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@O.V.Mosin
The role of deuterium in molecular evolution

1. SUMMARY
The role of deuterium in molecular evolution is most interesting question
of nowdays science comprises two points mainly: the evolution of deuterium
itself as well as the chemical processes going with participation of
deuterium. It is believed the big bang produce the universe that was much
denser and hotter than it is now and made almost entirely of two main
elements - hydrogen and helium. Deuterium itself was made only at a second
stage of the beginning of the universe, namely through the collision of one
neutron with one proton at a temperature of about one billion degrees;
furthemore the two formed deuterons in turn stuck together into helium
nuclei, which contain two protons and two neutrons. It is considered, that
during the formation of helium nuclei, almost all the deuterons combined to
form helium nuclei, leaving a tiny remant to be detected today so that only
one in 10.000 deuterons remained unpaired.
Thus, deuterium serves as a particularly important marker. The quantity of
deuterium in contemporary nature is approximately small and measured as no
more than 0.015% (from the whole number of hydrogen atoms) and depends
strongly on both the uniformity of substance and the total amount of matter
formed in course of early evolution. One may suggest, that the very
reliable source of producing of deuterium theoretically may to be the
numerical explosions of nova stars, but deuterium itself is very readily
destroyed in those stars. If it was so, perhaps this was the answer to the
question why the quantity of deuterium increased slitely during the global
changes of climate for worming conditions.
The second point is the chemical processing of deuterium as a result of
this the 2H2O on the first hand may be formed from gaseous deuterium and
atomic oxyden at very high temperature. Pretty interesting with chemical
point of view seems our own idea proposed recently about the possible small
enrichment of primodial environment with 2H2O. We supposed, that this fact
if really existed, may be conditioned by a powerful electrical discharges
taken place in premodial atmosphere laking the natural shield of ozone and
may be resulting in electrolysis processes of H2O, e.g. those ones are now
used for the enrichment of 2H2O. But the realization of this process with
practical point of view seems unlikely. Nevertheless, if such process has
really occured, the some hydrophobic effects of 2H2O as well as chemical
isotopic effects should be taken into account while discussing the
chemico-physical properties of primodial environment. Perhaps, it is also a
big practical interest to study the properties of fully deuterated
membraine structures composed for example from fully deuterated lipids and
proteins. Either way or not, the model of deuterium evolution provides a
framework for predicting the biochemical consequences of such new
fascinating ideas.
Deuterium (2H), the hydrogen isotope with nuclear mass 2, was discovered
by Urey. In the years immediately following this discovery, there developed
a keen interest in development of methods for uniform biological enrichment
of a cell with 2H, that may be best achived via growing of an organism on
medium with high content of 2H2O (99% 2H), which since yet resulted in a
miscellany of rather confusing data (see as an example Katz J., Crespy H.
L. 1972).
The main resolute conclusion that can be derived from the most competent
and comprehensive of the early studies is that high concentrationsof 2H2O
are incompatible with life and reproduction and furthemore could even
causing even lethal effects on a cell. However, today a many cells could be
adapted to 2H2O either via employing a special methods of adaptation which
of them we shall describe above, or using selected (or/and resistent to
2H2O) strains of bacterial and other origin.
In this connection the main interesting question arises-what is the nature
of this interesting phenomenon of biological adaptation to 2H2O and what is
the role of life important macromolecules (particularly DNA, individual
proteins, and/or enzymes) in this process? It is seems very likely, that
during adaptation to 2H2O the structure and conformation of [U -2H]labeled
macromolecules undergoing some modifications that are more useful for the
working in 2H2O-conditions. Unfortunately, there are a small number of
experiments carried out with fully deuterated cells, that could confirmed
that during the growth on 2H2O [U-2H]labeled macromolecules with difined
isotopical structures and conformations are formed, so that a discussion
about the role of deuterium on the structure and the conformation of
[U-2H]labeled macromolecules in course of biolodical adaptation to 2H2O is
still actual through more than four decades of years after the first
description of the biological consequences of hydrogen replacement by
deuterium.
To further discuss the matter, we should distingueshed mainly three
aspects of biological enrichment with deuterium: chemical, biological and
biophysical aspects, all of them are connected in some way with the
structure of [U -2H]labeled macromolecules. Theoretically, the presence of
deuterium in biological systems certainly could be manifested in more or
less degree by changes in the structure and the conformation of
macromolecules. Nevertheless, it is important namely what precise position
in macromolecule deuterium ocupied and dipending from that the primary and
secondary isotopic effects are distingueshied. For example, most important
for the structure of macromolecule the hydrogen (deuterium) bonds form
between different parts of the macromolecule and play a major part in
determining the structure of macromolecular chains and how these structures
interact with the others and also with 2H2O environment. Another important
weak force is created by the three-dimentional structure of water (2H2O),
which tends to force hydrophobic groups of macromolecule together in order
to minimize their disruptive effect on the hydrogen (deuterium)-bonded
network of water (2H2O) molecules.
On the other side the screw parameters of the proton helix are changed by
