Hey Gilbert Lewis: Has life evolved a use for deuterium? Or does it just tolerate it?

Andy Maloney in our lab has been studying solvent (water) isotope effects on kinesin and microtubules in the gliding motility assay.  He has data showing a speed slow down from both heavy-hydrogen water (D2O; deuterium oxide) and heavy-oxygen water (H₂¹⁸O; oxygen-18 water). The preliminary results are very exciting to me, because I think varying the water isotopes may be a useful new knob for studying kinesin, molecular motors, and other biomolecular systems such as protein-DNA complexes. In particular, I think oxygen-18 water may be a neat way of probing the kinetics of large surface area binding/unbinding events. Hopefully soon, I will blog about those results either here, or on our kochlab research blog.  If you want more info now, you can see our presentations and posters on nature precedings. 

But that's not what I want to talk about tonight.  Tonight, I want to ask the question:  Have life forms adapted a use for deuterium?  Or is it merely tolerated?  To talk about this, I will go back to really fun 1933 letter to JACS by Gilbert N. Lewis¹.  I found this paper because of our work with D2O in the lab and my quick realization that my initial assumptions about D2O were way off.  I had assumed that D2O was pretty much like regular water, just a bit denser.  I completely missed the point that D (deuterium: one proton, one neutron) is chemically very different from H (hydrogen or protium, one proton, zero neutrons).  This is because the reduced mass of an H-X bond is substantially different from a D-X bond, and thus the binding energy is substantially different.  (X refers to some other atom such as oxygen or carbon.)  It turns out that the chemistry of D is so much different from H that relatively pure D2O is toxic to eukaryotic lifeforms!  D2O also has many other amazing effects on life forms and biomolecules.  For example, it stabilizes microtubules², which is a major reason it is toxic³.  It also increases the thermostability of proteins⁴ or even whole fruit flies⁵.  Last fall, I summarized some of the things I learned about D2O in a group meeting.

So, while I was naive in 2009 (and likely continuing 2010->), Gilbert Lewis was amazingly prescient in 1933.  In his letter to JACS, he says, "Sir: Even before I had succeeded in concentrating the isotope of hydrogen, I predicted that H²H²O would not support life and would be lethal to higher organisms."  Don't you wish research letters today began so strikingly??? What a breath of fresh air(rogance)!  The letter goes on to describe a beautifully simple experiment that he was able to carry out to demonstrate the toxicity of D2O to tobacco seeds.  He used tobacco seeds because they are tiny and he only had a small amount of D2O (that he purified himself).  He showed that 6 tobacco seeds in regular water sprouted nicely over the course of two weeks.  Whereas 6 tobacco seeds in reasonably pure D2O did not sprout at all.  Tobacco seeds in 50/50 D2O / H2O (which would be 50% H¹H²O) sprouted, but slowly.  At the end of the letter, Lewis says, "I have long desired to determine the proportions of isotopes in living matter, in order to see whether the extraordinary selective power of living organisms, which is exemplified by their behavior toward optical isomers, might lead to a segregation of isotopes in some of the substances which are necessary to growth.  The marked biochemical differences between the two isotopes of hydrogen lends a further incentive to this search."  It's not exactly clear, but I have to guess that Lewis was wondering whether living organisms would use D and H differently, and whether he could detect this via deviations from the ratio of D to H in (standard mean) ocean water.

To me, this is a fascinating question.  Do cells use D for specialized purposes?  If not, do they use pumps, etc. to increase the concentration of H inside cells and reduce the toxic effects of D?  When I mention this to most scientists, it seems to set off their "crackpot" sensors, which is understandable.  I mentioned this to Steven Benner at a DTRA program review a few weeks ago and his crackpot sensor initially was triggered.  He said, "Deuterium is only 1 hundredth of a percent [of the total hydrogens in regular water]."  I said, "But that's 17 millimolar!"  He said, "OK, well you changed the units on me..." But then he changed his tune a bit and I think he considered it plausible. 

0.03% does sound trivial.  But the way I look at it, biology has somehow evolved to make use of different divalent cations in much lower concentration, such as magnesium, zinc, calcium, etc.  And it can distinguish between potassium (K+) and sodium (Na+).  How much more different are K+ and Na+ from each other, compared to the difference between D and H?  I actually don't know, but my intuition would say it's in the same ballpark.  I find it remarkable that regular water has 17 millimolar of deuterium "contamination" in it, and up until 2009 it never occured to me that it could matter!  As an example, if you're studying a proton pump at the single-molecule level, 1 out of six thousand events may be artificially slowed because D is in there instead of H.  This could make a difference, especially if studying events like pausing.  (There is a recent paper by Yuan and Berg that studies isotope effects in the bacterial rotary motor that I have not yet read carefully⁶.)

