The research proposal is based on a combination of microbiological and genetical research combaining with investigation of the structure and the conformation of macromolecules with using mainly NMR-technique applyed together with X-ray diffraction, IR-, laserand CD-spectroscopy: 3.1. Study of certain aspects of synthesis of [U -2H]labeled macromolecules by cells adapted to 2H2O; 3.2. Investigations of the structural and the functional properties of fully deuterated macromolecules; 3.3. The preparation of fully deuterated macromolecules (particularly individual proteins) with using adapted cells.

First, in frames of the biological research with deuterium the studies of incorporation of deuterium into the macromolecules are necessary. A biosynthetic introduction of deuterium in conjunction with mass spectrometry EIMS[1], FAB[2], and NMR[3] technigue has been used to study biological convertion of low molecular weight substrates ([U-2H]MetOH and 2H2O) to the amino acids, proteins and nucleotides by various strains of microbial and algae origin (green microalgae; Chlorella sp. and Dunaliella salina Teod., blue green algae Spirulina sp., halophilic bacterium Halobacterium halobium[4], methylotrophic bacteria; Methylobacillus flagellatum and Brevibacterium methylicum[5], bacills; Bacillus subtilis and Bacillus amiloliqufanciens[6] (Mosin O. V., Karnaukhova E. N., et all., 1993; Mosin O. V., Skladnev D. A., et all., 1996a). The influence of 2H2O and other [U -2H]labeled substrates (e.g. [U-2H]MeOH) on several growth characteristics of the microorganisms (time of generation, lag-phase, yield of biomass) and biosynthetic activity was also investigated in those studies.

Second, these microorganisms were investigated to growth and biosynthesis on media containing high concentrations of 2H2O (90-98 v/v.%[7]). For example, an adaptation method for the preparation of [U -2H]labeled cell protein highly enriched with 2H (93.0% 2H) based on using a methylotrophic bacterium B. methylicum grown on dM9-medium containing 2% (v/v) [U-2H]MeOH and 98% (v/v) 2H2O was developed and successfuly tasted on most microoorganisms (Mosin O. V., et all., 1996d)[8]. The method called a “step by step” adaptation method, consists in incubation of cells by the plating on agaric media with 2 v/v. [U-2H]MeOH and increasily content of 2H2O (starting from ordinary water, then taking 24.5, 49.0, 73.5 up to 98% (v/v) 2H2O) with following selection of cells colonies grown on those media (the colonies resistant to action of 2H2O were selected)[9]. As obtained the cells stored asseptically in 50% glyserol solution (in 2H2O) at -140C to prevent a possible dillution with environmental H2O. The phenomenon of adaptation may implay a considerable alteration in the proton-deuteron composition of the DNA and proteins and those effects may course a visible hindering of growth and proliferation in the presence of 2H2O[10]. On the other hand the results indicated, that decreasing of cell production of B. methylicum (not more than 1.5 fold) in highly deuterated media was accompanied by additional increasing of the lag-phase as well as by increasing of the cell generation time. The method developed is very suitable for the preparation of gram quantities of deuterated biomass from that a many valuable [U -2H]labeled macromoleculs with high levels of enrichment could be extracted. A second advantage is that the [U -2H]labeled biomass with protein content approximately 55.0% (from dry weight) obtained via multistep adaptaition could be used after the hydrolsis[11] as a mixture of [U -2H]labeled substrates for growing the other strains.

Third, various microorganisms were investigated for the efficient biosynthetic preparation of [U -2H]labeled cell compounds. For example, [U -2H]labeled amino acids[12], proteins[13] and nucleosites[14] with levels of enrichment (90.0-97.5% 2H[15]) were obtained from adapted cells of Chlorella sp. (cell protein) (Egorova T. A., Mosin O. V., et all., 1993), H. halobium (bacteriorhodopsin) (Mosin O. V., Karnaukhova E. N., Pshenichnikova A. B.; 1994), B. methylicum (cell protein and phenylalanine)(Skladnev D. A., Mosin O. V., et all; 1996), and B. subtilis (inosine) (Shvets V. I., Yurkevich A. M., Mosin O. V.; 1995) and isolated in gram quantities with using a big varaity of standard methods including centrifugation, precipitation, crystallization, extraction, electrophoresis as well as an ion exchange chromatography and a reverse-phase HPLC method adaptable for the isolation of [U -2H]labeled compounds [16] (Egorova T. A., Mosin O. V., et all., 1993)

