A stabilizing action on the nuclear membrane and gel structures, i.e., aster, spindle, and peripheral plasmagel layer of the cytoplasm, can be detected. Prophase and metaphase cells in 80% (v/v) 2H2O remain frozen in the initial state for at least 30 minutes. Furrowing capacity probably is not abolished by 2H2O. The  2H2O-block is released on immersion in 2H2O although cells kept in deuterium-rich media for long periods show multipolar and irregular divisions after removal to 2H2O, and may subsequently cytolyze. The inhibition of mitosis in the fertilized egg is not the only interesting effect of deuterium. The unfertilized egg also responds. It was described by Gross that deuterium parthenogenesis in Arbacia in the following graphic terms: if an unfertilized egg is placed in 2H2O, there appear in the cytoplasm, after half an hour, a number of cytasters. The number then increases with time. If, after an hours immersion in  2H2O, eggs are transferred to normal sea water, a high proportion (80% of the population) raises a fertilization membrane, which gives evidence that activation has occurred.

Deuterium genetics is, for the most part, like genetics itself, conveniently divisible into dipteran mutation studies, the genetics of microorganisms, and miscellaneous studies of which those of Gross and Harding, and Flaumenhaft et al. are examples. The customary procedure in most of the dipteran and bacterial investigations so far reported has been to administer 2H2O to the organism and then to test it for mutation or other chromosomal change. The results obtained by such an investigation have seldom been striking. For example, many researchers found an increase in sex-linked lethals in the sperm of flies that had been exposed to deuterium, either by way of injection into their pupae, or by the inclusion of 2H2O in their food. They introduced 2H2O into Drosophila melanogaster larvae both by feeding and by injection. The males which matured from these larvae were tested for mutation by CIB method. But the test showed no increase in the mutation rate. It was assumed by these scientists that the deuterium which was used in dilute form entered the DNA molecule.

De Giovanni and Zamenhof  have carried out the most comprehensive investigations on the genetic effects of deuterium in bacteria. The results are of considerable interest. For example, they found a several mutants of E. coli, including a so called rough mutant 1/D which is more resistant to 2H2O than its parent strain, were isolated from E. coli grown in 2H2O media. The spontaneous frequency of occurerence of this mutant was 10-4, and the mutation rate could be increased 300-fold by ultraviolet irradiation. This mutant was derived only from the strain E. coli 15 thymidine, and no similar mutant was observed in other strains of E. coli or B. subtilis. By application of a fluctuation test, De Giovanni then was able to show convincingly that this mutation to increased deuterium resistance occurred spontaneously and not in response to the mutagenic effect of 2H2O. Back mutations in some instances do seem to occur at higher rates in 2H2O. Reversion from streptomycin dependence to streptomycin sensitivity in E. coli strain Sd/4, or from thymine dependence to thymine independence in strain 1 occurs with higher frequency in 2H2O, but 2H2O does not cause a discernible increase in mutation in the wild type.

De Giovanni further found that deuteriated purines and pryrimidines had no effect upon the growth and back mutation rates of specific base-requiring strains. Thymine containing deuterium in two of the four nonexchangeable positions adequately supplied the requirement for thymine with no concominant genetic changes. It would appear therefore that the preponderance of the evidence from these studies with bacteria is in favor of the view that 2H2O is not a strong mutagenic agent.

It was reported by many researchers a series experiments designed to test the ability of deuterium to produce mutation and nondisjunction. Deuterium like tritium appear to increase nondisjunction, but either agent separately is less effective than the two acting together. Hughes and Hildreth exposed male flies which had been grown on a 20% (v/v) 2H2O diet to an irradiation of 1000 r. of X-rays. It was found that there was not significant difference in the frequency of observed mutations between 2H2O flies and normal flies subjected to the same radiation.