the presence of deuterium so that ordinary proteins dissolved in 2H2O
exhibit a more stable helical structure (Tomita K., Rich A., et all.,
1962). While 2H2O probably exerts a stabilizing effect upon the
three-dimentional hydrogen (deuterium)-bonded helix via forming many
permanent and easily exchangeable hydrogen (deuterium) bonds in
macromolecule in the presence of 2H2O (as an example the following types of
bonds -COO2H; -O2H; -S2H; -N2H; N2H2 et.), the presence of nonexchangeable
deuterium atoms in amino acid side chains could only be synthesized de novo
as the species with only covalent bonds -C2H, causes a decrease in protein
stability.
These opposing effects do not cancel with the case of protein
macromolecule, and fully deuteration of a protein often results in the
destabilization. As for the deuteration of DNA macromolecule, today there
are not reasonable considerations that such negative effect of 2H2O on the
structure and function is really existiting. Nevertheless, deuterium
substitution can thus be expected to modify by changes in the structure and
the conformation of both [U- 2H]labeled DNA and protein, not only the
reproductionl and division systems of a cell, and cytological or even
mutagenical alterations of a cell, but to a greater or lesser degree of an
order of a cell.
It should be noted, however, that not only these functions but also the
lipid composition of cell membrane are drastically changed during
deuteration. The lipid composition of deuteriated tissue culture cells has
been most complitely investigated by a certain scientists (Rothblat et
all., 1963, 1964). As it is reported in these articles mammalian cells
grown in 30% (v/v) 2H2O contain more lipid than do control cells. THe
increase in the lipids of 2H2O grown cells is due primarily to increased
amounts of triglycerids and sterol esters. Radioisotope experiments
indicate that the differens are due to an enhanced synthesis of lipid.
Monkey kidney cells grown in 25% (v/v) 2H2O and or irradiated with X-rays
likewise showed increases of lipid. The 2H2O grown cells contained more
squalene, sterol esters, sterols, and neutral fat than did either the
control of X-irradiated cells. Phospholipid levels were equal for all
groups of cells. Thus the effects of 2H2O on lipid synthesis are
qualitatively quite similar to those of radiation damade. An interisting
observation that deserves further scrutiny relates to the radiation
sensitivity of deuterated cells. Usually, cells grown and irradiated in
2H2O shown much less sensivity to radiation than ordinary cells suspended
in water. Suspension of ordinary cells in 2H2O did not have any effect on
the reduced sensitivety became apparent.
A serious alteration in cell chemistry must be reflected in the ability of
the cells to divide in the presence of 2H2O and in the manner of its
division. However, a many statements suggesting that 2H2O has a specific
action on cell division are common since today. Probably it may be true
that rapidly proliferating cells are highly sensitive to 2H2O, but that
deuterium acts only to prevent cell division is unlikely.
The rabbit cells grown on medium containing the various concentrations of
2H2O shown, that 2H2O caused a reduction in cell division rate, and this
effect increased as the concentration of 2H2O or duration of exposure, or
both, were increased (Lavillaureix et all., 1962). With increasing
concentration of 2H2O the frequency of early metaphases increased,
accompanied by proportional decreases in the other phases.
It was suggested that 2H2O blocks mitosis in the prophase and the early
metaphase of many cells grown in 2H2O. The blockage, however, was overcome
if the initial concentration of 2H2O was not too high and the exposure time
not too long. In experiments with eggs of the fresh water cichlid fish
Aequidens portalegrensis, they observed that in 30% 2H2O only one-fifth of
the eggs hathed and in 50% (v/v) 2H2O none did so. Segmentation in
fertilized frog eggs developed normally for 24 hours in 40% (v/v) 2H2O,
after which the embryos died. It was also found by Tumanyan and Shnol that
2H2O disturbed embryogenesis in Drosophila melanogaster eggs (Lavillaureix
et all., 1962. Feeding female flies with 20% (v/v) 2H2O caused a
significant increase in the proportion of nondeveloped eggs, whether males
were deuterated or not.
As pointed out by many researches, carried elsewhere, the reason for the
cessation of mitotic activity from exposure to 2H2O is not clear. Certain
microorganisms have been adapted to grow on fully deuterated media.
However, higher plants and animals resist adaptation to 2H2O. Even in
microorganisms, however, cell division appears initially to be strongly
inhibited upon transfer to highly deuterated media.
After the adaptation, however, cellular proliferation proceeds more or
less normally in 2H2O, but this stage is not reached in higher organisms.
No ready explanation in terms of the present understanding of mitosis
suggests itself. In Arbacia eggs antimitotic action of 2H2O is manifested
almost immediately at all stages of the mitotic cycle and during
cytokinesis (Gross P. R., et all., 1963, 1964).
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 2Н2О 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 2Н2О 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 2Н2О. On the contrary, bacills
(eubacteria) and methylotrophs (gram-negative bacteria) worse adapted to
2Н2О.
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 2Н2О 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 2Н2О. At the same time the effect of
convertibility of growth on Н2О/2Н2О - does not theoretically exclude an
opportunity that this attribute is stably kept when cells grown on 2Н2О,
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 2Н2О 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 2Н2О 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 2Н2О. 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 2Н2О 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 2Н2О as
solvent (density, viscosity) in comparison with Н2О 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, рН, 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 2Н2О
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

