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
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.
SUMMARY:
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).
Table. Isotope components of growth media
and characteristics of bacterial growth of Brevibacterium methylicum
|
Media components, % (v/v)
H2O 2H2O MetOH
[U -2H]
MetOH
|
Lag-phase (h)
|
Yield of
biomass (%)
|
Generation time (h)
|
Production of phenylalanine (%)
|
(a)
|
98
|
0
|
2
|
0
|
20
|
100.0
|
2.2
|
100.0
|
(b)
|
73.5
|
24.5
|
0
|
2
|
34
|
85.9
|
2.6
|
97.1
|
(c)
|
49.0
|
49.0
|
0
|
2
|
44
|
60.5
|
3.2
|
98.8
|
(d)
|
24.5
|
73.5
|
0
|
2
|
49
|
47.2
|
3.8
|
87.6
|
(e)
|
98.0
|
0
|
2
|
60
|
30.1
|
4.9
|
37.0
|
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
|
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.
6.
LITERATURE
Campbell I. D., and Dwek. Biological Spectroscopy.
Benjamin/Cummings, Menlo Park, Calif. 1990.
Covington A. K., Robinson
R. A., and Bates R. G. // J. Phys. Chem. 1966. V. 70. P. 3820.
Еgorova T. A., Mosin O. V., Shvets V.
I., et al.
// Biotechnologija. 1993. ¹.8. P. 21-25.
Fesic S. W. and Zuiderweg
E. R. //
Quarterly Reviews of Biophysics. - 1990. - V.23. - N.2. - P. 97-131.
Johnson W. C. Protein secondary
structure and circular dichroism: A practical guide. Proteins Struct. Funct.
Genet. 1990. 7:205-214.
Glasoe P. K., and Long F.
A. // J.
Phys. Chem. 1960. V. 64. P. 188.
Hogan C. J. // Scientific American.
December 1996. P. 36-41.
Karnaukhova E. N., Mosin
O. V., and Reshetova O. S. // Amino Acids. 1993. V.5. ¹.1.P.125.
Katz J., Crespy H. L. // Pure Appl. Chem. 1972.
V. 32. P. 221-250.
Lewis G. N. // Science. 1934. V. 79.
P. 151.
Mathews C. K., van Holde
K. E. Biochemistry Benjamin/Cummings, Menlo Park, Calif. 1996. P. 204-210.
Mosin O. V., Karnaukhova
E. N., Skladnev D. A., et al. // Biotechnologija. 1993. ¹.9. P. 16-20.
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.
Mosin O. V., Karnaukhova
E. N., and Skladnev D. A. Preparation of 2H-and 13C-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.
Mosin O. V., Skladnev D.
A., Egorova T. A., Yurkevich A. M., Shvets V. I. // Biotechnologija. ¹3. 1996a.
P. 3-12.
Mosin O. V., Egorova T.
A., Chebotaev . B., Skladnev D. A., Yurkevich A. M., Shvets V. I. // Biotechnologija. 1996b.
¹ 4. P. 27-34.
Mosin O. V., Kazarinova L.
A., Preobrazenskaya K. A., Skladnev D. A., Yurkevich A. M., Shvets V. I. // Biotechnologija.
1996c. ¹ 4. P. 19-26.
Mosin O. B., Skladnev D.
A., Egorova T. A., Shvets V. I // Bioorganicheskaja khimia. 1996d. V. 22. N 10-11. P.
861-874.
Skladnev D. A., Mosin O.
V., Egorova T. A., Eremin S. V., Shvets V. I. Methylotrophic bacteria as sourses
of 2H-and 13C-amino acids. // Biotechnologija. ¹5.
1996. P. 14-22.
Shvets V.
I., Yurkevich A. M., Mosin O. V., Skladnev D. A // Karadeniz Journal of Medical
Sciences. 1995. V.8. ¹ 4. P.231-232.
Thomson J. F. Biological Effects of
Deuterium. 1963. Pergamon, New York.
Tomita K.,
Rich A., de Loze C., and Blout E. R. // J. Mol. Biol. 1962. V. 4. P. 83.
Wiberg K. B. // Chem. Rev. 1955. V.
55. P. 713.