Tumanyan and Shnol also found no mutagenic effect of 2H2O on recessive and dominant lethal marks in D. melanogaster, inbred line Domodedovo 18. Flaumenhaft and Katz grew fully deuteriated E. coli in 99,6% (v/v) 2H2O with fully deuteriated substrates, and found that the mutation rate after ultraviolet irradiation was distinctly lower than that of nondeuteriated organisms. The simultaneous presence of both deuterium and protium in nearly equal proportions in the constituent molecule of an organism could conceivably create difficulties for the organism since the rate pattern would be seriously distorted. They further found that cells grown in 2H2O and then transferred to 2H2O showed an enhanced susceptibility to ultraviolet irradiation. This suggests that organisms containing both hydrogen or deuterium, but it leaves unanswered the question of why serial subculture in H2O-2H2O media is required for adaptation of many organisms.

Many researchers studied the growth of phage T4 in E. coli cells which were cultivated in media containing various concentrations of  2H2O from zero to 95% (v/v). No significant increase in forward mutation in this phage could be observed, but the rate for reverse mutation was increased, and reached a maximum in phage grown in 50%  (v/v) 2H2O. Although it was reported that a further increase in H2O concentration up to 90% (v/v) producers little augmentation of the reversion index, the actual data presented by Konrad indicates a decided increase in reverse mutation rate in phage exposed to more than 50% (v/v) 2H2O.

There have been carried out a big deal of cytochemical study of fully deuteriated microorganisms grown autotrophically for very long periods in 2H2O (Flaumenhaft E., Conrad S. M., and Katz J. J., 1960a, 1960b). The main conclusion that could be made from these studies is that the nucleus of deuterated cells was much larger than that of nondeuterated cells, and it contained greater amounts of DNA. Also present were much greater amounts of rather widely scattered cytoplasmic RNA within the cells. It was found also, that deuterated cells stained much more darkly for proteins, indicating higher concentrations of free basic groups. Both fluorescence and electron microscopy indicated that deuteration results in readily observable morphological changes. For example, the chloroplast structure of deuteriated plants organisms was more primitive in appearance, less well-differentiated, and distinctly less well-organized. The very interesting conclusion was made, then a low or/and high temperature grown organisms implied the morphological consequences of extensive isotopic replacement of hydrogen by deuterium so that in some respects resemble with the effects produced by reduction or/and increase in temperature of growth.

But, paradoxically as shown numerious studies on biological adaptation to 2H2O, a many cells of bacterial and algae origin could, nevertheless, well grown on absolute 2H2O and, therefore, to stabilize their biological apparatus and the structure of macromolecules for working in the presence of 2H2O. The mechanism of this stabilization nor at a level of the structure of [U-2H]labeled macromolecules or at a level of their functional properties is not yet complitely understood. We still don’t know what possibilities a cell used for adaptation to 2H2O. We can only say, that probably, it a complex phenomenon resulting both from the changes in structural and the physiological level of a macrosystem. That is why there is every prospect that continued investigation of deuterium isotope effects in living organisms will yield results of both scientific and practical importance, for it is precisely. For example, the studies of the structure and the functioning of biolodical important [U -2H]labeled macromolecules obtained via biological adaptaition to high concentrations of 2H2O are most attract an attention of medical scientists as a simple way for creating a fully deuterated forms of DNA and special enzymes could well be working in a certain biotechnological processes required the presence of 2H2O. Secondly, if the structure of fully deuterated proteins may be stabilized in 2H2O in a view of duarability of deuterated bonds, it would be very interesting to study the thermo-stability of [U -2H]labeled proteins for using them directly in processes going at high temperatures.

It would be very perspective if someone could create the thermo-stable proteins simply via deuteration of the macromolecules by growing a cell-producent on 2H2O wit 99% 2H. Third, particular interest have also the studies on the role of primodial deuterium in molecular evolution. The solution of these obscure questions concerning the biological adaptation to 2H2O should cast a new light on molecular evolution in a view of the preferable selection of macromolecules with difined deuterated structures. Thus, the main purpose of the present project is the studies of the structure and the function of fully deuterated macromolecules (particularly DNA and individual  proteins and/or enzymes) obtained via biological adaptation to high concentrations of 2H2O.