Secondary effects may still be of importance in biological systems
sensitive to kinetic distortions. Deuterium also affects equilibrium
constants, particularly the ionization constants of weak acids and bases in
composition of macromolecules dissolved in heavy water (see a Table below).
Acid strength of macromolecules in 2H2O is decreased by factors of 2 to 5,
and consequently, the rates of acid-base catalyzed reactions may be greatly
different in 2H2O as compared to H2O. Such reactions frequently may be a
faster in 2H2O than H2O solution (Covington A. K., Robinson R. A., and
Bates R. G., 1966; Glasoe P. K., and Long F. A., 1960).

4.2. The chemical isotopic effect of 2H2O.
The effect of isotopic replacement that has particularly attracted the
attention of chemists is the kinetic isotope effect (Thomson J. F., 1963).
The substitution of deuterium for hydrogen in a chemical bond of
macromolecules can markedly affect the rate of scission of this bond, and
so exert pronounced effects on the relative rates of chemical reactions
going in 2H2O with participation of macromolecules. This change in rate of
scission of a bond resulting from the substitution of deuterium for
hydrogen is a primary isotopic effect. The direction and magnitude of the
isotope effect will depend on the kind of transition state involved in the
activated reaction complex, but in general, deuterium depresses reaction
rates. The usual terminology of the chemist to describe the primary kinetic
effect is in terms of the ratio of the specific rate constants kh/kd. The
maximum positive primary kinetic isotopic effect which can be expected at
ordinary temperatures in a chemical reaction leading to rupture of bonds
involving hydrogen can be readily calculated, and the maximum ratio kh/kd
in macromolecules is in the range of 7 to 10 for C-H versus C-2H, N-H
versus N-2H, and O-H versus O-2H bonds. However, maximum ratios are seldom
observed for a variety of reasons, but values of kh/kd in the range of 2 to
5 are common (Wiberg K. B., 1955). Deuterium located at positions in a
macromolecule other than at the reaction locus can also affect the rate of
a reaction. Such an effect is a secondary isotope effect and is usually
much smaller than a primary isotope effect.
In general, when the macromolecules transfer to deuterated medium not only
water due to the reaction of an exchange (Н2О -2Н2О) dilutes with
deuterium, but also occurs a very fast isotopic (1Н-2Н)-exchange in
hydroxylic (-OH), carboxilic (-COOH), sulfurhydrilic (-SH) and nitrogen
(-NH; -NH2) groups of all organic compounds including the nucleic acids and
proteins. It is known, that in these conditions only С-2Н bond is not
exposed to isotopic exchange and thereof only the species of macromolecules
with С-2H type of bonds can be synthesized de novo. This is very probably,
that the most effects, observed at adaptation to 2Н2О are connected with
the formation in 2Н2О [U -2H]labeled molecules with conformations having
the other structural and dynamic properties, than conformations, formed
with participation of hydrogen, and consequently having other activity and
biophysical properties.
So, according to the theory of absolute speeds the break of С-1H-bonds can
occur faster, than С-2H-bonds (C-2H-bonds are more durable than C-1 ,
mobility of an ion 2H+ is less, than mobility of 1Н+, the constant of
ionization 2Н2О is a little bit less than ionization constant of 2Н2О.
Thus, in principle, the structures of [U -2H]labeled macromolecules may to
be more friable that those are forming in ordinary H2O. But, nevertheless,
the stability of [U -2H]labeled macromolecules probably depending on what
particular bond is labeled with deuterium (covalent bonds -C2H that causing
the instability or hydrogen bonds causing the stabilization of conformation
of macromolecules via forming the three-dimentional netwok of
hydrogen(deuterum) bonds in macromolecule) and what precise position of the
macromolecule was labeled with deuterium. For example, the very valuable
and sensitive for deuterium substitution position in macromolecule is the
reactive center (primary isotopic effects). The non-essential positions in
macromolecule are those ones that situated far away from the reactive
