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Deuterium is present in all naturally occurring water, and accounts for 1 in 6420 hydrogen atoms. Seemingly insignificant, that ratio is actually quite impactful in life sciences. Pure deuterium oxide (heavy water) has been found to be toxic to eucharyotic organsims, and has been found to be inhibitory in smaller doses. Here I present results that demonstrate that in even smaller doses the concentration of deuterium has a dramatic effect on life processes. I also show that at the molecular level, deuterium has a stabilizing effect. By simply changing the properties of the solvent, there can be dramatic effects that impact a variety of experiments. For the entire story, please refer to my dissertation: https://docs.google.com/document/d/1p9-qqF8oUcwNGJjZG3CoKL2xvIhbqKCFQ7_faegNPIU/edit?usp=sharing
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The Biophysical EffectsOf
Heavy WaterBy Anthony Salvagno
History Effects on Life Effects on molecules
What is heavy water?
deuteriumhydrogen
Normal water - H2O
Density 0.998 g/ml
Freezing Point 0°C
Boiling Point 100°C
Viscosity 1.002
pH 7.00
Heavy water -
Density 1.101 g/ml
Freezing Point 3.8°C
Boiling Point 101.4°C
Viscosity 1.247
pH 7.43
²H2O D2O
The Biophysical Effects of Heavy Water
Lewis, GN. The biochemistry of water containing hydrogren isotope (1933).
Lewis, GN. The biology of heavy water (1934).
“It is not inconceivable that heavy hydrogen, which exists in small amounts in all natural water, may actually be essential to some plants or animals.” Gilbert N. Lewis
Heavy water-life science experimentation(1933-1970)
Simple organisms
E. coli
Yeast
P. aeruginosa
Various pathogens
Various algae
Paramecium
Spirogyra
Tobacco
Arabidopsis
Wheat
Peas
Clover
Radish
Kentucky Bluegrass
Plants
Flies
Mice
Dogs
Tadpoles
Planaria
Fish
Quail
Humans?
Animals
Every experiment reported the same results:
1. Pure heavy water is toxic to most organisms
2. Heavy water at lower concentrations inhibits biological processes
But no one explored Lewis’ hypothesis...
...until now
Helen Crumley et al. (1950)
Day 3 Day 7 Day 11
33% D2O
What’s the difference?
DI water: 0.0155% D2ODDW: 0.0000% D2O
...popcorn break!
DI water: 0.0155% D2ODDW: 0.0000% D2O1% D2O: 1.0000% D2O
Tobacco Morphology
DDW DI water tap water
lots of seeds in DDW
Limitations with tobacco yield experiments with Arabidopsis thaliana
1. Everyone studies Arabidopsis.
2. Arabidopsis grows FAST!
Why use Arabidopsis?
Repeating Crumley with Arabidopsis
Root phenotypes in DDW
Arabidopsis growth after 7 weeks
DDW 10% D2O 60% D2O
Arabidopsis growth in pure D2O?
Plants and animals cannot survive in pure heavy water...
...but simple organisms can!
0.5
1.0
1.5
2.0
2.599.9% D2O
80% D2O
60% D2O
40% D2O
20% D2O
DI water
543210
Hours
Abs
orba
nce
Saccharomyces cerevisiae (baker’s yeast) growth in D2O
DI YPD 20% D2O YPD 40% D2O YPD
80% D2O YPD60% D2O YPD 99.9% D2O YPD
25um
Yeast cellular morphology in D2O
Yeast in 99.9% D2OYeast in DI water
Yeast in 99.9% D2O
DI YPD 20% D2O YPD 40% D2O YPD
60% D2O YPD 80% D2O YPD 99.9% D2O YPD
250um
Yeast colony morphology
Escherichia coli exhibits even stranger behavior...
0.0
0.2
0.4
0.6
0.8
1.099.9% D2O
60% D2O
30% D2O
DDW
DI
76543210
Hours
Abs
orba
nce
E. coli growth in D2O
E. coli adaptation to D2OA
bsor
banc
e
Days
0.5
1.0
1.5
2.0
2.5
3.0
11 25 39
Adapted E. coli growth in D2O
0.5
1.0
1.5
2.0
2.5
3.0Proj. WT in DI
D2O cells in DDW
D2O cells in D2O
6543210
Hours
Abs
orba
nce
Adapted E. coli cellular morphology
Adapted cells in DI water Adapted cells in D2O
Normal cells in DI water Normal cells in D2O
Adapted E. coli cellular morphology
Normal cells in DI water Adapted cells in D2O
Normal cells in D2O Adapted cells in DI water
Summary of deuterium effects on life
1. New phenotypes found in very low deuterium enviornments: a. Root hairs in tobacco b. Earlier germination in tobacco c. Curly roots in arabidopsis2. Optimal deuterium concentration above normal amounts a. Tobacco root growth b. Arabidopsis plant growth3. D2O adaptation produces new phenotypes a. E. coli “brainy” colonies b. E. coli joining c. Faster growth
History Effects on Life Effects on molecules
Deuterium chemical effects
covalent bonding hydrogen bonding pH or pD?
DOH
These chemical effects can be detected physically!
fourier transforminfrared spectroscopy
(FT-IR)
cavity ring-downspectroscopy
(CRDS)
mass spectroscopy nuclear magnetic resonance(NMR)
...evidence of hydrogen-deuterium replacement!machine use provided by Dr. Krishna
assistance provided by Stephen Myers and Alex Haddad
HD exchange
Why should we worry?
In low and high D environments, HD exchange/replacementcan affect experimental results.
Growth in <99% D2O
To quantify HD replacement, use CRDS.
picarro.com
Long-time HD exchange
You can also detect bond strength...
...indirectly.
Dynamic Light Scattering
Detector
Detector
4030 50 60 70 80 90
1
2
3
4
5
6
7
8
D2O
H2O
Inte
nsity
(Mill
ions
of C
ount
s)
Temp ˚C
Catalase Aggregation
machine use provided by Dr. Osinskiassistance provided by Kenji Doering
YPD deterioration in D2O
D2ODI water
Even DNA is affected by D2O...
The optical tweezers
Using optical tweezers to overstretch DNA
tweezer design and development by Pranav Rathi
DNA Length (nm)
Forc
e (p
N)
DNA Length (nm)
Forc
e (p
N)
DNA Length (nm)
Forc
e (p
N)
55
60
65
70
75H2O
Mean Force
Std Error
D2O
Forc
e (p
N)
DNA tethers
67.6
65.1
Average DNA overstretch force
Summary of deuterium effects on molecules
1. D2O stabilizes molecules. a. Better for single molecule and in-vitro studies b. It could be used for long term storage of biomolecules.
2. Good probe for solvent effects. a. Just changing the water affected DNA-DNA interations!
3. HD exchange/replacement: a. Important to pay attention in low D experiments. a. Good way to measure local atmospheric isotope ratios.
1. Low D2O concetration effects: -Deuterium use vs. tolerance -Lots of potential new discoveries
2. High D2O concentration effects: -Useful to study rare events
3. Solvent effects: -Under-utilized in studied of biomolecular interactions
4. HD replacement: a. Expand studies with CRDS.
Future work
Ackowledgements
Dr. Steven Koch -and all of KochLabDr. Mary Ann Osley -and the entire Osley LabDr. Robert OlendorfDr. David Dunlap
Dr. Abhaya Datye -NSMS IGERTDr. Maggie Werner-Washburne -IMSDDr. Krishna, Dr. Osinski, and Dr. Sharpe
Mom and Dadmy brother JasonAll my friends!