H-D exchange (or D-exchange as I’ve sometimes referred to it) has been a problem I’ve dealt with in the lab for some time. It is essentially something that I need to minimize but can never actually stop. It is also a process that I know almost nothing about other than it happens, it occurs somewhat instantaneously, but may be mediated by evaporation rates (when dealing with DDW and 99% D2O). Now I’m perusing the internet looking for some information. Come take a walk with me:
- At ScienceOnline 2013, I met a person who pointed me in the direction of a Dr. Richard Zare. She told me he was very knowledgable in the field and so I looked up his papers. He had five papers relevant to exchange reactions, all of which are way above my head. So I’ll start on Wikipedia and work my way up.
- For those unaware, hydrogen-deuterium exchange is a reaction where a covalently bonded hydrogen is replaced with a deuterium atom. In the case of my experiments this happen with water. If I have deuterium depleted water and I keep it in contact with the environment (which has deuterium in it at around 16mM), eventually it will reach equilibrium and the deuterium level (of the DDW) will rise. The mechanism that causes this, I presume, is D-exchange.
- Apparently the reaction is pH dependent. It can be quenched around pH 2.6, but seems to work best at pH 7.0-8.0. That’s really interesting to me. The pH levels that I’m normally working around are optimal for these reactions. Unfortunately there is nothing I can do about that.
- The reaction may also be quite slow. According to Wikipedia, exchange is slow/unlikely intramolecularly, but is quite rapid via exposed surface hydrogen bonds. For the purposes of NMR, in vivo deuterium incorporation would be valuable and is hard to attain by dissolving proteins in D2O.
- The first person to measure H-D exchange was Kaj Ulrik Linderstrøm-Lang, and I would read some of his stuff right now but they are locked down. The mechanism he used to study D-exchange was pretty interesting and involved a density gradient tube. If I read this correctly, KULL filled a long tube with oils of different desities. He could place a drop of water, with a small amount of proteins in the drop, into the oils to determine the density. As reactions occur, the density would change and the drop would move accordingly.
Ok I’ve reached the limit of what I can do tonight without using the UNM network for access to papers. I’ll try to look for more information this weekend. The quest for a basic understanding of H-D exchange continues…
by Oscar Richards, Journal of Bacteriology. Here is the link.
Before we get to the notes, I have to say I have no idea what journal this paper comes from. I have no details about it other than the fact that this paper comes from some lab at Yale and was received in 1934. Hmmm… anyways, some notes:
- They use water with a specific gravity of 1.000061. After looking it up, specific gravity is the ratio of densities of one substance to a reference substance. In our case it’s probably D2O to H2O. Water should have a SG of 1 at same temp and pressure. The SG of pure D2O is something like 1.107. After thinking about this a lot, there is almost nothing you can infer from that number. They do however provide the ppm (part per million) which is 500 (they say 1 in 2000). Compared to SMOW, that is roughly 3.2 times more D. If I had to guess, I’d say they didn’t discover anything! But luckily I don’t have to guess. They wrote a paper about it.
- Some other interesting numbers I thought about while comparing their SG to SMOW:
- 500ppm is 0.05% D
- 99.9% D2O (which is what I normally use) is 999,000ppm
- For refresher, my DDW is <1ppm D and SMOW is 155.76ppm D
- I inquired with Sigma-Aldrich about the ppm of their D2O, because 99.9% is not terribly descriptive when compared to the other isotope counts.
- This paper is maddeningly frustrating! The data in table one is yeast grown in heavy water compared to yeast grown in DI water, but they don’t provide the reference details. They just give everything as a percentage compared to DI water. WHHHHYYY?!?!?!
- They state “…Nor were significant differences found in the …mean cell size of the two populations of yeast grown under these conditions.”
- Table 1 is full of the most uselessly descriptive information. They chart age, volume and weight of cells and the whole time I’m thinking it’s individual cells but turns out it’s a count of all the cells at once.
- They use this device: photoelectric nephelometer to measure cell counts
- Interesting sentence, “As with most poisonous substances, less injury, some stimulation, and finally no effect should be obtained with increasing dilution of the heavy water.” I’ve read that sentence probably 50 times and I still don’t understand what it says.
- I’ll have to think about their results some more, but if they are accurate in their findings, this study may indicate there is some optimal amount of D2O. This is something we have suggested when analyzing the tobacco seed growth when looking at germination rates.
- They also say that an optimal amount of deuterium may lie somewhere around 1 in 4000. And they state that for growth in bulk low concentrations of deuterium may be a stimulant.