            To carry out the studies with fully deuterated macromolecules one must firstly to obtain the appropriate deuterated material with high level of enrichment for isolation of pure DNA and individual proteins to whom the various methods of stable isotope detection further can be applyed. For example, the three-dimentional NMR combined together with the method of X-ray diffraction,  infrared (IR)-, laser spectrometry and circular dichroism (CD) is a well proved method for the studies of the structure and the functioning of [U -2H]labeled macromolecules, and for investigations of various aspects of their biophysical behavior. Taking into account the ecological aspect of using [U -2H]labeled compounds, it should be noted in conclusion, that the preferable properties of applying deuterium for biochemical studies are caused mainly by the absence of radioactivity of deuterium that is the most important fact for carrying out the biological incorporation of deuterium into organism.

2. SCIENTIFIC ACTUALITY OF THE RESEARCH

A special attention is to be given to the investigation of biological adaptation to 2H2O allowing cells to synthesize a deuterated forms of macromolecules (particulary interest have DNA and short-chain individual proteins both with well known amino acid sequence and conformation) with a certain structure allowing their functioning in 2H2O environment.

Firstly, in this connection it would be very interesting to know, how the structure of fully deuterated macromolecules could be changed neganively or positively in a course of biological adaptation to 2H2O requiring the presence of high concentrations of 2H2O in growth media.

Secondly, if a cell will be growing on media containing the stepwise increasing concentrations of 2H2O, for example starting up from zero up to 100% (v/v) 2H2O, will the changes in the structure of [U -2H]labeled macromolecules to be corresponding to the 2H2O content in media and what is a limit concentration of 2H2O when the macromolecular structure keeps a stable constancy and how this fact corresponds with a limit of biological resistance to 2H2O? For answers to these questions a number of modern consideration at the levels of the structure (primary, secondary, tertiary) and conformation of [U -2H]labeled DNA and individual proteins with using the methods of a special sequencing and modifications of deuterated macromolecules combined together with gel electrophoresis method as well as such powerful methods as NMR-spectroscopy to which will be taken a most part of proposed research, X-ray diffraction, IR-, laser- and CD-spectroscopy will be further involved.

An investigation will necessary mainly into the structure of [U -2H]labeled macromolecules in order to find at what level of macromolecular hierarchy a substitution of hydrogen atoms with deuterium ensued the consequence on the differences in the structure and the conformation of macromolecules and, therefore, the functional properties of the macromolecules in 2H2O. In the frames of proposed research the developing of methods of biological adaptation to obtain [U -2H]labeled biological material with high levels of enrichment are also of a big interest. For this purpose the special biotechnological approaches based on using the strains with improved properties when growing on 2H2O for obtaining fully deuterated DNA and individual proteins should be applied for allowing to prepare [U -2H]labeled macromolecules in gram scale quantities.

3. DISCUSSION

3.1. The methods for analyzing the structure and the conformation of [U -2H]labeled macromolecules.

The biological labelling with deuterium is an useful tool for investigating the structure and the conformational properties of macromolecules. The fundamental objectives have meant that living models have retained their importance for functional studies of such biological important macromolecules and can be used to obtain structural and dynamic information about the [U -2H]labeled macromolecules.

The method of X-ray diffraction should be noted as a indespencible tool for determing the details of the three-dimentional structure of globular proteins and other macromolecules (Mathews C. K., van Holde K. E., 1996). Yet this technique has the fundamental limitation that it can be employed only when the molecules are crystallized, and crystallization is not always easy or even possible. Furthermore, this method cannot easily be used to study the conformational changes in response to changes in the molecules environment. 

Other methods, for example IR-spectroscopy, can provide direct information concerning the macromolecular structure. For example, the exact positions of infrared bands corresponding to vibrations in the polypeptide backbone are sensitive to the conformational state (a helix, b sheet et.) of the chain (Campbell I. D., and Dwek R. A., 1984). Thus, the studies in this region of the spectrum are often used to investigate the conformations of protein molecules.

