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Published in the Russian Federation
European J ourn al of Molecular Biotechn ology
H as been issued sin ce 20 13.ISSN: 2310 -6255
Vol. 3, No. 1, pp. 11-24, 20 14
DOI: 10 .1318 7/ issn .2310 -6255
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B io lo gica l In flu e n ce o f D e u te riu m o n Pro ca rio tic a n d Eu ka ryo tic Ce lls
1 Oleg Mosin 2 Ign at Ign atov
1 Moscow State Un iversity of Applied Biotechn ology, Russian Federation Sen ior research Fellow of Biotechn ology Departm en t, Ph. D. (Chem istry) 10 3316, Moscow, Talalihin a ulitza, 33
E-m ail: m osin-oleg@yan dex.ru
2 T h e Scien tific Research Cen ter of Medical Biophysics (SRC MB), Bulgaria Professor, D. Sc., director of SRC MB.
1111, Sofia, N. Kopern ik street, 32 E-m ail: m [email protected]
Abs tra ct. Biologic in fluen ce of deuterium (D) on cells of various taxon om ic groups of prokaryotic and eukaryotic m icroorganism s realizin g m ethylotrophic, chem oheterotrophic, photo-organ otrophic, and photosyn thetic ways of assim ilation of carbon substrates are in vestigated at growth on m edia with heavy water (D2О). The method of step by step adaptation technique of cells to D2О was developed, consisting in plating of cells on 2 % agarose nutrient media containing increasin g gradien t of concen tration of D2О (from 0 up to 98 % D2O) an d the subsequen t selection of stable to D2O cells. In the result of that techn ique were obtain ed adapted to m axim um con centration of D2O cells, biological m aterial of which in stead of hydrogen con tain ed deuterium with levels of en richm en t 92– 97,5 at.% D.
Ke yw o rd s : deuterium ; heavy water; adaptation ; isotopic effects; bacteria; blue-green algae.
In tro d u ctio n .
The most interesting biological phenomenon is the ability of some microorganisms to grow on heavy water (D2O) media in which all hydrogen atoms are replaced with deuterium [1, 2]. D2O has high environm ental poten tial due to the absen ce of radioactivity an d poccebility of detectin g the deuterium label in the m olecule by high-resolution m ethods as NMR, IR, an d m ass spectrom etry that facilitates its use as a tracer in biochem ical an d biom edical research [3]. The average ratio of H / D in nature m akes up approxim ately 1:570 0 [4]. In m ixtures of D2O with Н2O the isotopic exch ange occurs with high speed with the form ation of sem i-heavy water (H DO): D2O + H2O = H DO. For this reason deuterium presen ts in sm aller con ten t in aqueous solution s in form of НDO, while in th e higher content – in form of D2O.
The chem ical structure of D2O m olecule is analogous to that on e for Н2O, with sm all differences in the length of the covalent H – O-bonds and th e angles between them . The m olecular m ass of D2O exceeds on 10 % that one for Н2O. The difference in nuclear m asses stipulates the isotopic effects, which m ay be sufficiently essential for H / D pair [5]. As a result, physical-ch em ical properties of D2O differ from H2O: D2O boils at 10 1,44 0С, freezes at 3,8 2 0С, has m axim al den sity at 11,2 0С (1,10 6 g/ cm3) [6]. The ch em ical reactions in D2O are som ehow slower com pared to Н2O.
D2O is less ionized, the dissociation constan t of D2O is sm aller, an d the solubility of the organ ic an d inorgan ic substances in D2O is sm aller com pared to these on es in Н2О [7]. Due to isotopic effects the hydrogen bonds with the participation of deuterium are slightly stron ger th an those on es form ed of h ydrogen .
For a lon g tim e it was con sidered that heavy water is in com patible with life. Experim en ts with the growin g of cells of differen t organism s in D2O show toxic in fluen ce of deuterium . The high concentrations of D2O lead to the slowin g down the cellular m etabolism , m itotic in h ibition of the prophase an d in som e cases – som atic m utation s [8 ]. This is observed even while usin g n atural water with an increased conten t of D2O or H DO [9]. Bacteria can en dure up to 90 % (v/ v) D2О, plant cells can develop norm ally up to 75 % (v/ v) D2О, while an im al cells – up to n ot m ore than 30 % (v/ v) D2О [10 ]. The decrease of the deuterium con ten t in water to 25 % (v/ v) of the physiological level stim ulates th e cellular m etabolism .
With the developm en t of new m icrobiological approaches, there appears an opportun ity to use adapted to deuterium cells for preparation of deuterated n atural com poun ds [11, 12]. The traditional m ethod for production of deuterium labelled com poun ds con sists in the growth on m edia containing m axim al concen tration s of D2O an d deuterated substrates as [D]m ethan ol, [D]glucose etc. [13]. Durin g growth of cells on D2O are syn thesized m olecules of biologically im portant n atural com poun ds (DNA, protein s, am in o acids, n ucleosides, carbohydrates, fatty acids), which hydrogen atom s at th e carbon backbon es are com pletely substituted with deuterium . They are bein g isolated from deuterated biom ass obtain ed on growth m edia with high D2O con ten t and deuterated substrates with usin g a com bin ation of physico-chem ical m ethods of separation – hydrolysis, precipitation and extraction with organ ic solven ts an d chrom atographic purification by colum n chrom atography on differen t adsorben ts. Th ese deuterated m olecules eviden tly un dergo structural adaptation m odification s necessary for the n orm al fun ction in g in D2O.
The adaptation to D2O is interested n ot on ly from scien tific poin t, but allows to obtain th e un ique biological m aterial for the studyin g of m olecular structure by 1H -NMR [14]. Tren d towards the use of deuterium as an isotopic label are stipulated by the absen ce of radioactivity an d possebility of determ in ation the deuterium localization in the m olecule by high resolution NMR spectroscopy [15], IR spectroscopy [16] an d m ass spectrom etry [17]. The recen t advan ces in the techn ical an d com putin g capabilities of an alytical m ethods have allowed to con siderable in crease the efficien cy of de n ovo biological studies, as well as to carry out structural-fun ction al studies with deuterated m olecules on a m olecular level.