center of macromolecule (secondary isotopic effects). It is also possible
to make a conclusion, that the sensitivity of various macromolecules to
substitution on 2Н bears the individual character and depending on the
structure of macromolecule itself, and thus, can be varried. From the point
of view of physical chemistry, the most sensitive to replacement of 1Н+ on
2H+ can appear the apparatus of macromolecular biosyntesis and respiration
system, those ones, which use high mobility of protons (deuterons) and high
speed of break of hydrogen (deuterium) bonds. From that it is posible to
assume, that the macromolecules should realize a special mechanisms (both
at a level of primary structure and a folding of macromolecules) which
could promote the stabilizition of the macromolecular structure in 2H2O and
somewhat the functional reorganization of their work in 2H2O.
A principal feature of the structure of such biologically important
compounds as proteins and nucleic acids is the maintenance of their
structure by virtue of the participation of many hydrogen bonds in
macromolecule. One may expect that the hydrogen bonds formed by of many
deuterium will be different in their energy from those formed by proton.
The differences in the nuclear mass of hydrogen and deuterium may possibly
cause disturbances in the DNA-synthesis, leading to permanent changes in
its structure and consequently in the cells genotype. The multiplication
which would occur in macromolecules of even a small difference between a
proton and a deuteron bond would certainly have the effect upon its
structure.
The sensitivity of enzyme function to structure and the presumed
sensitivity of nucleic acids function (genetic and mitotic) to its
structure would lead one to expect a noticeable effect on the metabolic
pattern and reproductive behavior of the organism. And next, the changes in
dissociation constants of DNA and protein ionizable groups when transfer
the macromolecule from water to 2H2O may perturb the charge state of the
DNA and protein. Substitution of 1H for deuterium also affects the
stability and geometry of hydrogen bonds in apparently rather complex way
and may, through the changes in the hydrogen bond zero-point vibrational
energies, alter the conformational dynamics of hydrogen (deuterium)-bonded
structures within the DNA and protein in 2H2O.

5. CONCLUSION
The successful adaptation of organisms to high concentration of 2H2O will
open a new avenues of investigation with using [U- 2H]labeled
macromolecules could be isolated from these organisms. For example, fully
deuterated essential macromolecules as proteins and nucleic acids will give
promise of important biological, medical and diagnostical uses. Modern
physical methods of study the structure of [U- 2H]labeled macromolecules,
particularly three-dimentional NMR in a combination with crystallography
methods, X-ray diffraction, IR-, and CD- spectroscopy should cast new light
on many obscure problems concerning with the biological introduction of
deuterium into molecules of DNA and proteins as well as the structure and
the function of macromolecules in the presence of 2H2O. The variety of
these and other aspects of biophysical properties of fully deuterated
macromolecules in the presence of 2H2O remain an interesting task for the
future.
First, I hope that the structural and the functional studies of [U-
2H]labeled macromolecules can provide us to the useful information about a
many aspects of the synthesis of fully deuterated macromolecules and their
biophysical behaviour in 2H2O.
Second, the extensive body of available structural data about a cell
protection system (at the level of the structure and the functioning of [U-
2H]labeled DNA and enzymes) will also form the basis for a particularly
useful model for the study of biological adaptation to 2H2O in aspect of
molecular evolution of macromolecules with difined isotopic structures.
Finally, we also believe, the research can make a favour the medicine and
biotechnology, especially for creating a fully deuterated analogues of
enzymes and DNA having something different properties then the protonated
species and working in the presence of 2H2O.