Although, IR-, and absorption spectroscopy can be helpful in following molecular changes, such measurements are difficult to interpret directly in terms of changes of secondary structure. For this purpose, techniques of circular dichroism involving polarized light have become important (Johnson W. C., 1990). For example, if a protein is denatured so that its native structure, containing a helix and b sheet regions, is transformed into an unfolded, random-coil structure, this transformation will be reflected in a dramatic change in its CD spectrum. Circular dichroism can be used in another way, to estimate the content of  a helix and b sheet in native proteins. The contributions of these different secondary structures to their circular dichroism at different wavelenghths are known, so we may attempt to match an observed spectrum of protein by a combination of such contributions.

Although circular dichroism is an extremely useful technique, it is not a very discriminating one. That is, it cannot, at present, tell us what is happening at a particular point in a protein molecule. A method that has the great potential to do so is nuclear magnetic resonance. This advance now make it possible to use NMR to study a big varieties of DNA and proteins with more complex biological functions functioning in natural liquid environment. Often these proteins have more than one domain and more than one site of interaction. Allosteric systems, receptors and small molecule ligand-modulated DNA-binding proteins and DNA are some examples of the molecular systems which can now be analysed in molecular detail. For example, due to the development of two-dimentional Fourier transformation techniques, NMR spectroscopy has become a powerful tool for determining the protein structure and conformation (Fesic S. W. and  Zuiderweg E. R., 1990).

3.2. The preparation of [U- 2H]labeled macromolecules.

Through technical advances of biotechnology, many macromolecules, for example a certain individual proteins are successfuly cloned and can be obtained in large quantities by expression in microbial and/or mammalian systems, so that an ever-increasing number of individual [U- 2H]labeled macromolecules from various biological objects are becoming commercially available. It should be noted, however, that the application of various methods for the preparation of [U -2H]labeled macromolecules (chemical or biosynthetical) often results in obtaining the forms of molecules with different number of protons substituted by deuterium, the phenomenon that is known as heterogenious labelling, so that the special methods for the preparation of [U -2H]labeled macromolecules should be applyed to minimaze this process. For example, the proteins containing only deuterium atoms in polypeptide chain of macromolecule can be produced biotechnologically with using the special genetically constructed strains of bacteria carrying the mutations of geens excluding the metabolic exchange between the parterns of unlabeled intermediators during the biosynthesis of [U -2H]labeled macromolecules.

I may briefly indicate three possibilities for deuterium enrichment:

(1) to grow the organism on a minium salt medium with content of 2H2O 99% 2H; 

(2) To grow the organism on a medium supplemented with 99% 2H2O and [U -2H]labeled amino acid mixture.

(3) the isotopic exchange of susceptible protons in amino acid residues already incorporated into protein.

Method 1 is very useful for the preparation of [U- 2H]labeled macromolecules if only applyed strains of bacterial or different origin could well be grown on minimal media in the presence of high concentrations of 2H2O. Very often in this case the biological adaptation to 2HO is required. Method 2, while generally applicable, is limited by the difficulty and expense of preparing fully deuterated amino acid mixtures from algae grown on 2H2O. However, recently we proposed to use a fully deuterated biomass of methlotrophic bacterium B. methylicum  with protein content about 55% (from dry weight) obtained via multistep adaptaition to 98% (v/v) 2H2O and 2% (v/v) [U-2H]MetOH as growth substrates for growing the other bacterial strains to prepare a gram quantities of [U -2H]labeled amino acids, proteins and nucleosites with high levels of enrichment (90.0-97.5% 2H) (Mosin O. V., Karnaukhova E. N., Pshenichnikova A. B.; 1994; Skladnev D. A., Mosin O. V., et all; 1996; Shvets V. I., Yurkevich A. M., Mosin O. V.; 1995).

 Method 2 is also necessary when the organism will not grow on a minimal medium as it was in the case with the applying the bacteria requiring the complex composition media for their growth. This approach will also be necessary for the labeling of proteins expressed in systems other than E. coli (e.g. yeast, insect, and mammalian expression systems) which may be important for the proper folding of proteins from higher organisms. Since the protons of interest in proteins are most often carbon bound and thus do not exchange under mild conditions, method 3 is severely limited by stability of proteins under the harsh conditions necessary for (1H-2H) exchange.