Finally, to follow an early started research on the structure and the function of 2H-labeled membrane protein bacteriorhodopsin, the efficiency of site-specific incorporation of certain amino acids important for the forming the three-dimentional structure of bacteriorhodopsin as [2,3,4,5,62H5]phenylalanine (90% 2H), [3,52H2]tyrosine (96% 2H), [2,4,5,6,7-2H5]tryptophan (98% 2H) into the protein as well an attempt to obtain the [U -2H]labeled bacteriorhodopsin with high level of enrichment (90% 2H) were studied (Mosin O. V., 1996b). For this aim we used as growth substrates for the preparation of [U -2H]labeled bacteriorhodopsin[17] the hydrolysate of [U -2H]labeled biomass of B. methylicum obtained through the adaptation of the microorganism on a medium with 2% (v/v) [U2H]MeOH and 98% (v/v) 2H2O (Mosin O. V., et all., 1996a, Mosin O. V., et all., 1996a).


In the present investigation the following key questions (points) will be considered: 3.1. The studies of deuterium incorporation into biological macromolecules issuing the proposed chandes in the structure and the conformation of the [U -2H]labeled macromolecules; 3.2. The efficiency of using the adapted cells for the technological preparation of fully deuterated macromolecules.


The main goal of the project is the biological adaptation to 2H2O for the growing of cells in the presence of high concentration of 2H2O as a main stage to obtain a gram quantities of biological deuterated material to isolate [U -2H]labeled DNA and individual proteins for further their structural and functional studies.

The concrete goal is a). the NMR-research combined (as far as it is possible) with other complimentary methods for analyzing the structure and the conformation of [U -2H]labeled macromolecules (particulary DNA and short-chain individual proteins with well known protonated structure, but obtained from the specifically producents adapted to high concentrations of 2H2O; b). The evaluation of isotopic enrichment of [U -2H]labeled macromolecules.

The second point is the study of functional properties of [U -2H]labeled macromolecules.


I plan to investigate the following questions: 5.1. How cells by the changes in the structure and the conformation of fully deuterated macromolecules could, nevertheless, adapt the biological apparatus to working in the presence of high concentration of 2H2O and 5.2. How functionate the deuterated macromolecules in 2H2O?

The main objects will be the different kinds of biological objects synthesizing a big quantities of intracellular DNA and proteins (in a case with proteins the specially cells or/and strains of bacteria producing the individual proteins should be applied) both of microbial, plants and/or animal origin, that are available elsewhere. Cells will be adapted to highly concentrations of 2H2O by “step-by-step” adaptation method developed ealier so that to obtain the preparative quantities of fully deuterated biological material (biomass)[19] from whom the neccessary fraction containing the [U -2H]labeled macromolecules will be isolated[20] and then separated on group of individual protein and DNA to allow the macromolecules retain its native properties and function unchanged. The structure of the deutereted macromolecules will be studied together by applying a special sequencing methods, modification and/or degradation by chemical agents (Edman sequencing in case with proteins and Maxam-Gilbert sequencing in case with DNA) and/or enzymes cleavage (trypsin, chymotrypsin in case with protein cleavage and restriction enzymes such as endoand exo-nucleases in case with DNA) and gel electrophoresis so that the information about the conformation of [U -2H]labeled macromolecules obtained from powerful NMR-technique combined together with X-ray diffraction, IR-, and CD-spectroscopy (which are only complimentary methods to our studies) could be then true estimated and verified with a knowledge about the primary structure of [U -2H]labeled macromolecules.

Genetic manipulation with deuterated cells is the subject of special research. In particular I would like to get in touch with the facultative methylotrophic bacteria B. methylicum adapted to growth on minimal salt dM9-medium containing 98% (v/v) 2H2O and 2% (v/v) [U-2H]MetOH. I plan to use this bacterium together with other bacterial strains for construction a gene expressing systems to prepare the individual [2H]labeled proteins, which now are out of this project.

When all above listed questions will be studied and successfully cleared up, quantitative preparation of individual [U -2H]labeled proteins will be considered to evaluate technological parameters of large scale isotopic production. Ecological purity of such production and utilization of wastes will be considered in frames of the complex research[21].