This study is a contin uation of our research for the practical utilization of differen t cells of bacteria an d m icroalgae for the synthesis of deuterium labeled com poun ds in deuterated growth m edia with D2O. Th e purpose of our research was studyin g the physiological in fluen ce of deuterium on the cells of different taxon om ic groups of m icroorgan ism s an d m icroalgae realizin g m ethylotrophic, chem oheterotrophic, photo-organ otrophic an d photosyn thetic pathways of carbon assim ilation .
Ma te ria l a n d m e th o d s .
The objects of the study were various m icroorgan ism s, realizin g m ethylotrophic, chem oheterotrophic, photo-organ otrophic, an d photosyn thetic ways of assim ilation of carbon substrates. The initial strains were obtain ed from the State Research In stitute of Gen etics an d Selection of Industrial Microorgan ism s (Moscow, Russia):
1. Brev ibacterium m ethy licum B-5652, a leucin e auxotroph Gram -positive strain of facultative m ethylotrophic bacterium , L-phen ylalan in e producer, assim ilatin g m ethan ol via the NAD+ dependent m eth an ol dehydrogenase varian t of ribulose-5-m on ophosphate cycle (RuMP) of carbon fixation.
2. Bacillus subtilis B-3157, a polyauxotrophic for histidin e, tyrosin e, aden in e, an d uracil spore-form ing aerobic Gram -positive chem oheterotrophic bacterium , in osin e producer, realizin g hexose-6-m ono-phosphate (GMP) cycle of carbohydrates assim ilation .
3. H alobacterium halobium ET-10 0 1, photo-organotrophic caroten oid-con tain in g strain of extrem e halobacteria, synth esizing the phototransform in g transm em bran e protein bacteriorhodopsin .
4. Chlorella v ulgaris B-8 765, photosynthesizing single-cell blue-green algae.
For preparation of growth m edia was used D2O (99,9 at.%) an d DСl (95,5 аt.%) received from the “Isotope” Russian Research Cen tre (St. Petersburg, Russian Federation ). In organ ic salts an d glucose were prelim inary crystallized in D2О an d dried in vacuum before usin g. D2O distilled over KMnO4 with the subsequen t con trol of isotope en richm en t by 1H -NMR-spectroscopy on a Brucker WM-250 device (“Brucker”, Germ an y) (workin g frequen cy 70 MH z, in tern al stan dard Me4Si).
For cell cultivation an d adaptation were used various growth m edia with an in creasin g gradient of D2O concen tration from 0 ; 24,5; 49,0 ; 73,5 up to 98 % (v/ v) D2O. Cultivation of m ethylotrophic bacteria was carried out on m in im al salt M9 m edium (g/ l): KH2PO4 – 3; Na2H PO4 – 6; NaCl – 0 ,5; NH4Cl – 1. Cultivation of chem oheterotrophic bacteria was carried out on H W m edium (g/ l): glucose – 12; yeast extract or hydrolyzed deuterated biom ass of B. m ethy licum – 2,5; NH4NO3 – 3; MgSO4.7H2O – 2; CaCO3 – 2. Cultivation of photo-organ otrophic bacteria was carried out on SM m edium (g/ l): yeast extract or hydrolyzed deuterated biom ass of B. m ethy licum – 2,5; NaCl − 250 ; MgSO4.7H2O − 20 ; KСl − 2; NH4Cl − 0 ,5; KNO3 − 0 ,1; KH2PO4 − 0 ,0 5; K2H PO4 − 0 ,0 5; Na+-citrate − 0 ,5; Mn SO4.2H2O − 3.10-4; CaCl2.6H2O − 0 ,0 65; Zn SO4.7H2O − 4.10-5; FeSO4.7H2O − 5.10-4; CuSO4.5H2O − 5.10-5; glycerol − 1,0 . Blue-green algae C. v ulgaris grew on m in eral growth m edium (g/ l): KNO3 – 5,0 ; MgSO4.7H2O – 2,5; KH2PO4 – 1,25; FeSO4 – 0 ,0 0 3; Mn SO4.2H2O − 3.10-4; CaCl2 6H2O − 0 ,0 65; Zn SO4.7H2O − 4.10-5; CuSO4.5H2O − 5.10-5, CоCl2.6H2O − 5.10-6.
For adaptation were used solid 2 % (w/ v) agarose m edia with gradually in creasin g concentration of D2O, com bin ed with the subsequen t selection of in dividual cellular colon ies resistant to deuterium . As a source of deuterated growth substrates for the growth of chem oheterotrophic bacteria an d photo-organ otrophic bacteria was used the deuterated biom ass of facultative m ethylotrophic bacterium B. m ethy licum , obtain ed via a m ulti-stage adaptation on solid 2 % (w/ v) agarose M9 m edia with an in creasin g gradien t of D2О (from 0 ; 24,5; 49,0 ; 73,5 up to 98 % (v/ v) D2О). Raw deuterated biom ass (output, 10 0 gram of wet weight per 1 liter of liquid culture) was suspended in 10 0 m l 0 ,5 M 2H Cl (in D2O) an d autoclaved for 30 – 40 m in at 0 ,8 atm . The suspension was neutralized with 0 ,2 M KOH (in D2O) to pH = 7,0 an d used as a source of growth substrates for growing of chem oheterotrophic bacterium B. sublilis an d photo-organotrophic bacterium H . halobium .