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Информация об авторе: Олег Викторович Мосин, кандидат химических наук.
Закончил в 1996 году Московскую Государственную академию тонкой химической
технологии им. М.В. Ломоносова по специальности "биотехнология". Область
научных интересов -биотехнология биологически активных соединений (БАС)
(протеины, аминокислоты, нуклеозиды, жиры и сахара), меченных стабильными
изотопами 2Н, 13С и 15N; практическое применение генетически
сконструированных микробных объектов различной таксономической
принадлежности для препаративного получения 2H-, 13C-и 15N-меченных БАС;
клеточный метаболизм стабильных изотопов и функциональная активность
меченых БАС в искусственно сконструированных средах с высокими
концентрациями стабильных изотопов; генетические и физиологические
клеточные механизмы биологической адаптации к тяжелой воде и изотопные
эффекты дейтерия в биологических системах.
По данным исследований к.х.н. О. В. Мосина опубликовано около 30-ти научных
работ и монографий. Изучен механизм физиологической адаптации микробных
объектов различной таксономической принадлежности к тяжелой воде;
осуществлен биосинтез и выделение 2H-, 13C-и 15N-меченных БАС высокого
уровня изотопной чистоты за счет биоконверсии 2H-, 13C-и 15N-меченных
ростовых субстратов в генетически сконструированных клетках
С1-утилизирующих метилотрофных факультативных и облигатных бактерий
Brevibacterium methylicum и Methylobacillus flagellatum; осуществлен
препаративный биосинтез аминокислот фенилаланина и лейцина клетками
факультативной метилотрофной бактерии B. methylicum и облигатной
метилотрофной бактерии M. flagellatum в присутствии высоких концентраций
тяжелой воды; выполнен полупрепаративный биосинтез дейтерированного
мембранного белка бактериородопсина, выполняющего роль АТФ-зависимой
транслоказы в клеточной мембране солелюбивой галофильной бактерии
Halobacterium halobium и изучено функционирование дейтерированного
бактериородопсина; осуществлен биосинтеза 2Н-меченого пуринового
рибонуклеозида инозина бактерией Bacillus subtilis на средах с тяжелой
водой; изучены ростовых, биосинтетических параметров адаптированных к
тяжелой воде биологических объектов и мониторинг уровня дейтерированности
биологического материала и распределения метки в углеродных скелетах
молекул методами масс-спектрометрии и спектрометрии ядерного магнитного
резонанса.
Автор лауреат Президентской Премии, член Японского общества биологии,
биотехнологии и агрохимии
Места работы 21.07.1996-12.09.1997, каф. биотехнологии Московской
государственной академии тонкой химической технологии им. М.В. Ломоносова,
117571, Москва, Вернадского пр-т, 86.
14.09.1997-21.04.1998. Государственный центр генетики и селекции
промышленных микроорганизмов, 113545, Москва, 1-й
Образование и квалификация: Высшее, инженер-химик технолог.
Дополнительные специальности:
1. экономист (специальность «мировая экономика») Московская академия
экономики и права
2. юрист – (специальность «гражданское право») Институт правового
обеспечения Московского государственного университета сервиса.
Научная работа: Роль дейтерия в молекулярной эволюции на примере изучения
функционирования в искусственно созданных максимальных изотопных средах
моделей адаптированных к тяжелой воде биологических объектов, включая
бактерии, микроводоросли и дрожжи различной таксономической
принадлежности.
Область научных интересов: Практическое применение генетически
сконструированных микробных объектов различной таксономической
принадлежности для препаративного получения 2H-, 13C-и 15N-меченных
биологически активных соединений (БАС): полипептиды, дезоксирибонуклеиновые
кислоты, амино-и жирные кислоты, сахара и нуклеозиды; клеточный метаболизм
стабильных изотопов и функциональная активность меченых БАС в искусственно
сконструированных средах с высокими концентрациями стабильных изотопов;
генетические и физиологические клеточные механизмы биологической адаптации
к тяжелой воде и изотопные эффекты дейтерия в биологических системах.