[1] For mass spectrometric evaluation of levels of enrichment the derivatization of amino acids to the volatile methyl esters of dansyl N-(Dns)-and carbobenzoxy(Z)-amino acids was used. For the preparation of Dns-amino acids to 200 mg of liophylised sample of growth medium or protein hydrolyzate in 2 ml. 2 N. NaHCO3 (0.01 mol) solution with рН 9-10, fractional portions of 50 mg. DnsCl (3x10-4 mol) in 2 ml acetone solution were added. A reaction mixture was kept under vigorous stirring at 400C. After 60 min the reaction was completed, 2 N. HCl was added to the solution to adjust pH 3.0, after that the Dns-amino acids were extracted ith 15 ml. of ethyl acetate (3x5 ml). The combined extracts were dried over sodium sulphate and concentrated in vacuum. The yield of Dns-amino acids was 89-95%. For the preparation of Z-amino acids to a solution containing 200 mg of liophylised sample of growth medium or protein hydrolyzate in 2 ml. 4 N. NaOH (4x10-3 mol), 100 mg ZCl (3.5x103 mol) and 2 ml. 4 N. NaOH (4x10-3 mol) were simultaneously added over a 40 min time period. The reaction mixture was kept at 00C under vigorous stirring. The pH was maintained between 10.0 and 12.0 by adjusting the flow rate of ZCl and NaOH. After the reaction was completed, the solution was extracted with 0.5 ml. of pentane to remove the excess of ZCl followed by cooling and acidification of the aqueous layer to congo red with 4 N. HCl. Then, the Z-amino acids were extracted ith 15 ml. of ethyl acetate (3x5 ml). The combined extracts were dried over sodium sulphate and concentrated in vacuum. The yield of Z-amino acids was 69-75%. These derivatives of amino acids were then preparative separated by the method of reverse phase HPLC performed on a Knauer chromatograph (Germany) with 150 x 3.3 mm Separon SGX C18 18.7 mm (Czech Republic) using mobile phase: (A) acetonitrile-trifluoracetic acid (100:0,1-0,5, v/v); (B) acetonitrile (100 v/v); gradient elution: from 20% В to 100% В for 30 min, at 100% В for 5 min, from 100% В to 20% В for 2 min, at 20% В for 10 min so that the yields of individual amino acids were 68-89%, chromatographic purity 96-99%. Mass spectrometry EIMS was performed on Hitachi MB 80 spectrometer (Japan) at ionizing energy 70 eV and an ion source temperature of 180 0C.

[2] Mass spectrometry FAB was performed on Hitachi MBA spectrometer (Japan) on glyserol template at potential 5 kV and ion streem 0.6-0.8 mA.

[3] Spectra PMR were registered in 2H2O on a Bruker WM-250 spectrophotometer (Germany) with working frequency 250 mHz, as the internal standard used tetramethylsilane (0.005 p.m.).

[4] In a case with H. halobium ЕТ 1001 a growing has carried out on a medium prepared from 100% (v/v) of 2H2O and supplemented with hydrolysate of [2H]labeled biomass B. methylicum obtained via “step-by-step” adaptation to medium containing 98% (v/v) 2H2O and 2% (v/v) [U2H]MetOH. Its composition (in g/liter): hydrolysate of [2H]labeled biomass B. methylicum 30; NaCl 250; MgSO4 x7H2O 20; KCl 2; NH4Cl 0.5; KNO3 0.1; Na cytrate 0.5; MnSO4x 2H2O 3x10-4; CaCl2 x6H2O 0.065; ZnSO4 x7H2O 4x10-5; FeSO4 x7H2O 5x10-4; CuSO4x5H2O 5x10-5; glyserol 1; growth factors (biothine 0.1x10-3; folic acid 10x10-3; vitamin В12 0.02x10-3). In usual for this bacterium growth conditions (370С, on light) the cells could synthesize a violet pigment that was under all spectral characteristics (UV-spectroscopy) resembled a native bacteriorhodopsin.