Cellular growth was carried out in 50 0 m l Erlen m eyer flasks con tain in g 10 0 m l of the growth m edium at 32– 34 0С an d vigorously aerated on an orbital shaker Biorad (“Biorad Labs”, Polan d). Photo-organ otrophic bacteria and blue-green algae were grown at illum in ation by fluorescen t m onochrom atic lam ps LDS-40 -2 (40 W) ("Alfa-Electro", Russia). Growin g of m icroalgae C.
v ulgaris was carried out at 32 0C in a photoreactor with CO
2 bubblin g. Bacterial growth was m on itored on ability to form individual colon ies on a surface of 2 % (w/ v) agarose m edia, an d on optical den sity of cell suspension m easured on Beckm an DU-6 (Beckm an Coulter, USA) spectrophotom eter at λ = 620 n m . After 6–7 days the cells were harvested an d separated by cen trifugation (10 0 0 0 g, 20 m in) on T-24 cen trifuge ("H eracules", Germ an y). Th e biom ass was washed with D2O and extracted with a m ixure of organ ic solven ts: chloroform -m eth an ol-aceton e = 2: 1: 1, % (v/ v) for isolating lipids an d pigm en ts. The resultin g precipitate (10–12 m g) was dried in vacuum an d used as a protein fraction , while th e liquid extract – as a lipid fraction . Th e exogen ious deuterated am ino acids and ribonucleosides were isolated from the liquid cultures of appropriate strain -producers. In osin e was isolated from LC of B. subtilis by adsorption / desorption on activated carbon as adsorben t with following extraction with 0 ,3 M NH4-form ate buffer (pH = 8 ,9), subsequent crystallization in 8 0 % (v/ v) ethan ol, an d ion exch an ge ch rom atography (IEC) on a colum n with cation exchan ge resin AG50 WX 4 equilibrated with 0 ,3 M NH4-form ate buffer an d 0 ,0 45 M NH4Cl (output, 3,1 g/ l (8 0 %); [α]D20 = 1,61 (eth an ol)). Bacteriorhodopsin was isolated from the purple m em bran es of photo-organ otrophic halobacterium H . halobium by the m ethod of D. Osterhelt, m odificated by the authors [18 ].
The an alysis of am ino acids from protein hydrolyzates was carried out on a Biotron ic LC-50 0 1 (230 × 3,2) colum n (“Eppen dorf– Nethleler– H inz”, Germ any) with a UR-30 (“Beckm an– Spin co”, USA) sulfon ated styren e (7,25 % cross lin ked) resin as a stationary phase; the gran ule diam eter was 25 μm ; 0 ,2 N sodium – citrate buffer (pH = 2,5) was used as an eluent; the workin g pressure – 50 – 60 atm ; the eluent input rate – 18 ,5 m l/ h; the ninhydrin input rate – 9,25 m l/ h; detection at λ = 570 an d λ = 440 n m (for prolin e).
Carbohydrates were an alyzed on a Kn auer Sm artlin e chrom atograph (“Kn auer”, Germ an y) equipped with a Gilson pum p (“Gilson In c.”, Germ an y) and a Waters K-40 1 refractom eter (”Water Associates”, Germ any) usin g Ultrasorb CN as a station ary phase: the colum n size – 250 × 10 m m ; the granule diam eter – 10 μm ; the m obile phase – aceton itrile– water (75 : 25, % (w/ w); the in put rate – 0 ,6 m l/ m in .
Fatty acids were an alyzed on a Beckm an Gold System (USA) chrom atograph, equipped with Model 126 Detector (USA). Station ary phase: Ultrasphere ODS, particel size 5 µm , 4,6 × 250 m m ; m obile phase: linear gradien t of 5 m M KH2PO4-aceton itrile, elution rate 0 ,5 m l/ m in , detection at λ = 210 n m .
For evaluation of deuterium enrich m en t levels EI an d FAB m ass spectrom etry was used. EI m ass spectra were recorded on MB-8 0 A device (“H itachi”, J apan ) with double focusin g (the en ergy of ion izin g electrons, 70 eV; th e acceleratin g voltage, 8 kV; the cathode tem perature, 18 0 – 20 0 0С) after am in o acid m odification in to m ethyl esters of N-5-dim ethylam in o(n aphthalen e)-1-sulfon yl (dansyl) am ino acid derivatives accordin g to an earlier elaborated protocol [19]. FAB-m ass spectra were recorded on pulse m ass spectrom eter VG-70 SEQ (“Fison s VG An alytical”, USA), supplied with caesium source Cs+ on a glyceric m atrix with acceleratin g pressure 5 кВ and an ionic current 8 мА.
IR-spectra were registered on Brucker Vertex (“Brucker”, Germ an y) IR spectrom eter (a spectral range: average IR – 370 – 78 0 0 cm-1; visible – 250 0 – 8 0 0 0 cm-1; the perm ission – 0 ,5 cm-1; accuracy of wave n um ber – 0 ,1 cm-1 on 20 0 0 cm-1).
Re s u lts a n d d is cu s s io n .
When biological objects bein g exposed to water with differen t deuterium con ten t, th eir reaction varies depending on the isotopic com position of water an d m agn itude of isotope effects determ in ed by the differen ce of constan ts of chem ical reaction s rates kH/ kD in H2O an d D2O. The m axim um kinetic isotopic effect observed at ordin ary tem peratures in chem ical reaction s leading to rupture of bonds involvin g hydrogen an d deuterium lies in the ran ge kH/ kD = 5– 7 for C– H versus C– D, N– D versus N– D, an d O– H versus O– D-bon ds. Isotopic effects have an im pact n ot on ly on the physical and chem ical properties of m acrom olecules, but also on the biological behaviour of biological objects in D2O. Experim en ts with D2O (Table 1) have shown , that green-blue algae is capable to grow on 70 % (v/ v) D2O, m ethylotrophic bacteria – 75 % (v/ v) D2O, chem oheterotrophic bacteria – 8 2 % (v/ v) D2O, an d photo-organ otrophic halobacteria – 95 % (v/ v) D2O.