Научное исследование: Биосинтетическое получение 2H-, 13C-и 15N-меченных
БАС высокого уровня изотопной чистоты за счет использования генетически
модифицированных адаптированных клеток микроорганизмов.
Практические достижения: По данным исследований к.х.н. О. В. Мосина
опубликовано около 30-ти научных работ и монографий. Изучен механизм
физиологической адаптации микробных объектов различной таксономической
принадлежности к тяжелой воде; осуществлен биосинтез и выделение 2H-, 13C-и
15N-меченных БАС высокого уровня изотопной чистоты за счет биоконверсии
2H-, 13C-и 15N-меченных ростовых субстратов в генетически сконструированных
клетках С1-утилизирующих метилотрофных факультативных и облигатных бактерий
Brevibacterium methylicum и Methylobacillus flagellatum; осуществлен
препаративный биосинтез аминокислот фенилаланина и лейцина клетками
факультативной метилотрофной бактерии B. methylicum в присутствии высоких
концентраций тяжелой воды; выполнен полупрепаративный биосинтез
дейтерированного мембранного белка бактериородопсина, выполняющего роль
АТФ-зависимой транслоказы в клеточной мембране солелюбивой галофильной
бактерии Halobacterium halobium и изучено функционирование дейтерированного
бактериородопсина; осуществлен биосинтеза 2Н-меченого пуринового
рибонуклеозида инозина бактерией Bacillus subtilis на средах с тяжелой
водой; изучены ростовых, биосинтетических параметров адаптированных к
тяжелой воде биологических объектов и мониторинг уровня дейтерированности
биологического материала и распределения метки в углеродных скелетах
молекул методами масс-спектрометрии и спектрометрии ядерного магнитного
резонанса.
Научные награды: Президентская стипендия, назначенная Президентом
Российской Федерации Б.Н. Ельциным за выдающиеся достижения в учебе и
научной работе (номер приказа от 6.11. 1993 № 613-pт, а также от 27.01.94
№71).
Членства в научных обществах: Член Японской Академии бионауки,
биотехнологии и агрохимии (Japan Society for Bioscience, Biotechnology and
Agrochemistry) с 6.10. 1997, номер членского билета № 9705509.
Общественная деятельность: в прошлом инструктор Гагаринского райкома ВЛКСМ
Места работы 21.07.1996-12.09.1997, каф. биотехнологии Московской
государственной академии тонкой химической технологии им. М.В. Ломоносова,
117571, Москва, Вернадского пр-т, 86.
14.09.1997-21.04.1998. Государственный центр генетики и селекции
промышленных микроорганизмов, 113545, Москва, 1-й Дорожный пр-д, 1.
Академическая история:
1.12.1992-3.06.1996. Кандидат химических наук, диплом к.х.н KT № 022522,
каф. биотехнологии Московской государственной академии тонкой химической
технологии им. М.В. Ломоносова, диссертационная работа на соискание уч.
степени к.х.н. “Разработка методов биотехнологического получения белков,
аминокислот и нуклеозидов, меченных 2Р и 13С с высокими степенями
изотопного обогащения” (работа выполнена под рук. д.х.н., члена корр. РАМН,
проректора МГАХТ проф. В.И. Швеца и д.б.н., вед. н.с. ГНИИ Генетика
Складнева Д.А).
1.09.1984-10.02.1992. Инженер химик технолог, диплом о высшем образовании
ЦВ № 237951, дипломная работа “Исследование биоконверсии изотопно-меченых
низкомолекулярных субстратов в клетках метилотрофных бактерий” (работа
выполнена под рук. д.х.н., члена корр. РАМН, проректора МГАХТ проф. В.И.
Швеца и д.б.н., вед. н.с. ГНИИ Генетика Складнева Д.А).
1.09. 1996-1998. Экономист. Московская академия экономики и права
1.09.2001 – 25.02.2005. Юрист. Институт правового обеспечения Московского
государственного университета сервиса.
Tехнические навыки: Методы генетических и биохимических исследований,
мутагенез и конструирование вектров экспрессии, культивирование
бактериальных штаммов на изотопных средах с выделением изотопно-меченых
соединений; аналитические методы разделения и детекции изотопно-меченых
соединений высокоэффективная жидкостная хроматография, ионообменная
хроматография, ядерный магнитный резонанс, масс-спектрометрия.
Публикации: автор имеет 40 научных работ и монографий.

