The role of deuterium in molecular evolution

The role of deuterium in molecular evolution



Oleg V. Mosin1


1 Department of Biotechnology, M. V. Lomonosov State Academy of
Fine Chemical Technology, Vernadskogo Prospekt 86, 117571, Moscow, Russia


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.



(2H), the hydrogen isotope with nuclear mass 2, was discovered by
. 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).

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.

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.

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)

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

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.

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.

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.

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.,
. With increasing concentration of 2H2O the
frequency of early metaphases increased, accompanied by proportional decreases in
the other phases.

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.

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.

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).

Table. Isotope components of growth media and characteristics of bacterial
growth of Brevibacterium methylicum


Media components, % (v/v)


2H2O           MetOH      [U -2H]



Yield of


time (h)

of phenylalanine (%)













































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
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.
, 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.
. 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.
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.
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.

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.



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

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



3.1. The methods for analyzing the structure and the
conformation of

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

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

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

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

I may briefly indicate three possibilities for deuterium

(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.
(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)



The imaginary principle of realization
of biological adaptation

I                                                           II

1 works           2 not work    not work          
2 works     



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.

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,
).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:

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

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;

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.   



Physical constant



d20 (g/c.c)



volume, V20 (ml/mole)



m20 (centipose)



point (0C)



point (0C)



of maximum density (0C)



product (25 0C)



of formation (cal/mole)



energy of formation (cal/mole)






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

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.



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

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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