0 10 20 30 40 50 60 70 80 90 100
Halobacteria Halobacterium halobium
Chemoheterotrophic bacteria Bacillus subtilis
Methylotrophic bacteria Brevibacterium
methylicum
Blue-green algae Chlorella vulgaris
D
eut
eri
um
c
ont
ent
s i
n
w
at
er,
vol
.%
Figure 1. Cell survival of studied m icroorganism s in water with differen t deuterium con ten t
In the course of the experim en t were obtain ed adapted to the m axim um con cen tration of D2O cells belon ging to differen t taxon om ic groups of m icroorgan ism s, realizin g m ethylotrophic, chem oheterotrophic, photo-organotrophic an d photosyn thetic pathways of assim ilation of carbon substrata, as facultative m ethylotrophic bacterium B. m ethy licum , ch em oheterotrophic bacterium B. subtilis, h alobacterium H . halobium an d blue-green algae C. v ulgaris.
Selection of m ethanol-assim ilatin g facultative m ethylotrophic bacterium B. m ethy licum was conn ected with the developm ent of new m icrobiological strategies for preparation of deuterated biom ass via biocon version of [D]m ethan ol an d D2O an d its further use as a source of deuterated growth substrates for th e growin g of other strain s-producers in D2O.
Choosin g of photo photo-organotrophic halobacteria H . halobium was stipulated by th e prospects of further isolation of retin al con tain ing tran sm em bran e protein bacteriorhodopsin (BR) – chrom oprotein of 248 am in o acid residues, con tain ing as a chrom ophore an equim olar m ixture of 13-cis-an d 13-tran s C20 caroten oid associated with a protein part of the m olecule via a Lys-216 residue [20 ]. BR perform s in the cells of halobacteria the role of ATP-depen den t tran slocase, wh ich creates an electroch em ical gradien t of H+ on the surface of the cell m em bran e, which en ergy is used by th e cell for the synth esis of ATP in the an aerobic photosyn thetic phosphorylation .
Usin g ch em oheterotrophic bacterium B. subtilis was determ in ed by preparative isolation produced by this bacterium deuterated ribon ucleoside – in osin e (total deuteration level 65,5 at.% D) for biom edical use [21], and the use of photosynthetic blue-green C. v ulgaris was stipulated by the study of biosynth esis of deuterated chlorophyll an d caroten oid pigm en ts (deuteration level 95– 97 at.% D) on growth m edia with high D2O-con ten t.
We used stepwise in creasing gradien t con cen tration of D2O in growth m edia, because it was assum ed that the gradual accustom ing of m icorgan ism s to deuterium would have a ben eficial effect upon the growth and physiological param eters. The strategy of adaptation to D2O is shown in Table. 1 on an exam ple of m ethylotrophic bacterium B. m ethy licum , which deuterated biom ass was used in further experim en ts as a source of deuterated growth substrates for growin g of chem oheterotrophic and photo-organ otrophic bacteria.
Table 1
Th e is o to p ic c o m p o s itio n o f gro w th m e d ia a n d gro w th ch a ra cte ris tics o f m e th ylo tro p h ic ba cte riu m B . m e t h y lic u m in th e p ro ce s s o f a d a p ta tio n to D2O* Experim en t
n um ber
Media com pon en ts, % (v/ v)
Lag-period, (h)
Yeald of
biom ass, gram from 1 liter of liquid culture
Cell gen eration tim e (h)
H2O D2O
1 98 ,0 0 20 ,0 ±1,40 20 0 ,0 2±1.40 20 ,0 ±1,40
2 73,5 24,5 34,0 ±0 ,8 9 171,8 ±1.8 1 2,6±0 ,23
3 49,0 49,0 44,0 ±1,38 121,3±1.8 3 3,2±0 ,36
4 24,5 73,5 49,0 ±0 ,91 94,4±1.74 3,8 ±0 ,25
5 0 98 ,0 60 ,0 ±2,0 1 60 ,2±1.44 4,9±0 ,72
6 0 98 ,0 40 ,0 ±0 ,8 8 174,0 ±1.8 3 2,8 ±0 ,30
Notes: * Th e data in Expts. 1– 5 is subm itted for B. m ethy licum at growin g on growth m edia, containing 2 % (v/ v) deutero-m ethan ol an d specified am oun ts (% , v/ v) of D2О.
The data in Expt. 6 is subm itted for adapted to D2О bacterium .
As the control used experim en t 1 wh ere used proton ated water an d m ethan ol.
The adaptation strategy to D2O consisted in platin g of in itial cells of m icroorgan ism s on Petri dishes with solid 2 % (w/ v) agarose growth m edia with stepwise in creasin g D2O-con ten t therein (0 ; 24,5; 49,0 ; 73,5 an d 98 % (v/ v) D2O), an d the subsequent selection of resistant cells to D2O. Cells grown on m edia with a low gradient of D2О concentration were con sequen tly transferred on to m edia with higher gradient, up to 98 % (v/ v) D2О. At th e final stage of this procedure on th e m axim ally deuterated growth m edium with 98 % (v/ v) D2O were isolated in dividual cell colon ies represen tin g the progeny of a single cell resistant to the action of D2O. Th en th e colon ies were transferred onto the liquid growth m edium of the sam e D2O-con ten t, prepared on the basis of 98 % (v/ v) D2O and grown for 5 days at 34 0C. The survival rate in the m axim al deuterated growth
m edium was not m ore than 40 %. The progress of adaptation was observed by th e chan ges of lag-tim e period, lag-tim e of cell gen eration an d yrald of m icrobial biom ass, as well as by the ability of cells to form single colonies on the surface of solid 2 % (w/ v) agarose m edia with 2H2O an d cell coun tin g.
All m icroorganism s adapted to D2O retain ed th e ability to grow on growth m edia with high conten t of D2O. Th e general feature of bacterial growth in D2О was th e proportion al in crease in duration of the lag-period and tim e of cellular gen eration an d sim ultan eous reduction of yealds of m icrobial biom ass. These param eter values were correlated with the con ten t of D2O in growth m edia with the lowest fixing values of these param eters on m axim um deuterated m edia. Th e added gradually in creasing con cen trations of D2O in to growth m edia caused the proportion al in creasin g lag-period and output of m icrobial biom ass in all isotopic experim en ts (Table 1). In con trast to th e adapted m icroorganism s, the growth of n on-adapted m icroorgan ism s on the m axim al deuterated m edia with D2O was inh ibited. Th e yealds of biom ass on deuterated growth m edia were varied 8 5– 90 % for different taxonom ic groups of m icroorgan ism s. Adapted m icroorgan ism s possessed slightly reduced levels of m icrobial biom ass accum ulation an d in creased cell gen eration tim es on m axim al deuterated m edia.