[5] In the frames of the research an opportunity of adaptation of various straines to growth on media with high content of 2H2O was investigated. For this purpose the representatives of various taxonimic groups of methylotrophic bacteria were employed including a leucine producing mutant of obligate methylotroph M. flagellatum KT, realizing the KDPGA/TA variant of ribulose monophosphate (RuMP) cycle of C1-assimilation and a phenylalanine producing facultative methylotroph B. methylicum BКПМ 5962, assimilating methanol as a source of carbon and energy via the RuMP cycle. Both microorganisms were mutagenized with the nitrosoguanidine (50 mg/ml) at the laboratory for Genetics of methylotrophs at Institute for Genetics and Selection of Industrial Microorganisms (Russia). The leasions were made to eliminate negative control over the main products in biosynthesis. The cells with altered amino acid metabolism were isolated on media supplemented with the analogues of phenylalanine (meta-fluoro-phenylalanine) and leucine (10 mg/l). The auxotrophic mutants obtained were used for the preparation of [U -2H]labeled phenylalanine and [U -2H]labeled leucine. The amounts of other metabolites as alanine, valine and leucine/isoleucine could be also produced and accumulated in growth medium in addition to the main product of biosynthesis. For realization of adaptation of these methylotrophic bacteria a step character of increase of 2H2O concentration in growth media was chosen, as we have assumed, that gradual habit of organism to 2H2O will render the favorable effect on the growth rate. Its composition (in g/liter): KH2PO4 3; Na2HPO4 6; NaCl 0.5; NH4Cl 1; supplemens of 10 mg/l leucine or isoleucine (depending of the strain used)). The physiological concentrations of exogenic [U2H]MetOH was, as it is usual for these strains in the limits of 2% v/v). It was found, however, that strain M. flagellatum has displaied an increased sensitivity to 2H2O: inhibition of growth rates of bacteria was observed when concentration of 2H2O in media was 73.5% (v/v). All attemps to growth of the bacteria on media containing higher concentration of 2H2O were failed. In this connection, this concentration of 2H2O wasassumed to be the high limit of 2H2O-tolerance for the strain M. flagellatum. On the contrary, we can easily adapt another methylotroph B. methylicum to media containing 98% (v/v) 2H2O and 2% (v/v) [U2H]MeOH.

[6] The ability to growth on 2H2O the various strains of baccils B. subtillis (his-, tyr-, ade-, ura-), and B. amyloliquefaciens (ade-, ura-), served as producents of inosine and thymidine was investigated. We have assumed, that the delay of growth of bacteria on the minimum medium, containing high concentrations of 2H2O could be caused by the occurrence of auxotrophisity of the bacterium on additional groth factors, e.g. amino acids. To check up this assumption, hereinafter we used a complex media, containing peptones or hydrolyzates of methylotrophic bacteria. As it was supposed, both strains it was possible to adapt to 2H2O by simple plating of cells on solid agaric media (2% -agarose), prepared from 100% (v/v) of 2H2O and supplemented with hydrolysate of [U -2H]labeled biomass B. methylicum, obtained via “step-by-step” adaptation to medium with 98% (v/v) 2H2O and 2% (v/v) [U-2H]MeOH. Its composition (in g/liter): glucose 120; hydrolysate of [U -2H]labeled biomass B. methylicum 25; NH4NO3 30; MgSO4 x7H2O 20; chalk 20. The cells have at once displayed the normal growth on 2H2O -media (judging by physiological and morphological properties of cells). Both straines B. subtilis and B. amyloliquefaciens where found to be kept a decreased (in 3-4 times) a level of production on [U -2H]labeled inosine and thymidine (3.9 and 3.0 grams per 1 liter of growth medium accordingly).

[7] To create a high isotopic content of 2H2O in growth media, 2H2O with trade marked isotopic purity 99.9 at.% 2H, was used. Futhermore, 2H2O was freshly distilled from KMnO4. The deuterium content of used 2H2O was verified with using NMR-technic so that the true content of deuterium of 2H2O was found to be nor more then 97.0% . The phosphate salts were several times preliminarily crystallyzed in 99.9 at% 2H2O and dried up in vacuum before using (the true content of deuterium in growth media after the sterillisation measured by the method of NMR was nevertheless less smaller on 8-10% then isotopic purity of initial 2H2O). The experiments were carried out in destilled dry solvents: [U-2H]MeOH was destilled from Mg (5 g/l), and stored on molecular sieves 3A0, acetone was destilled from P2O5, chlorophorm was destilled from P2O5 and stored on molecular sieves 3A0, diethyl ether was destilled from CaCl2 and stored on molecular sieves 4A0. L-[2,3,4,5,6-2H5] -Phe (90 % 2H), L[3,5-2H2] -Tyr (96 % 2H) and L[2,4,5,6,7-2H5] -Trp (98 % 2H) were obtained using a standard isotopic exchande (1H-2H) procedure (Griffiths D.V., 1976; Matthews H. R., 1977). All other chemicals were of reagent grade.