Рекомендации.
1. д.х.н., член корреспондент РАМН, проректор МГАХТ профессор Виталий
Иванович Швец, Московская государственная академия тонкой химической
технологии им. М.В. Ломоносова, 117751, Москва, Вернадского пр-т, 86.
2. д.х.н., профессор Александр Морисович Юркевич, Московская
государственная академия тонкой химической технологии им. М.В. Ломоносова,
117751, Москва, Вернадского пр-т, 86.
3. д.б.н., ведущий научный сотрудник ГНИИ Генетика Дмитрий Анатольевич
Складнев, 113545, Москва, 1-й Дорожный пр-д., 1, тел (095) 315 3710; (095)
314 3747; факс (095) 315 0501.
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List of publications:
1. Mosin O. V., Karnaukhova E. N., Skladnev D. A., et al. Biosynthetic
preparation of deuterated phenylalanine by methylotroph Brevibacterium
methylicum. // Biotechnologija. 1993. ¹.9. P. 16-20.

2. Еgorova T. A., Mosin O. V., Shvets V. I., et al. Preparative separation
of N-benzylcarboxy-derivatives of amino acids obtained from protein
hydrolysates. // Biotechnologija. 1993. ¹.8. P. 21-25.

3. Bekker G. D., and Mosin O. V. Amino acids labeled with stable isotopes.
Preparation and mass spectrometric control. in: 4th All-Russian Students
Scientific Conference. Problems of Abstract and Applied Chemistry. 20-22
April. 1994. Ural State University. Ekaterinburg. P.127.

4. Mosin O. V., Karnaukhova E. N., Skladnev D. A., Tsygankov Y. D. A strain
of facultative methylotrophic bacterium Brevibacterium methylicum -
producent of phenylalanine, 1993, Russian patent ¹ 93055824. Positive
solution ¹ 055610 from 17.11.1995 for obtaining Russian patent.

5. Kazarinova L. A, Mironov A. S., Mosin O. V., Skladnev D. A., Yurkevich
A. M. A method for the preparation of nucleosides and nucleotides labeled
with deuterium with high levels of isotopic enrichment, 1995, Application
for Russian patent ¹ 95118778.

6. Karnaukhova E. N., Mosin O. V., and Reshetova O. S. Biosynthetic
production of stable isotope labeled amino acids using methylotroph
Methylobacillus flagellatum. // Amino Acids. 1993. V.5. ¹.1.P.125.

7. Mosin O. V., Karnaukhova E. N., Pshenichnikova A. B., Reshetova O. S.
Electron impact spectrometry in bioanalysis of stable isotope labeled
bacteriorhodopsin. in: Sixth International Conference on Retinal Proteins.
19-24 June 1994. Leiden. The Netherlands. P.115.

8. Mosin O. V., Karnaukhova E. N., Skladnev D. A. Application of
methylotrophic bacteria for the preparation of stable isotope labeled amino
acids. in: 7th International Symposium on the Genetics of Industrial
Microorganisms. 26 June 1July 1994. Quebec. Canada. P. 163.

9. Matveev A. V., Mosin O. V., Skladnev D. A., and Yurkevich A. M.
Methylotrophic adaptation to highly deuterated substrates. in: 8th
International Symposium on Microbial Growth on C1 Compounds. 27 August-1
September 1995. San Diego. U.S.A. P. 79.

10. Mosin O. V., Karnaukhova E. N., and Skladnev D. A. Preparation of
2H-and 13C-labeled amino acids via bioconvertion of C1-substrates. in: 8th
International Symposium on Microbial Growth on C1 Compounds. 27 August-1
September 1995. San Diego. U.S.A. P. 80.