The result obtain ed in experim en ts on the adaptation of m ethylotrophic bacterium B. m ethy licum to D2О allowed to use hydrolysates of biom ass of this bacterium obtain ed in the process of m ulti-stage adaptation to D2О, as a source of deuterated growth substrates for th e growin g of chem oheterotrophic bacterium B. subtillis an d photo-organ otrophic halobacterium H . halobium . The assim ilation rate of m ethylotrophic biom ass by protozoa an d eukaryotic cells am oun ts to 8 5– 98 %, while the productivity calculated on the level of m ethan ol biocon version in to cell com pon ents m akes up 50 – 60 % [22]. While usin g deutereted biom ass of m ethylotrophic bacteria B. m ethy licum as a sourse of deuterated growth substrates it was taken in to accoun t th e ability of m ethylotrophic bacteria to syn thesize large am oun ts of protein (output, 50 % (w/ w) of dry weight), 15– 17 % (w/ w) of polysaccharides, 10 – 12 % (w/ w) of lipids (m ain ly, ph ospholipids), an d 18 % (w/ w) of ash [23]. The m ost im portan t fact is that ability is preserved on growth m edia containing D2О and [D]m ethan ol. To provide high outputs of these com poun ds an d m in im ize th e isotopic exchan ge (Н– D) in am in o acid residues of protein m olecules, the biom ass was hydrolyzed by autoclaving in 0 ,5 M DCl (in D2О) and used for the growin g of chem oheterotrophic bacterium B. subtillis an d photo-organotrophic halobacterium H . halobium .
Taking into accoun t the pathways of assim ilation of carbon substrates, th e adaptation of chem oheterotrophic bacterium B. subtilis an d photo-organ otrophic halobacterium H . halobium was carried out v ia plating of initial cells to separate colon ies on solid 2 % (w/ v) agarose m edia based on 99,9 at.% D2О and deuterated hydrolyzate biom ass of B. m ethy licum , with the followin g subsequen t selection of the colonies resistan t to D2О. On con trary to D2О deuterated substrates in com position of deuterated biom ass hydrolyzate had n o sign ifican t n egative effect on the growth param eters of the studied m icroorgan ism s. Output of deuterated in osin e at growth of B. subtilis on D2О-m edium was 3,9 g/ l, while the level of glucose assim ilation from the liquid culture was 40 g/ l. The fraction ation of inosine from liquid culture was perform ed by adsorption / desorption on the surface of activated carbon, extraction by 0 ,3 M NH4-form ate buffer (pH = 8 ,9) with subsequen t crystallization in 8 0 % (v/ v) ethan ol and colum n ion exchan ge chrom atography on cation exchan ge resin AG50 WX 4 equilibrated with 0 ,3 M NH4-form ate buffer with 0 ,0 45 M NH4Cl. Deuteration level of inosin e m olecule m eadsured by FAB m ass spectrom etry was five deuterium atom s (62,5 atm .% D) with th e in clusion of three deuterium atom s in the ribose an d two deuterium atom s in the m olecule fragm en ts hypoxan thin e.
For adaptation of blue-green algae C. v ulgaris was used liquid m in eral m edium con tain in g 25, 50 , 75 an d 98 % (v/ v) D2О. The levels of deuterium en richm en t of caroten oids were In the case of C. v ulgaris and H . halobium used fluorescen t illum in ation , as both m icroorgan ism s grown in the presence of light. Individual colonies of cells of these m icroorgan ism s resistant to D2О, allocated by selection were grown on liquid growth m edia of the sam e com position with 99,9 at.% D2О for producing the deuterated biom ass.
While growin g of photo-organotrophic halobacterium H . halobium on D2О-m edium cells synthesized the purple carotenoid pigm ent, iden tified as a n ative BR on the the spectral ratio of protein an d chrom ophore fragm ents in the m olecule (D28 0/ D568 = 1,5 : 1,0 ). The growth of this bacterium on D2О-m edium was slightly in hibited as com pared with the con trol on proton ated
growth m edium th at sim plifies th e optim ization of con dition s for the production of m icrobial biom ass, wh ich consists in the growing of this halobacterium on deuterated growth m edium with 2 % (w/ v) of deuterated biom ass h ydrolyzate of B. m ethy licum , isolation of purple m em bran e fraction, the separation of low- and high-m olecular im purities, cellular RNA, pigm en ts (preferably caroten oids) an d lipids, protein solubilization in 0 ,5 % (w/ v) SDS-Na, fraction ation of solubilized protein by m ethanol an d purification on Sephadex G-20 0 . The total level of deuterium en richm en t of the BR m olecule, calculated on deuterium en richm en t levels of am in o acids of the protein hydrolyzate was 95 at.% D.
Our studies indicated that the ability of adaptation to D2О for differen t taxon om ic groups of m icroorganism s is differen t, and stipulated by taxon om ic affiliation , m etabolic characteristics, pathways of assim ilation of substrates, as well as by evolution ary n iche occupied by the object. Thus, the lower the level of evolution ary organ ization of th e organ ism , the easier it adapted to the presen ce of deuterium in growth m edia. Th us, m ost prim itive in evolution ary term s (cell m em bran e structure, cell organization, resistan ce to en viron m en tal factors) of the studied objects are photo-organotrophic halobacteria related to archaebacteria, stan din g apart from both prokaryotic and eukaryotic m icroorganism s, exhibitin g in creased resistan ce to 2Н
2О an d practically n eeded n o adaptation to D2О, contrary to blue-green algae, which, bein g eukaryotes, are the m ore difficult adapted to D2О an d exhibit in hibition of growth on 70 – 75 % (v/ v) D2О.
