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

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.