11. Shvets V. I., Yurkevich A. M., Mosin O. V., Skladnev D. A. Preparation
of deuterated inosine suitable for biomedical application. // Karadeniz
Journal of Medical Sciences. 1995. V.8. ¹ 4. P.231-232.

12. Mosin O. V., Skladnev D. A., Egorova T. A., Yurkevich A. M., Shvets V.
I. The studying of amino acid biosynthesis by facultative methylotroph
Brevibacterium methylicum on media containing heavy water. //
Biotechnologija. ¹3. 1996. P. 3-12.

13. Mosin O. V., Egorova T. A., Chebotaev . B., Skladnev D. A., Yurkevich
A. M., Shvets V. I. Preparation of bacteriorhodopsin labeled with deuterium
on the residues of aromatic amino acids phenylalanine, tyrosine and
triptophan. // Biotechnologija. 1996. ¹ 4. P. 27-34.

14. Mosin O. V., Kazarinova L. A., Preobrazenskaya K. A., Skladnev D. A.,
Yurkevich A. M., Shvets V. I. The growth of the bacterium Bacillus subtilis
and biosynthesis of inosine on highly deuterated media. // Biotechnologija.
1996. ¹ 4. P. 19-26.

15. Skladnev D. A., Mosin O. V., Egorova T. A., Eremin S. V., Shvets V. I.
Methylotrophic bacteria as sourses of 2H- and 13C-labeled amino acids. //
Biotechnologija. ¹5. 1996. P. 14-22.

16. Mosin O. B., Skladnev D. A., Egorova T. A., Shvets V. I. Mass
spectrometric evaluation of 2H-and 13C enrichment of amino acids molecules
obtained from bacterial objects // Bioorganicheskaja khimia. 1996. V. 22. N
10-11. P. 861-874.

17. Mosin O. B., Skladnev D. A., Egorova T. A., Shvets V. I. The methods
for the preparation of proteins and amino acids labeled with stable
isotopes 2H, 13C, 15N, 18O // Biotechnologija. ¹ 10. P. 1-17.
18. Mosin O. B., Skladnev D. A., Egorova T. A., Shvets V. I. Biosynthesis
of deuterated bacteriorhodopsin by Halobacterium halobium. // 7th
International Conference “New developments in biotechnology”. 15-20 May
1996. Pushino. Russian Federation. P. 95.

19. Mosin O. B., Skladnev D. A. Shvets V. I Investigation of process of
physiological abaptation of methylotrophic bacteria to 2H2O. // Conference
“Autotrophic microorganisms”. Moscow. 17-20 April 1996. P. 113

20. Mosin O. V., Skladnev D. A., Egorova T. A., Shvets V. I.
Biotechnological potential of methylotrophs for the preparation of
deuterated amino acids. // 8th International Congress of Bacteriology and
Applied Microbiology Division. Jerusalem. Israel. August 18-23. 1996. P.
56

21. Kazarinova L. A., Preobrazhenskaja E. S.,Mosin O. B., Skladnev D. A.,
Yurkevich A. M., Shvets V. I. Production of deuterated inosine by Bacillus
subtilis. // 8th International Congress of Bacteriology and Applied
Microbiology Division. Jerusalem. Israel. August 18-23. 1996. P. 82

22. Mosin O. V., Skladnev D. A., Egorova T. A., Shvets V. I. The methods
for preparations of 2H-labeled proteins, amino acids and nucleosides. //
Conference in commemoration of Preobrazensky N. A., M. V. Lomonosov state
academy of fine chemical technology. November 1996. P. 89.

23. Mosin O. V., Skladnev D. A., Shvets V. I. Introduction of ino
[2,3,4,5,6 -2H]phenylalanine, [3,5 -2H]tyrosine and [2,4,5,6,7]tryptophane
into molecule of bacteriorhodopsine of Halobacterium halobium. //
Prikladnayia biokhimija microbiologija. 1999, N1, PP. 34-45.

24. Mosin O. V., Skladnev D. A., Shvets V. I. Biosynthesis of 2 H-labeled
phenylalanine by a new mutant of RuMP facultive methylotroph Brevibacterium
methylicum // Biosience, biotechnolody and bioengineering, 62, N 2, PP.
225-229, 1997.

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