The com position of growth m edia eviden tly plays an im portan t role in process of adaptation to D2О, because the reason of in hibition of cell growth an d cell death can be chan ges of the parity ratio of synthesized m etabolites in D2О-m edia: am in o acids, protein s an d carbohydrates. It is n oted that adaptation to D2О occures easier on com plex growth m edia than on the m in im al growth m edia with full substrates at a gradual increasin g of deuterium con ten t in the growth m edia, as th e sen sitivity to D2О of differen t vital system s is differen t. As a rule, even highly deuterated growth m edia contain rem aining protons from 0 ,2– 10 at.%. These rem ain in g proton s facilitate th e restructurin g to th e changed con dition s durin g the adaptation to D2О, presum ably in tegratin g in to those sites, which are the m ost sensitive to the replacem en t of hydrogen by deuterium . Also deuterium induces physiological, m orphological an d cytological alteration s on the cell. There were m arked th e significant differences in the m orphology of the proton ated an d deuterated cells of blue-green algae C. v ulgaris. Cells grown on D2O-m edia were 2– 3 tim es larger in size an d had thicker cell walls, than the con trol cells grown on a con ven tion al proton ated growth m edia with ordinary water, th e distribution of DNA in them was n on-un iform . In som e cases on on the surface of cell m em bran es m ay be observed areas con sistin g of tightly packed pleats of a cytoplasm ic m em bran e resem blin g m ezosom s – intracytoplasm ic bacterial m em bran e of vesicular structure and tubular form form ed by th e invasion of cytoplasm ic m em bran e in to the cytoplasm (Fig. 2). It is assum ed th at m ezosom s in volved in the form ation of cell walls, replication an d segregation of DNA, nucleotides and other processes. Th ere is also eviden ce that the m ajority n um ber of m ezosom s being absen t in norm al cells is form ed by a chem ical action of som e extern al factors – low and high tem peratures, fluctuation of pH an d and other factors. Furtherm ore, deuterated cells of C. v ulgaris were also characterized by a drastic chan ge in cell form an d direction of their division . The observed cell division cytodieresis did n ot en d by the usual divergen ce of the daughter cells, but led to the form ation of abnorm al cells, as described by other authors [24]. Th e observed m orphological changes associated with the in hibition of growth of deuterated cells were stipulated by th e cell restructuring during the process of adaptation to D2О. The fact that the deuterated cells are larger in size (apparen t size was of 2– 4 tim es larger than the size of the proton ated cells), apparen tly is a general biological phenom en n proved by growin g a n um ber of other adapted to D2О prokaryotic an d eukaryotic cells.
Figure 2. Electron m icrographs of M icrococcus ly sodeikticus cells: a) – proton ated cells obtain ed on H2O-m edium ; b) – deuterated cells obtain ed on D2O-m edium [24]. Th e arrows show the
tightly-packed portion s of the m em bran es
Our data gen erally con firm a stable n otion that adaptation to D2О is a phen otypic phen om enon as the adapted cells eventually return back to the n orm al growth after som e lag-period after their replacem ent back onto H2O-m edium . At the sam e tim e the effect of reversibility of growth on H2O/ D2О does not exclude an opportun ity that a certain gen otype determ in es the displaying of the sam e phenotypic attribute in D2О-m edia with m axim um deuterium con ten t. At placin g a cell on to D2О-m edia lackin g protons, not on ly H2O is rem oved from a cell due to isotopic (H – D) exchan ge, but also there are occurred a rapid isotopic (H – D) exchan ge in hydroxyl (-OH ), sulfohy dry l (-SH ) and am ino (-NH2) groups in all m olecules of organ ic substan ces, includin g proteins, n ucleic acids, carbohydrates and lipids. It is known , that in these con dition s only covalent C– H bon d is n ot exposed to isotopic (H – D) exch an ge an d, thereof on ly m olecules with bonds such as C– D can be synthesized de n ovo [25]. Depen din g on the position of the
deuterium atom in th e m olecule, th ere are distin guished prim ary an d secon dary isotopic effects m ediated by in term olecular interactions. In this aspect, the m ost im portan t for the structure of m acrom olecules are dyn am ic short-lived hydrogen (deuterium ) bon ds form ed between th e electron deficient H (D) atom s an d adjacent electron egative O, C, N, S- heteroatom s in the m olecules, actin g as acceptors of H -bon d. The hydrogen bon d, based on weak electrostatic forces, don or-acceptor interactions with charge-tran sfer an d in term olecular van der Waals forces, is of the vital im portance in the chem istry of in term olecular in teraction s an d m ain tain in g the spatial structure of m acrom olecules in aqueous solutions. An other im portan t property is defin ed by the three-dim en sion al structure of D2О m olecule havin g the ten den cy to pull together hydrophobic groups of m acrom olecules to m inim ize their disruptive effect on th e h ydrogen (deuterium )-bon ded n etwork in D2О. This leads to stabilization of the structure of protein an d n ucleic acid m acrom olecules in the presen ce of D2О. Th at is why, the structure of m acrom olecules of protein s an d n ucleic acids in the presen ce of D2О is som ehow stabilized [26].
