event occurs in
vivo is still under much debate. We demonstrated that while several proteins are able to
reduce nitrite to NO, the relatively high concentration of Hb in the vasculature casts
doubt on the role other proteins play in nitrite bioactivation, at least by red blood cells.

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