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]labeledmacromolecules.
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).