Eviden tly the cell im plem en ts a special adaptive m echan ism s prom otin g the fun ction al reorgan ization of vital system s in D2О. Thus, for the n orm al syn thesis an d fun ction in D2О of such vital com poun ds as nucleic acids an d protein s con tributes to the m ain ten an ce of th eir structure by form ing hydrogen (deuterium ) bonds in the m olecules. The bon ds form ed by deuterium atom s are differed in stren gth an d en ergy from sim ilar bon ds form ed by hydrogen . Som ewhat greater strength of D–O bond com pared to H–O bon d causes the differen ces in the kin etics of reaction s in H2O and D2О. Thus, according to th e theory of a chem ical bon d th e breakin g up of сovalen t H–C bon ds can occur faster than C–D bon ds, the m obility of D3O+ ion is lower on 28,5 % than Н3O+ ion , and ОD- ion is lower on 39,8 % than OH- ion , the con stan t of ion ization of D2О is less than that of H2O [27]. These ch em ical-physical factors lead to a slow down in the rates of en zym atic reaction s in D2О [28 ]. H owever, there are also such reaction s which rates in D2О are high er than in H2O. In gen eral these reaction s are catalyzed by D3O+ or Н3O+ ion s or OD- an d OH- ion s. The substitution of H with D affects the stability and geom etry of hydrogen bon ds in an apparen tly rather com plex way an d m ay, through the chan ges in the hydrogen bon d zero-poin t vibration en ergies, alter th e conform ation al dynam ics of hydrogen (deuterium )-bon ded structures of DNA an d protein s in D2O. It m ay cause disturban ces in the DNA-syn th esis, leadin g to perm an en t chan ges on DNA structure and consequen tly on cell genotype. Isotopic effects of deuterium , which would occur in m acrom olecules of even a sm all differen ce between hydrogen an d deuterium , would certain ly have the effect upon the structure. The sen sitivity of en zym e fun ction to the structure an d the sen sitivity of nucleic acid fun ction (gen etic an d m itotic) would lead to a n oticeable effect on the m etabolic pathways and reproductive behaviour of an organ ism in the presen ce of D2O. An d n ext, th e chan ges in dissociation con stants of DNA an d protein ion izable groups when tran sferrin g the m acrom olecule from H2O to D2O m ay perturb the charge state of the DNA an d protein m olecules. All this can cause variations in n ucleic acid syn thesis, which can lead to structural chan ges an d fun ction al differences in the cell an d its organ elles. Th us, th e structural an d dyn am ic properties of the cell m em bran e, which depends on qualitative an d quan titative com position of m em bran e fatty acids, can also be m odified in the presen ce of D2O. The cellular m em bran e in the bacteria is on e of the m ost im portant organ elles for m etabolic regulation , com bin in g apparatus of biosyn th esis of polysaccharides, transform ation of en ergy, supplyin g cells with n utrien ts an d in volvem en t in the biosynth esis of proteins, nucleic acids an d fatty acids. Obviously, th e cell m em bran e plays an im portant role in the adaptation to D2O. But it has been n ot clearly kn own what occurs with th e m em bran es − how they react to the replacem en t of H to D an d how it concern s the survival of cells in D2O-m edia devoid of proton s.
Com parative analysis of the fatty acid com position of deuterated cells of chem oheterotrophic bacteria B. subtilis, obtained on the m axim um deuterated m edium with 99,9 at.% D2O, carried out by H PLC m ethod, revealed significan t quan titative differen ces in the fatty acid com position com pared to the control obtain ed in ordinary water (Fig. 3 a, b). Characteristically, in a deuterated sam ple fatty acids havin g retention tim es at 33,38 ; 33,74; 33,26 and 36,0 3 m in are n ot detected in H PLC-chrom atogram (Fig. 3b). This result is apparently due to th e fact that the cell m em bran e is on e of the first cell organ elles, sen sitive to the effects of D2O, and thus com pen sates the chan ges in rheological properties of a m em brane (viscosity, fluidity, structuredness) not on ly by quantitative but also by qualitative com position of m em bran e fatty acids. Sim ilar situation was observed with
the separation of other natural com poun ds (protein s, am in o acids, carbohydrates) extracted from deutero-biom ass obtain ed from m axim ally deuterated D2O-m edium .
Figure 3. H PLC-chrom atogram s of fatty acids obtain ed from proton ated (a) an d deuterated (b) cells B. subtilis on the m axim ally deuterated D2O-m edium . The peaks on chrom atogram s with retention tim e 3,75 m in (instead of 3,74 m in utes in the con trol), 4,10 ; 4,27; 4,60 (in stead of 4,0 8 ; 4,12; 4,28 in the con trol), 5,0 7 (in stead of 4,98 in con trol) 12,57; 12,97 (in stead of 12,79; 13,11; 13,17
in con trol) 14,0 0 (in stead of 14,59 in the con trol), 31,8 7 (in stead of 31,8 3 in the con trol); 33,38 ; 33,74; 33,26; 36,0 3; 50 ,78 ; 50 ,99 (in stead of 51,0 3; 51,25 for con trol) correspon d to in dividual
intracellular fatty acids
Am ino acid analysis of protein hydrolysates and intracellular carbohydrates isolated from deuterated cells of B. subtilis, also revealed the differences in quantitative com position of am in o acids syn thesized in D2O-m edium . Protein hydrolyzates contains fifteen iden tified am in o acids (except proline, which was detected at λ = 440 nm) (Table 2). An indicator that determines a high efficiency of deuterium inclusion into am ino acid m olecules of protein hydrolyzates are high levels of deuterium enrich m ent of am ino acid m olecules, which are varied from 50 at.% for leucine/ isoleucin e to 97,5 at.% for alanine
Table 2
Am in o a cid co m p o s itio n o f th e p ro te in h yd ro lys a te s o f B . s u b t i lis , o bta in e d o n th e m a x im u m d e u te ra te d m e d iu m a n d le ve ls
o f d e u te riu m e n rich m e n t o f m o le cu le s * Am in o acid Yield, % (w/ w) dry weight per 1
gram of biom ass
Num ber of deuterium
atom s in corporated in to the carbon backbon e of a m olecule**
Level of deuterium
en richm en t of m olecules, % of the
total n um ber of hydrogen atom s***
Protonated sam ple (con trol)
The sam ple obtain ed in 99,9 % D2O
Glycin e 8 ,0 3 9,69 2 90 ,0
Alanin e 12,95 13,98 4 97,5
Valin e 3,54 3,74 4 50 ,0
Leucin e 8 ,62 7,33 5 50 ,0
Isoleucin e 4,14 3,64 5 50 ,0
Phenylalan in e 3,8 8 3,94 8 95,0
Tyrosin e 1,56 1,8 3 7 92,8
Serin e 4,18 4,90 3 8 6,6
Threonin e 4,8 1 5,51 − −
Methion in e 4,94 2,25 − −
Asparagin e 7,8 8 9,59 2 66,6
Glutam ic acid 11,68 10 ,38 4 70 ,0
Lysin e 4,34 3,98 5 58 ,9
Argin in e 4,63 5,28 − −
H istidine 3,43 3,73 − −
Notes:
* The data obtain ed by m ass spectrom etry for the m ethyl esters of N-(dim ethylam in o) n aphthalen e-1-sulfon yl chloride (dan syl) am in o acid derivatives.