[8] Strains of Chlorella sp, Dunaliella salina Teod. and Spirulina sp. were adapted through the growth on liquid media containing increaselly 2H2O content. No special selection was made for these straines. For straines H. halobium ET 1001, M. flagellatum, B. methylicum, B. subtilis and B. amiloliqufanciens adaptation to 2H2O was accomplished using agaric media (2%-agarose) containing 100% (v/v) 2H2O with following selection of cells could be well grown and reproduced on these media. For that the simple plating procedure of cells on agaric media with 100% (v/v) 2H2Owas used for strain H. halobium ET 1001 and B. subtilis and B. amiloliqufanciens as well as a special “step-by-step” adaptation technique using agaric minimal salt media containing the increasily content of 2H2O was applied for strain B. methylicum. For strain H. halobium ET 100 adaptation was carried out on 2H2O-medium in the presence of light.

[9] I plan to use this method further for carry out the adaptation of other strains of bacteria.

[10] Each highly deuterated medium containing about 0.1-0.5% and even more so called remanent protons. When protonated cells were used for growth on 2H2O-media, cells taken up the small amount of these remanent protons from media, so that the intracellular content of deuterium was little decreased when at the same time the content of deuterium in media was proportionally increased. On the contrary, when we used already deuterated (adapted) cells for growth in those conditions, cells preferably taken up deuterium and the content of deuterium inside the cell was little increased, when the content of deuterium in media was decreased.

[11] For the preparation of the mixture of [U -2H]labeled growth substrates, the methylotrophic bacterium B. methylicum was grown in completely deuterated mineral medium dM9 in mass culture as described above. Deuterated cells were precipitated by centrifugation (280 g, 10 min), washed once with 2H2O and stored at -14 0C. Periodically, 10 g (wet weight) portions are thawed, suspended in 0.5 N. 2HCl solution (in 2H2O) and autoclaved at 120 0C for 30 min. After adjusting pH till 7.0-7.2 with KOH, the hydrolysate was used as a substrate for the growth of B. subtilis and B. amiloliqufanciens.

[12] For the isolation of secreted [U -2H]labeled amino acids from growth media, to five grams of lyophilized growth media 40 ml of iso-PrOH was added, acidified to pH 2.0 using 5 N. solution of 2HCl (in 2H2O). The salts and other purities were removed by centrifugation (300 g, 10 min) and the supernatant was then evaporated, individual amino acids were recrystallised from EtOH or n-ButOH. Individual amino acids were also isolated from growth media as Dns,-and Z-derivatives with a yields of 68-89% and a purity of 96-99%.

[13] [U -2H]Labeled proteins were isolated as individual ones (bR) as well as total protein. For the isolation of total protein the deuterated cells were suspended in 2H2O and collected by centrifugation (600 g, 5 min). The washing procedure was repeated twice. The cells were sonificated at 35 kHz for 2 min (4x30 sec) while in an ise saline bath for cooling. Cell material was precipitated by centrifugation, lipids were removed from the wet pellet (about 5-6 mg of dry weight) by extraction with a chloroform-MetOH (1:2, v/v) mixure. For the analyical purposes, the resulting delipidized protein was dried under reduced pressure and hydrolyzed in 4 N. Ba(O2H)2 (in 2H2O) or in 6 N. 2HCl (in 2H2O) in sealed ampules at 110 0C for 24 h. After cooling the ampules were opened, the contents were dissolved in one volume of hot dist. water and filtrated. In case with Ba(OH)2 hydrolysis the pH of the solution was adjusted to 7.0 by addition of 2 N. H2SO4. The hydrolizates were evaporated to dryness and the resulting mixture of [U -2H]labeled amino acids was derivatized with DnsCl and ZCl.