- This is a fascinating statement, “Until more heavy water becomes available it will not be possible to determine the optimal concentration, the effects on fermentation and respiration, and the extent to which the yeast may be adapted to greater concentrations of the heavy isotope of hydrogen.” It hadn’t occurred to me until just now that this paper was written in 1934, which is just a few years after the purification of D2O! Well I hope I can shed some more light on their original studies, seeing as how the experiment was left unfinished some ~80 years ago.
K. Unno, T. Kishido, and S. Okada, “Effect of over-expressed Hsp26 on cell growth in yeast,” Biol. Pharm Bull., vol. 21, pp. 631–633, 1998.
In an attempt to further understand the poorly written paper I summarized yesterday I’m going back to read the paper they cited pretty frequently. Hopefully this paper will answer some of the questions I had yesterday. Note time:
- The motivation for this study is that there has been no observation of the effects of the over-expression or depression of hsp26. Apparently hsp26 is induced during the transition of log-phase growth to stationary phase in yeast growth.
- As they mentioned in yesterday’s paper (this one is earlier by a few years), the yeast cells used were ssa1ssa2 and have a slower growth rate than the wt.
- Like the methods from yesterday, they picked clones of ssa1ssa2. However, this time the clones they chose had different doubling times from each other. Yesterday they chose clones whose doubling times were comparable to the wt.
- cells were incubated at 25C in a minimal dextrose medium (YPd? hahaha) supplemented with nutrients but lacking uracil. WHY???
- They plot the doubling time of the clones of ssa1ssa2 and the wt. It didn’t occur to me that the growth rate of clones could be different. This may be interesting to explore for my D2O variant strain. It should be noted that the doubling time of the wt strain had little variance amongst clones. What is the mechanism for this?
- Exactly what is the difference between clones of the wt and clones of ssa1ssa2???
- they then relate the expression of hsp26 to doubling time and compare that to other proteins (hsp70, hsp90, hsp104, Ssb, and Kar2). They find that hsp26 is more closely correlated to doubling time than the other 3 hsp’s and make no claim about Ssb and Kar2. But they do state that the amounts of Ssb and Kar2 in the clones is similar to the wt. This is sort of true.
So based on subtle information in this paper, it seems the mutant ssa1ssa2 lacks the genes Ssa1 and Ssa2 which make the hsp70 enzyme. But this paper mentions that Ssa4 is inducible and also produces hsp70 when induced. So in yesterday’s paper when they said there was no hsp70 but then they got hsp70, I’m guessing they induced the Ssa4 gene to get some hsp70. Good thing they mentioned that!
In this paper the induced hsp70 levels are less than the wt, and their data shows that hsp70 levels increase with increased doubling time. So yeast that lack Ssa1 and Ssa2 but have Ssa4 have higher levels of hsp26 (than wt) and almost normal levels of hsp70 are at risk for longer growth rates (doubling time). So the proteins may be linked in the extreme case that the Ssa1 and 2 genes are off and the Ssa4 gene is on (both produce hsp70).
But how does this information relate to the paper yesterday? It doesn’t really, but it may explain why they were seemingly contradicting the amount of hsp70 they had in their cells.
Ok just quickly rereading the first few pages of yesterday’s paper and I still have no idea what is going on. No matter. Their results claim to show that some clone of ssa1ssa2, which is beyond arbitrary, is D2O tolerant. I’m not too sure that their methods apply to what I’m doing, since they are just looking for deuterium resistance and not necessarily an organism that is made of D instead of H. I will go over yesterday’s paper again later tonight/early tomorrow to make sure things still make no sense (but maybe I’ll have a moment of clarity?).
K. Unno, T. Kishido, and M. Morioka, “Increased expression of Hsp70 for resistance to deuterium oxide in a yeast mutant cell line,” Biological and …, vol. 26, no. June, pp. 799–802, 2003.
When I started my search for papers to read, I was basically looking for something just like this. Let’s hope this leads me to some interesting finds or at least cites work that is useful to me. To the notes:
- the intro is quite interesting. they speak about using deuterated molecules for NMR (which I’ve read about before) and using microorganisms can be a way to create macromolecules in this way. having an organism that is resilient in and resistant to D2O would go a long way in this regard, hence the motivation for adapting yeast for this purpose. I don’t really care about the purposes, I just think the science is fascinating, but it helps to have some avenues of growth for other people.
- look up citation 8
- “An inhibitory effect of D2O seems to be due to inhibition of tubulin polymerization and effects on microtubule-organizing centers and other structures governing the formation of the mitotic spindle. 9—11” – This is right in line with our research involving kinesin. Could be useful for Nadia and (former grad student now resident bad ass at UT Austin) Andy.