** While calculatin g the level of deuterium en richm en t proton s (deuteron s) at the carboxyl (COOH -) an d NH2-groups of am in o acid m olecules are n ot taken in to accoun t because of their easy dissociation in H2O/ D2O
*** A dash m eans absen ce of data.
Qualitative and quan titative com position of the in tracellular carbohydrates of B. subtilis obtain ed on m axim ally deuterated D2O-m edium shown in Table. 3 (th e n um berin g is given to th e sequence of their elution from the colum n ) con tain ed m on osaccharides (glucose, fructose, rham nose, arabinose), disaccharides (m altose, sucrose), an d four other un iden tified carbohydrates with reten tion tim e 3,0 8 m in (15,63 %); 4,26 m in (7,46 %); 7,23 m in (11,72 %) an d 9,14 m in (7,95 %) (n ot shown ). Yield of glucose in deuterated sam ple m akes up 21,4 % by dry weight, i.e. higher than for fructose (6,8 2 %), rham n ose (3,47 %), arabin ose (3,69 %), an d m altose (11,62 %). Their outputs are not significantly differen t from con trol in H2O except for sucrose in deuterated sam ple that was not detected (Table 3). The deuterium en richm en t levels of carbohydrates were varied from 90 ,7 at.% for arabin ose to 8 0 ,6 at.% for glucose.
Table 3
Qu a lita tive a n d q u a n tita tive c o m p o s itio n o f in tra c e llu la r ca rbo h yd ra te s o f B . s u b t ilis o bta in e d o n th e m a x im a lly d e u te ra te d m e d iu m a n d le ve ls o f d e u te riu m e n ric h m e n t
o f m o le cu le s *
Carbohydrate Con ten t in the biom ass, % of the dry weight of 1 g biom ass
Level of deuterium en richm en t, % of the total n um ber of hydrogen atom s***
Protonated sam ple (control)
The sam ple obtain ed in 99,9 % D2O**
Glucose 20 ,0 1 21,40 8 0 ,6
Fructose 6,12 6,8 2 8 5,5
Rham nose 2,91 3,47 90 ,3
Arabinose 3,26 3,69 90 ,7
Maltose 15,30 11,62 −
Sucrose 8 ,62 ND −
Notes:
* Th e data were obtained by IR-spectroscopy. ** ND − n ot detected
** A dash m ean s the absence of data.
Co n clu s io n s .
Our experim en ts dem onstrated that the effects observed at the cellular growth on D2O possess a com plex m ultifactor character con n ected to ch an ges of physiological param eters – m agnitude of the lag-period, tim e of cellular gen eration , outputs of biom ass, a ratio of am in o acids, protein , carbohydrates an d fatty acids syn thesized in D2O, an d with an evolution ary level of organ ization of investigated object as well. Th e cell eviden tly im plem en ts th e special adaptive m echanism s prom oting fun ctional reorgan ization of work of the vital system s in the presen ce of D2O. Th us, the m ost sensitive to replacem en t of Н on D are the apparatus of biosyn thesis of m acrom olecules an d a respiratory chain , i.e., th ose cellular system s usin g high m obility of proton s an d high speed of breakin g up of hydrogen bon ds. Last fact allows the con sideration of adaptation to D2O as adaptation to the n onspecific factor affectin g sim ultan eously the fun ction al con dition of several n um bers of cellular system s: m etabolism , ways of assim ilation of carbon substrates, biosyn th etic processes, and transport fun ction , structure an d fun ction s of m acrom olecules. It seem s to be reason able to choose as biom odels in these studies m icroorgan ism s, as they are very well adapted to the environm en tal con dition s an d able to withstan d high con cen tration s of D2O in the growth m edia.
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УДК 579.8 71.0 8 +577.38 5.4.0 8
Биологическое влияние дейтерия на клетки прокариот и эукариот
1 Олег Викторович Мосин 2 Игнат Игнатов
1 Московский государственный университет прикладной биотехнологии, Российская Федерация Старший научный сотрудник кафедрыбиотехнологии, канд. хим. наук
103316, Москва, ул. Талалихина, 33 E-mail: [email protected]
2 Научно-исследовательский центр медицинской биофизики (РИЦ МБ), Болгария
Профессор, доктор наук Европейской академии естественных наук (ФРГ), директор НИЦ МБ 1111, София, ул. Н. Коперника, 32/ 6
Аннотация. Изучено биологическое воздействие дейтерия (D) на клетки различных
таксономических групп прокариотических и эукариотических микроорганизмов, реализующих метилотрофные, хемогетеротрофные, фото-органогетеротрофные и фотосинтетические способы ассимиляции углеродных субстратов при росте на средах с тяжёлой водой (D2О). Разработан метод ступенчатой адаптации клеток к D2О, заключающийся в их рассеве на чашках Петри с твердыми (2 %-ный агар) питательными средами при ступенчатом увеличении градиента концентрации D2О (от 0 до 98 % D2O) и последующей селекции устойчивых к D2O клеток. В результате этой техники на максимально дейтерированной среде с 98 % D2О получены адаптированные к D2О клетки, биологический материал которых вместо атомов водорода содержит атомы дейтерия с уровнем дейтерированности молекул 92–97,5 ат.% D.
Ключевые слова: дейтерий; тяжелая вода; изотопные эффекты; адаптация;
бактерии; сине-зеленые водоросли.