[14] For the isolation of [U -2H]labeled inosine from growth medium a MetOH solution in 2H2O (50 v/v %, 20 ml) was added to a lyophilized growth medium, containing inosine. The mixture was allowed to - 4 0C and after 10 h purities of protein were precipitated and removed by centrifugation (300 g, 10 min). MetOH was evaporated under reduced pressure. The resulting mixture was dissolved in 2 H2O (30 ml) and 5 g of activated carbon was added. After keeping the mixture for 24 h at -4 0C, the inosine, eluting with ammonia, was concentrated and twice recrystallized from MetOH. The purity of the product was judged by using controls of normal inosine, and running mixed chromatograms with graded amounts of the neighboring nucleosides.

[15] Under the term of levels of enrichment it is meant the number of hydrogen atoms (bind with carbon atoms) in molecule could be substituted on deuterim de novo (easily exchangeable on deuterium in 2H2O protons at amino -NH2 groups, as well as protons at -OH, -SH, -NH, and -COOH groups in molecules were not discussed).

[16] To exclude the dilution with hydrogen from H2O-environment, all reactions for isolation of [U -2H]labeled compounds from biological material were carried out in the presence of [U -2H]labeled substrates and chemicals with high levels of isotopic purity.

[17] Cells, received after the growth of strain H. halobium are precipitated in centrifuge (300 g, 10 min.), then twice washed in distillar water. The pellet was destroyed by sonification at 35 kHz 3 min (6x30 s) cooling on ice bath and again centrifugated. The pigments were removed by low temperature extraction with acetone-heptane (1:1, v/v, 3x10 ml), lipids-by extraction with with chloroform-MetOH (1:2, v/v, 3x10 ml). The remaining material was washed once in distillar water and dried in vacuum. A dry pellet (30-40 mg) were suspended in 100 ml of buffer tris-HCl (рН 7.5), added 1 mg DNA-ase and incubated a mixture for 5-6 h. at 370C. After that the volume was diluted with distillar water up to 200 ml, and incubated for 15 h. at 40C. The supernatant was many times washed by water.The control of purity of purple membrane (PM) was carried out on spectrophotometer Beckman DU-6 (USA) using a specific extinctions of absorption D280nm/D568nm (e280=1,1 x 10-4Мсм and e568=6,3 x 10 -5Мсм). The isolaion of BR consists in further delipidization of the PM yielding in apoprotein, which is hydrolysed either in 6 N. 2HCl (in 2H2O) or/and in 4 N. Ba(OH)2. In the first step of the procedure 4-5 mg of PM was suspended in 0.5 ml of 0.2% (v/v) SDS solution, and stirring in the dark for 24 h at room temperature in glass vials. After the protein was precipitated with 2 ml of MetOH, the pellet was separated and washed twice with 2 ml of dist. water and centrifuged (300 g for 30 min at 4 0C).

[18] Since the aims of present proposal are very large and variable, I suggest firstly and globaly to study the structure of fully deuterated proteins, so that to carry out in second step the research concerning with the structure of DNA.

[19] Previously adapted strains of methylotrophic bacteria, bacills and halophilic bacteria will also be used.

[20] For the isolation of individual protein depending on its properties it should be applied the methods of extraction in salt solutions, acid treatment fractionation, the method of detergent solubilization (SDS, et.), the precipitation of protein by treatment of organic solvents (MetOH, EtOH, acetone), precipitaition in isoelectric poit pH, as well as salting-out method with using salt anions for example (NH4)2SO4 and dialysis or filtration through fine-pored granuled gels with following gel chromatography method are very reasonable. For the isolation of DNA the commonly used methods of SDS treatment of crude fraction of DNA with extraction of DNA by phenol-chlorophorm (1:1) mixture and following isolation of pure DNA by the centrifugation in step increasing gragient concentration of CsCl should be used.

[21] The process for the preparation of [U -2H]labeled macromolecules technologically is simple and could be considered as without wastes. [U -2H]Labeled biomass of methylotrophic bacterium B. methylicum obtained from multiadaptation process to 2H2O and after the hydrolisis should be firstly used directly for first fermentation of strain producent. [U -2H]Labeled biomass of the strain producent once obtained will be used after the hydrolisis for further fermentations of this strain again, so that the strain will be grown on its own biomass many times. 2H2O in composition of growth media obtained after fermentation in the turn could be separated and redistillated for the using again in the composition of growth media. The second point, is that the adapted cells could be further used for biological utilization of radioactive wastes obtained from nuclear reactors containing purities of 2H2O and 3H2O.