4. ADAPTATION TO 2H2O AND BIOPHYSICAL PROPERTIES OF [U -2H]LABELED MACROMOLECULES

FIGURE

The imaginary principle of realization of biological adaptation

    I                                                           II

1 works           2 not work    not work           2 works     

ordinaryenvironment(A)                   2H2O (B)

4.1. The main hypothese.

We proposed that a cell theoretically could in principle synthezise a big number of forms of [2H]labeled macromolecules with somewhat different structures and conformations, so that a cell could easily select a preferable one from al these species in a course of adaptation to 2H2O, that is the best suitable namely for that conditions. A simple imaginary principle I am going to discuss here perhaps somewhat may explain this probable mechanism. Let us suppose, for example that there are at least two imadinary structural systems - ordinary (normal) system call it a system 1 and unordinary (adaptive) system 2 (see a Figure above). Supporse, that the environment is a homoginious substanse and compose from ordinary substance A (H2O) (situation 1). The necessarely condition for the normal working of this model in natural H2O environment is that system 1 works and system 2 stay in background (situation 2). Supporse, that the environment have changed for substance B (2H2O). Then the system 2 will work, while the system 1 will stay in background (situation 2). When environment will be the natural again, the system 1 will begin the work again, while the system 2 will stay in background. Admitt, that the two systems both presented at the time being and could be regulated in such way that they may switch bitween each other during the working so that the model system does not undergoing the considerable alterations.

4.2. Phenomenon of biological adaptation to 2H2O.

Our research has confirmed, that ability to adaptation to 2Í2Î is differed for various species of bacteria and can to be varried even in frames of one taxonomic family (Mosin O. V. et al., 1996a, 1996b).From this, it is possible to conclude, that the adaptation to 2Í2Î is determined both by taxonomic specifity of the organism, and peculiarities of the metabolism, as well as by functioning of various ways of accimilation of hydrogen (deuterium) substrates, as well as evolutionary level, which an object itself occupies. The less a level of evolutionary development of an organism, the better it therefore adapts itself to 2H2O. For example, there are halophilic bacteria that are being the most primitive in the evolutionary plan, and therefore, they practically not requiring to carry out a special adaptation methods to grow on 2Í2Î. On the contrary, bacills (eubacteria) and methylotrophs (gram-negative bacteria) worse adapted to 2Í2Î.

At the same time for all tested cells the growth on 2H2O was accompanied by considerable decrease of a level of biosynthesis of appropriated cellular compounds. The data obtained confirm that the adaptation to 2Í2Î is a rather phenotypical phenomenon, as the adapted cells could be returned to a normal growth and biosynthesis in protonated media after lag-phase (Mosin O. V. et al., 1993).

However, when the adaptive process goes continuously during the many generation, the population of cells can use a special genetic mechanisms for the adaptation to 2H2O. For example, mutations of geens can be resulted in amino acid replacements in molecules of proteins, which in turn could cause a formation of a new  isoenzymes, and in the special cases - even the anomal working enzymes of a newer structure type. The replacements of these compounds can ensure a development of new ways of regulation of enzymic activity, ensuring more adequate reaction to signals, causing a possible changes in speeds and specifity of metabolic processes.

Despite it, the basic reactions of metabolism of adapted cells probably do not undergo essential changes in 2Í2Î. At the same time the effect of convertibility of growth on Í2Î/2Í2Î - does not theoretically exclude an opportunity that this attribute is stably kept when cells grown on 2Í2Î, but masks when transfer the cells on deuterated medium.

However, here it is necessary to emphasize, that for realization of biological adaptation to 2H2O the composition of growth medium plays an important role. In this case it is not excluded, that during the adaptation on the minimal medium, containing 2Í2Î there are formed the forms of bacteria, auxotrophic on a certain growth factors (for example amino acids et) and thereof bacterial growth is inhibited while grown on these media. At the same time the adaptation to 2Í2Î occurs best on complex media, the composition of which coul compensate the requirement in those growth factors.