- The rest of that paragraph seems pretty important for sources as well: “Other effects of D2O on energy production, such as lowered ATP/ADP ratios (12), and on membrane receptors, such as impaired Ca2+ channels and Na+ -K+ AT-Pase, have also been reported (13—15). D2O also affects heat- sensitivity (16—18) and the longevity of singlet oxygen (19—21). These effects suggest that cultivation in D2O is stressful for cells. The expression and induction of molecular chaperones (heat shock protein, Hsp) might be important for growth in D2O.”
- they used the cell line ssa1ssa2 – strain A1630. I’ll have to figure out what that is and where they got it from and how that got produced. “cells were kindly donated by Dr. S. Lindquist of the University of Chicago.” Maybe Dr. Linquist can kindly donate some cells to me as well? I’m a little bummed that this paper cheated and used premutated cells.
- They’re cell cultivation isn’t all too different from mine: place cells in liquid media and stick in incubator.
- Going back through the intro, they choose this cell line because increased Hsp70 production leads to enhanced stress resistances. They make it seem like they were hoping that D2O resistance was one of the stress tolerances, and they find out it was.
- this paper is classic scientist needs to use big and exclusive words to validate his claims. ALL THAT DOES IS MAKE IT TAKE LONGER FOR PEOPLE TO READ YOUR PAPER!
- I understand the role of Hsp70 in relation to this project now: Hsp70 is a heat shock trascriptional factor inhibitor. So this cell line doesn’t produce Hsp70 which prevents the transcription factor from deactiving and thus makes lots of heat shock proteins.
- after reading a little about yeast log-phase growth I wonder if my cell growth measurements are based on the dormancy phase of growth and i’m comparing that to mid-log phase growth. hmmm…
- they do 2-d electrophoresis! still don’t know too much about their methods here though…
- so it seems from the strain ss1ss2, they compare the growth of a bunch of clones and pick two (named S-10 and S-11) whose doubling time matches the wild type strain in H2O.
- from here they discovered that the amount of Hsp70 was higher in S-11 than in the wild type (wt). which is interesting because they wanted to have as little Hsp70 as possible based on the introduction. But they then state that the expression of Hsp70 (induction is the word they use) might provide D2O resistance.
- Their data shows that these S-11 cells grow as well as the wt and the ssa1ssa2 strain (still having a hard time understanding the difference between this line and the S-11 line) in H2O, but in D2O only the S-11 line exhibits growth.
- and in H2O the amounts of Hsp70 in S-11 were several times higher than the wt, but in D2O it was only slightly more.
- The grammar of these sentences confuse me: “As the level of Hsp70s was changed among ssa1ssa2 clones(24), the clone cells with low level of Hsp70s were used. However, the increased level of Hsp70s was not observed in D2O. An increased level of Hsp70, especially one previously induced, might be important for cell growth in D2O.” It seems like a contradictory statement. They state earlier in the paper that the clone they chose over-expressed Hsp70, “We isolated a clone named S-11 that grew as fast as the wt and over-expressed Hsp70.” Which is directly opposite what they say in the first quoted text above. Then they say that Hsp70 level increases were not observed in D2O, while saying “Cultivation in D2O slightly increased the level of Hsp70 in S-11… The induction of Hsp70 was significantly found in the wt and S-10 cells.” Am I looking at the wrong things? Is this paper just very poorly written? If anyone wants to read this paper, I’d be happy to share it with them via dropbox or Mendely.
- I suppose the takeaway from this is that Hsp70 production leads to D2O resistance.
- “As a next step for obtaining highly deuterated compounds, it might be needed to culture cells using D-labeled dextrose and amino acids.” I find this interesting. I suppose I’ve taken for granted the fact that mixing YPD with 99.9% D2O doesn’t automatically mean that my peptone, and dextrose will be mostly Deuterium. I was hoping that the amount of deuterium in solution would force d-exchange toward replacing most hydrogen on the non-water molecules. Hmm. I know I can get deuterated dextrose, but can I get deuterated peptone? Should I bother?
- Citation 24 seems pretty important to read, and may shed some light on issues that confuse me.
- Unno K., Kishido T., Okada S., Biol. Pharm. Bull., 21, 631—633 (1998).
The results of this experiment seem to indicate the same thing that yesterday’s paper indicated: D2O adaptation is more a tolerance than an adoption of deuterium. Organisms that tolerate deuterium may not incorporate deuterium in vital molecules. I think this because both papers reveal that D2O adapted organisms grow normally in the presence of H2O. The difference between the two is that this paper doesn’t experiment in water after testing for D2O resistance. The yeast here is just kinda good to grow in D2O but also grows in H2O.