So, at some level it seems important to remember that there's a lot of deuterium in regular "pure" water. But more interesting to me is whether life has adapted uses for this deuterium.  I think if Lewis had had easy access to deuterium-depleted water, he would have investigated this right away.  But as far as I know, deuterium-depleted water didn't become readily available until many decades later (I could be wrong on this).  I think Lewis' tobacco seed experiments are the perfect place to start studying this effect.  My hypothesis is: tobacco seeds in deuterium-depleted water (<1 part per million D; Aldrich product no. 195294) will sprout more slowly than in "regular" water with approximately 150 parts per million D.  To test this, we only need:

• Tobacco seeds (cheap, right?)
• Deuterium-depleted water $25
• 500 microliter microfuge tubes, a camera (maybe microscope?), and some time

Doesn't this sound like a really fun experiment to try out?  I've been wanting Andy to give it a whirl, but since he's got so many other things to do to finish his PhD, I can't in good conscience force him to do it.  I'm pretty tempted to try it out myself...especially if I could team up with an 8th grader for the science fair.  :)

I'm obviously not the first person to think of this.  I'm sure Lewis thought of it, but didn't have the resources.  D-depleted water is cheap nowadays, probably because of its use in NMR.  A google search for "deuterium-depleted water" is ruined by a huge amount of links to products about the use of D-depleted water for curing all kinds of cancer.  This is unfortunate.  Digging around, I was able to find a couple peer-reviewed papers investigating the effects of deuterium depletion on life.  The first I found is by Somlyai et all in FEBS 1993⁷.  They claim to prove that "naturally-occurring deuterium is essential for normal cell growth."  This paper has been cited 10 times, and of those 10, I think only two investigate deuterium-depletion effects.  Those two are by the same research group.  Their results would be striking, but it is not convincing for at least two reasons.  First, their level of depletion was not substantial: 150 ppm to 30-40 ppm (compared to <1 ppm which we can achieve now).  Secondly, they investigate the effects on human cell lines.  As far as I know, human cell lines are finicky...much, much more finicky than tobacco seeds.  I think it was a mistake to jump right into cell lines.

The results of Somlyai et al. may be true, and if so would be exciting.  Not, in my opinion, as an immediate cure for cancer, but rather as a fascinating new area of cell biology to study.  I think a quick extension of Lewis' beautiful tobacco seed experiments is a great first step.  If we don't see an effect?  Try again!  Maybe try mustard seed too.  Steven Benner, indicated the next step (paraphrasing from fuzzy memory): grow yeast or E. coli in D-depleted water.  Check for which genes are mutated.  Those genes are candidates for encoding proteins that utilize deuterium for the benefit of the cell.  Or proteins that sequester D, I suppose.

So, what do you think?  Should we try this experiment?  I think so, and I'll write up a proposed protocol in my OpenWetWare notebook soon (I hope).

PS: In addition to the short letter by Lewis I talked about above, I highly recommend reading a longer letter to Science in 1934⁸.  In his first paragraph he says, "Several months ago the experiments were interrupted, and since there may be no immediate opportunity of resuming them it seems best to publish the somewhat sporadic results so far obtained."  Don't you wish you could publish your own sporadic results in Science?  It's a very interesting paper where he describes further tobacco seed experiments as well as microorganisms, flatworms, and mice.

References (Send me a note if you'd like the PDF for the ref. 1)
1. Gilbert N. Lewis (1933). THE BIOCHEMISTRY OF WATER CONTAINING HYDROGEN ISOTOPE Journal of the American Chemical Society, 55 (8), 3503-3504 DOI: 10.1021/ja01335a509

2. Panda, D., Chakrabarti, G., Hudson, J., Pigg, K., Miller, H. P., Wilson, L., et al. (2000). Suppression of microtubule dynamic instability and treadmilling by deuterium oxide. Biochemistry, 39(17), 5075-81. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/10819973.

3. I think there is a study from 1935 that looks at cells in D2O and sees larger spindle apparatus in mitotic cells, but I can't find the reference now.

4. Sen, A., Balamurugan, V., Rajak, K. K., Chakravarti, S., Bhanuprakash, V., Singh, R. K., et al. (2009). Role of heavy water in biological sciences with an emphasis on thermostabilization of vaccines. Expert review of vaccines, 8(11), 1587-602. doi: 10.1586/erv.09.105.

5.Pittendrigh, C. S., & Cosbey, E. S. (1974). On the Very Rapid Enhancement by D2O of the Temperature-Tolerance of Adult Drosophila. Proceedings of the National Academy of Sciences of the United States of America, 71(2), 540-543. Retrieved from http://www.pnas.org/content/71/2/540.abstract. 

6. Yuan, J., & Berg, H. C. (2010). Thermal and solvent-isotope effects on the flagellar rotary motor near zero load. Biophysical journal, 98(10), 2121-6. Biophysical Society. doi: 10.1016/j.bpj.2010.01.061. 

7. Somlyai, G., Jancsó, G., Jákli, G., Vass, K., Barna, B., Lakics, V., et al. (1993). Naturally occurring deuterium is essential for the normal growth rate of cells. FEBS letters, 317(1-2), 1-4. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/8428617. 

8. Lewis, G. N. (1934). THE BIOLOGY OF HEAVY WATER. Science (New York, N.Y.), 79(2042), 151-153. doi: 10.1126/science.79.2042.151. 
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