It is possible also to assume, that the macromolecules realize the special mechanisms, which promote a stabilization of their structure in 2H2O and the functional reorganization for best working in 2Í2Î. Thus, the distinctions in nuclear mass of hydrogen atom and deuterium can indirectly to be a reason of distinctions in synthesis of deuterated forms of DNA and proteins, which can be resulting in the structural distinctions and, hence, to functional changes of  [2H]labeled macromolecules. Hawever, it is not excluded, that during incubation on 2Í2Î the enzymes do not stop the function, but changes stipulating by isotopic replacement due to the primary and secondary isotopic effects as well as by the action of 2Í2Î as solvent (density, viscosity) in comparison with Í2Î are resulted in changes of speeds and specifics of metabolic reactions.

In the case with biological adaptation to 2H2O we should inspect the following types of adaptive mechanisms:

1. adaptation at a level of macromolecular components of cells: It is possible to allocate mainly two kinds of such adaptation:

(a). Differences of intracellular concentration of macromolecules;

(b). The forming in 2H2O the deuterated macromolecules with other conformations, which could be replaced the ordinary protonated macromolecules synthesized by cells in normal conditions.

We suppose, that in principle, any protein macromolecule could adopt an almost unlimited number of conformations. Most pilypeptide chains, however, fold into only one particular conformation determined by their amino acid sequence. That is because the side chains of the amino acids associate with one another and with water (2H2O) to form various weak noncovalent bonds. Provided that the appropriate side chains are present at crucial positions in the chain, large forces are developed that make one particular conformation especially stable.

These two strategies of adaptation could possible to be distinqueshed accordinly as "quantitative" and "qualitative" strategies;

2. adaptation at a level of microenvironment in wich macromolecules are submerged: the essence of this mechanism is, that the adaptive change of structural and conformational properties of [2H]labeled macromolecules is conditioned both by directional action of 2H2O environment on a growth of cells and by its physico-chemical structure (osmotic pressure, viscosity, density, ðÍ, concentration of 2H2O).

2H2O appeared to stabilize the plasmagel structure of biological microenvironment. The external pressure required to make the cells assume a spherical shape increased 3.6 kg/cm2 for each per cent increase in the presence of 2H2O. It thus seems well established that deuteration can affect the mechanical properties of cytoplasm, and that this factor must be taken into account in assessing the consequences of isotopic substitution of macromolecules. In model experiments with gelatin structure, it was demonstrated that in 2H2O there is a greater protein-protein interaction than in H2O (Scheraga J. A; 1960).

A progressive increase in the melting temperature of the gel in 2H2O is observed accompanied by an increase in the reduced viscosity. That 2H2O can have marked effects on the physical properties of proteins has been known for some time. Consequently it is natural to attribute changes in the mechanical properties of cell structures induced by 2H2O to protein response. Nevertheless, the effects of deuterium on proteins, while real, must be only a partial explanation of the situation. The interaction of proteins with solvent water is extraordinarily complex, and the exact nature of the protein is crucial in determining the magnitude of changes resulting from the replacement of H2O by  2H2O.

This mechanism has extremely large importance and supplements the macromolecular adaptation; 3. adaptation at a functional level, when the change of an overall performance of macromolecular systems, is not connected with a change of a number of macromolecules being available or with the macromolecules of their types. Adaptation in this case could provide the changes by using the already existing macromolecular systems - according to requirements by this or that metabolic  activity.   

TABLE

Some physical constants of ordinary and heavy water

Physical constant

H2O

2Í2Î

Density, d20 (g/c.c)

0,9982

1,1056

Molecular volume, V20 (ml/mole)

18,05

18,12

Viscosity m20 (centipose)

1,005

1,25

Melting point (0C)

0,1

3,82

Boiling point (0C)

100,0

101,72

Temperature of maximum density (0C)

4,0

11,6

Ion product (25 0C)

10-14

0,3x10-14

Heat of formation (cal/mole)

-68,318

-70,414

Free energy of formation (cal/mole)

-56,693

-58,201

Entropy (e.u/mole)

45,14

47,41