This is a summary I gave at group meeting today on what I'd learned about D2O (aka "heavy water" aka "deuterium oxide") and its effects on biochemistry/biophysics of enzymes and proteins.

1. Steve’s group meeting summary of what he’s learned about deuterium oxide aka D2O aka heavy water over the past few days.Special emphasis is on tubulin / microtubules and stabilization of proteins in D2O. November 5, 2009

2. Types of “heavy water” Typically “heavy water” = D2O “Deuterium Oxide” Approx: $100 for 100 ml of purity we’re using now Reportedly is “rather dirty” as far as colloid science goes Should keep in mind that 1H18O is also available More “physics” less “chemistry” (Steve’s guess) I don’t think 18O will exchange with proteins H-bonding of solvent may still be affected, but less I’d think Very expensive I also find it hilarious that “ultrapure” water is actually contaminated with 8 mM deuterium (1 out of 6600) Everything natural is heterogeneous! Yikes

3. What happens when you put a protein in D2O? “protonatable” groups “rapidly” exchange with D. Since concentration of protein is << concentration of D, effectively every exposed group gets deuterated“rapidly” on timescale of motility (I think)Kinetic Isotope Effect (KIE); Deuterium IE (DIE) Buried groups can remain hydrogen on long timescale (I think) Hydrogen bonding with solvent and protein surface is different (reference Julio Fernandez SM work and references therein) “Physics” properties of solvent different (viscosity, diffusivity) Kinetic Solvent Isotope Effect (KSIE); also DIE? “Deuterium isotope effects on noncovalent interactions between molecules.” Wade 1999 Strength of hydrophobic affect changes

4. Prior work with tubulin/MTs and D2O 1935! : Study of cell growth in heavy water. Increased number and length of spindle MTs.Heavy water is toxic to eukaryotic cells(Another paper also claimed deuterium-depleted water affects cell growth) 1989, 1990: Himes group at Kansas: Stabilization of tubulin at 4C Promotion of MT polymerization Stabilization of MTs / reduction of GTPase activity 2008: Chakrabarti (India; out of Himes group) Details of tubulin stabilization by D2O 2005: “D2O promotes assembly and bundling of FtsZprotofilaments by increasing hydrophobic interactions between the protofilaments.” As far as I can tell, no prior work with kinesin and D2O

5. Just a couple theory papers I ran across Voth group paper 2009 Quantum Differences between Heavy and Light Water.Phys Rev Lett. 2008 Aug 8;101(6):065502. Soper AK, Benmore CJ. ISIS Facility, STFC Rutherford Appleton Laboratory Molecular dynamics of hydrogen bonds in protein-D2O: the solvent isotope effect. J Phys Chem A. 2008 Feb 7;112(5):797-802. Sheu SY, Schlag EW, Selzle HL, Yang DY. Department of Life Sciences, National Yang-Ming University, Taipei 112, Taiwan

6. Some more papers and full references (incomplete list of papers I could find in the past few days) Wade D. Deuterium isotope effects on noncovalent interactions between molecules. Chemico-biological interactions. 1999;117(3):191-217. Panda D, Chakrabarti G, Hudson J, et al. Suppression of microtubule dynamic instability and treadmilling by deuterium oxide. Biochemistry. 2000;39(17):5075-81. Available at: http://www.ncbi.nlm.nih.gov/pubmed/10819973. ChakrabartiG, Kim S, Gupta ML, Barton JS, Himes RH. Stabilization of tubulin by deuterium oxide. Biochemistry. 1999;38(10):3067-72. Available at: http://www.ncbi.nlm.nih.gov/pubmed/10074359. SenA, Balamurugan V, Rajak KK, et al. Role of heavy water in biological sciences with an emphasis on thermostabilization of vaccines. Expert review of vaccines. 2009;8(11):1587-602. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19863251. Dougan L, Koti AS, Genchev G, Lu H, Fernandez JM. A single-molecule perspective on the role of solvent hydrogen bonds in protein folding and chemical reactions. Chemphyschem : a European journal of chemical physics and physical chemistry. 2008;9(18):2836-47. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19058277. Cleland WW. The use of isotope effects to determine enzyme mechanisms. Archives of biochemistry and biophysics. 2005;433(1):2-12. Available at: http://www.ncbi.nlm.nih.gov/pubmed/15581561. SchowenKB, Schowen RL. Solvent isotope effects of enzyme systems. Methods in enzymology. 1982;87:551-606. Available at: http://www.ncbi.nlm.nih.gov/pubmed/6294457. SomlyaiG, Jancsó G, Jákli G, et al. Naturally occurring deuterium is essential for the normal growth rate of cells. FEBS letters. 1993;317(1-2):1-4. Available at: http://www.ncbi.nlm.nih.gov/pubmed/8428617.