The Biophysical Effects of Heavy Water


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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:

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The Biophysical Effects of Heavy Water

  1. 1. The Biophysical Effects Of Heavy Water By Anthony Salvagno
  2. 2. History Effects on Life Effects on molecules
  3. 3. What is heavy water? hydrogen deuterium ²H2ONormal water - H2O Heavy water - D2ODensity 0.998 g/ml Density 1.101 g/mlFreezing Point 0°C Freezing Point 3.8°CBoiling Point 100°C Boiling Point 101.4°CViscosity 1.002 Viscosity 1.247pH 7.00 pH 7.43
  4. 4. The Biophysical Effects of Heavy Water Lewis, GN. The biochemistry of water containing hydrogren isotope (1933).
  5. 5. “It is not inconceivable that heavy hydrogen,which exists in small amounts in all naturalwater, may actually be essential to some plantsor animals.” Gilbert N. Lewis Lewis, GN. The biology of heavy water (1934).
  6. 6. Heavy water-life science experimentation (1933-1970)Simple organisms Plants Animals E. coli Tobacco Flies Yeast Arabidopsis Mice P. aeruginosa Wheat Dogs Various pathogens Peas Tadpoles Paramecium Clover Planaria Spirogyra Radish Fish Various algae Kentucky Bluegrass Quail Humans?
  7. 7. Every experiment reported the same results:1. Pure heavy water is toxic to most organisms2. Heavy water at lower concentrations inhibits biological processes But no one explored Lewis’ hypothesis...
  8. 8. ...until now
  9. 9. Helen Crumley et al. (1950)
  10. 10. 33% D2ODay 3 Day 7 Day 11
  11. 11. What’s the difference? DI water: 0.0155% D2O DDW: 0.0000% D2O
  12. 12. ...popcorn break!
  13. 13. DI water: 0.0155% D2ODDW: 0.0000% D2O1% D2O: 1.0000% D2O
  14. 14. Tobacco MorphologyDDW DI water tap water
  15. 15. lots of seeds in DDW
  16. 16. Limitations with tobacco yield experiments with Arabidopsis thaliana Why use Arabidopsis? 1. Everyone studies Arabidopsis. 2. Arabidopsis grows FAST!
  17. 17. Repeating Crumley with Arabidopsis
  18. 18. Root phenotypes in DDW
  19. 19. Arabidopsis growth after 7 weeksDDW 10% D2O 60% D2O
  20. 20. Arabidopsis growth in pure D2O?
  21. 21. Plants and animals cannotsurvive in pure heavy water... ...but simple organisms can!
  22. 22. Saccharomyces cerevisiae (baker’s yeast) growth in D2O 2.5 99.9% D2O 80% D2O 2.0 60% D2OAbsorbance 1.5 40% D2O 20% D2O 1.0 DI water 0.5 0 1 2 3 4 5 Hours
  23. 23. Yeast cellular morphology in D2O DI YPD 20% D2O YPD 40% D2O YPD 25um60% D2O YPD 80% D2O YPD 99.9% D2O YPD
  24. 24. Yeast in DI water Yeast in 99.9% D2O
  25. 25. Yeast in 99.9% D2O
  26. 26. Yeast colony morphology 250um DI YPD 20% D2O YPD 40% D2O YPD60% D2O YPD 80% D2O YPD 99.9% D2O YPD
  27. 27. Escherichia coli exhibits even stranger behavior...
  28. 28. E. coli growth in D2O 1.0 99.9% D2O 0.8 60% D2O 30% D2OAbsorbance 0.6 DDW 0.4 DI 0.2 0.0 0 1 2 3 4 5 6 7 Hours
  29. 29. E. coli adaptation to D2O 3.0 2.5Absorbance 2.0 1.5 1.0 0.5 11 25 39 Days
  30. 30. Adapted E. coli growth in D2O 3.0 Proj. WT in DI 2.5 D2O cells in DDW D2O cells in D2OAbsorbance 2.0 1.5 1.0 0.5 0 1 2 3 4 5 6 Hours
  31. 31. Adapted E. coli cellular morphologyAdapted cells in DI water Adapted cells in D2ONormal cells in DI water Normal cells in D2O
  32. 32. Adapted E. coli cellular morphologyNormal cells in DI water Adapted cells in D2ONormal cells in D2O Adapted cells in DI water
  33. 33. Summary of deuterium effects on life1. 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
  34. 34. History Effects on Life Effects on molecules
  35. 35. Deuterium chemical effectscovalent bonding hydrogen bonding pH or pD? DOH
  36. 36. These chemical effects can be detected physically! mass spectroscopy nuclear magnetic resonance (NMR) fourier transform cavity ring-down infrared spectroscopy spectroscopy (FT-IR) (CRDS)
  37. 37. ...evidence of hydrogen-deuterium replacement! machine use provided by Dr. Krishna assistance provided by Stephen Myers and Alex Haddad
  38. 38. HD exchange Why should we worry?In low and high D environments, HD exchange/replacement can affect experimental results.
  39. 39. Growth in <99% D2O
  40. 40. To quantify HD replacement, use CRDS.
  41. 41. Long-time HD exchange
  42. 42. You can also detect bond strength... ...indirectly.
  43. 43. Dynamic Light ScatteringDetectorDetector
  44. 44. 8 Catalase Aggregation 7 6Intensity (Millions of Counts) 5 H2O 4 D2O 3 2 1 30 40 50 60 70 80 90 Temp ˚C machine use provided by Dr. Osinski assistance provided by Kenji Doering
  45. 45. YPD deterioration in D2O DI water D2O
  46. 46. Even DNA is affected by D2O...
  47. 47. The optical tweezers
  48. 48. Using optical tweezers to overstretch DNA tweezer design and development by Pranav Rathi
  49. 49. Force (pN) DNA Length (nm)Force (pN) DNA Length (nm)Force (pN) DNA Length (nm)
  50. 50. Average DNA overstretch force D2O 75 H2O Mean Force Std Error 70 67.6Force (pN) 65 65.1 60 55 DNA tethers
  51. 51. Summary of deuterium effects on molecules1. 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.
  52. 52. Future work1. Low D2O concetration effects: -Deuterium use vs. tolerance -Lots of potential new discoveries2. High D2O concentration effects: -Useful to study rare events3. Solvent effects: -Under-utilized in studied of biomolecular interactions4. HD replacement: a. Expand studies with CRDS.
  53. 53. AckowledgementsDr. Steven Koch Dr. Abhaya Datye -and all of KochLab -NSMS IGERTDr. Mary Ann Osley Dr. Maggie Werner-Washburne -and the entire Osley Lab -IMSDDr. Robert Olendorf Dr. Krishna, Dr. Osinski, andDr. David Dunlap Dr. Sharpe Mom and Dad my brother Jason All my friends!