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!