C. R. Bhatia and H. H. Smith, “Adaptation and Growth Response of Arabidopsis thaliana to Deuterium,” Planta, vol. 80, no. 2, pp. 176–184, 1968.
Looking for papers on yeast adaptation (of which I’ve found precisely one!) I came across this paper where they attempted to adapt arabidopsis to D2O. I was instantly intrigued and downloaded the paper for brain consumption. I will now take notes on the paper with the intent of setting up an experiment that can run with minimal effort on my part.
- A paper that is mentioned in the intro that reviews all the genetic and cytological effects of D2O, seems like an interesting place to check out next for work with the yeast project.
- Flaumenhaft, E., S. Bose, H. L. Crespi, and J. J. KATZ: Deuterium isotope effects in cytology. Int. Rev. Cytol. 18, 313–361 (1965).
- “Concerning the possible biological action of deuterium, it is known
that substitution of deuterium in hydrogen bonds of essential macro- molecules (nucleic acids and proteins) changes the properties of these molecules by increasing bond strength and also by a general retardation of reaction rates.“
- seeds were surface steralized in 3% hydrogen peroxide and 90% ethanol (a 1:1 mixture) for one minute
- seeds were sown into mineral media mixed with 0.78% agar – I’ll have to find out what mineral media is available in 2012. Also I find it strange that in this paper they say what products they use, except for the mineral media. If it’s made in house, why don’t they say what the composition is?! Argh!! It’s always the important detail that is left out…
- seeds were grown in test tubes, and closed with glass caps.
- samples were cold treated for 5 days at 4C and then moved to a climate controlled box maintained at 24C and under constant illumination (they use some fluorescent lighting and I’m assuming they are 1960′s grow lamps)
- they did 2 experiments: one was a germination experiment much like the experiments I did in the repeating crumley series, the other was the full adaptation experiments, in all experiments seeds were grown at 0, 10, 30, 50, 70, and 90% D2O
- in both experiments they looked for morphological differences from normal plants
- in the first generation of plants, they found germination rates to be increasingly delayed with increasing concentrations of D2O. This is pretty consistent with with the results I linked to above, although they counted germination from the emergence of the first leaves, whereas I looked for any emergence from the seed coat.
- they also discovered that the green-ness of the plant decreased with increasing D2O conc. and the survival rate of the plants also decreased. No plant produced seeds above 50% D2O in this first generation.
- flowering rates were also lower in samples with higher d2o amounts. the authors subtracted out the germination delay to determine an average flowering time and saw that that increased with increasing d2o concentration.
- as for the adaptation experiment, they say they screened over 850 plants, but only have growth information for plants grown in 50% D2O. They find that after 6 generations of growth on 50% D2O seeds can survive to maturity at 70% D2O and seeds obtained from these plants grow normally in the first generation on H2O media. They conclude that the D2O adaptation is not genetic because of this. I would like to add that I’m highly skeptical of the adaptation efforts included in this paper. There are no counts of the number of plants per generation, the number of seeds planted, etc.
- they hint that they would are taking measures to develop mutants that can grow at concentrations of D2O that are above 70%, I’m guessing they failed because the papers that cite this paper are not by the authors.
- This paper has been cited twice according to the internet:
- Brown, B. T. (1972) A new screening procedure for detecting plant growth regulating compounds. Pesticide Science 3(2)
- Foston, Marcus B. (2012) Deuterium incorporation in biomass cell wall components by NMR analysis. The Analyst 137(5)
This paper must have been one of the last studies on organism development in D2O, especially because the papers that cite this paper are in unrelated fields and this is one of the most recent papers I’ve seen in this field. With that said, I’m a little skeptical of the effort to obtain D2O adapted arabidopsis.
I am impressed that they wanted to answer the question of whether H2O affects D2O adaptations in similar ways that D2O affects H2O adaptations. I don’t think their study was thorough enough and growing plants in 50% D2O still leaves the room for plants to get the H2O requirements. I think growth at levels above 70% would be key to obtaining true D2O adapted plants.
Their methods seem pretty simple and I could work on my own version of this experiment pretty easily and have already looked up product information regarding plant growth in the lab:
- and a protocol: http://www.biosci.ohio-state.edu/~plantbio/Facilities/abrc/handling.htm
I’ll order those things tomorrow morning. And tomorrow I read a paper about D2O-Yeast adaptation!
Sorry if this makes no sense to anyone but Anthony Salvagno: