Xenon-Water Interaction in Bacterial Suspensions as Studied by NMR

Abstract

Xenon is a perspective gas for creation of oxygen free environment for different applications of biomaterials. To use xenon in suspensions and products properly it is necessary to know the molecular mechanisms of its interactions with water and cells. This work reports the study of bacterial suspensions of Escherichia coli in the presence of xenon using nuclear magnetic resonance (NMR). The work studied how the spin-lattice relaxation times of water protons in suspension change under xenon conditions. Xenon is able to form clathrate hydrates with water molecules at a temperature above the melting point of ice. The work studied NMR relaxation times which reflect the rotation freedom of water molecules in suspension. Lower relaxation times indicate reduced rotational freedom of water. Single exponential behavior of spin-lattice relaxation of protons in the suspensions of microorganisms has been registered. A recovery of longitudinal magnetization in cell suspensions with xenon clathrates has been characterized by two peaks in T1-distribution. Fast relaxing T1-component was related to the intracellular water and depended on the amount of xenon clathrates. The obtained results elucidate how the NMR method can monitor the process of clathrate formation and how the xenon atoms and hydrates interact with cells.

Full text

International Journal of Biochemistry and Biophysics 5(1): 26-36, 2017 http://www.hrpub.org
DOI: 10.13189/ijbb.2017.050104
Xenon-Water Interaction in Bacterial
Suspensions as Studied by NMR
Victor Rodin1,*, Alexander Ponomarev2, Maxim Gerasimov3, Leonid Gurevich4
1Institute of Organic Chemistry, Johannes Kepler University Linz, Altenbergerstraße 69, 4040 Linz, Austria
2Department of Medical Cell Technologies, Ural State Medical University, Ekaterinburg, Russian Federation
3Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
4Department of Physics and Nanotechnology, Aalborg University, Aalborg, Denmark
*Corresponding Author: victor.rodin@jku.at
Copyright© 2017 by authors, all rights reserved. Authors agree that this article remains permanently open access under the
terms of the Creative Commons Attribution License 4.0 International License
Abstract Xenon is a perspective gas for creation of
oxygen free environment for different applications of
biomaterials. To use xenon in suspensions and products
properly it is necessary to know the molecular mechanisms
of its interactions with water and cells. This work reports
the study of bacterial suspensions of Escherichia coli in the
presence of xenon using nuclear magnetic resonance
(NMR). The work studied how the spin-lattice relaxation
times of water protons in suspension change under xenon
conditions. Xenon is able to form clathrate hydrates with
water molecules at a temperature above the melting point of
ice. The work studied NMR relaxation times which reflect
the rotation freedom of water molecules in suspension.
Lower relaxation times indicate reduced rotational freedom
of water. Single exponential behavior of spin-lattice
relaxation of protons in the suspensions of microorganisms
has been registered. A recovery of longitudinal
magnetization in cell suspensions with xenon clathrates has
been characterized by two peaks in T1-distribution. Fast
relaxing T1-component was related to the intracellular water
and depended on the amount of xenon clathrates. The
obtained results elucidate how the NMR method can
monitor the process of clathrate formation and how the
xenon atoms and hydrates interact with cells.
Keywords Bacterial Suspensions, Water, Xenon,
Clathrates, Nuclear Magnetic Resonance
1. Introduction
Despite their inertness, noble gases possess a number of
interesting properties, including their anaesthetic effect
[1–4]. They are very soluble in lipids and may bind to both
membrane lipids and proteins [3–5]. Because of the high
correlation between lipid solubility and anaesthetic potency
(known as the Meyer-Overton correlation) it was suggested
that the molecular mechanisms of general anaesthetics are
associated with the interactions between anaesthetic agents
and a hydrophobic interior of the lipid bilayers [2, 4, 5].
Although different theories have been discussed in the
literature for long time, the exact mechanism of anaesthetic
action of noble gases is still a mystery [5, 6]. It is not clear
which of the different mechanisms suggested is most likely
to be the main mechanism of action of anaesthetic agents.
Xenon is the most potent anaesthetic of all the noble gases.
The high polarizability of xenon compared to the other noble
gases increases its affinity to hydrophobic environments.
Thus xenon can be substantially partitioned into the
hydrophobic core of the membrane bilayer. The solubility of
xenon in human serum albumin solutions, hemoglobin
solutions, human blood, and other biological solutions and
homogenates was studied in the Ref. [7]. For example, at 298
K the amount of xenon dissolved in 1 g human albumin at
0.1013 MPa of total pressure was found to be 0.2382 ml.
According to the data [3] a blood-gas partition coefficient for
xenon is 0.115 whereas the solubility in water measured
identically was 0.096.
The anaesthetic gas xenon is attractive for many
applications in biotechnology and medicine [8–10]. Xenon is
also the perspective gas for preservation of biological
materials including different enzymes and cells [11–16].
This noble gas is able to replace lighter gases of air, e.g.
oxygen and nitrogen [17, 18]. This inert gas was already
applied for many biological and medical systems in creation
of oxygen free environment, in particular, for bacterial cell
suspensions and erythrocytes of blood [9, 10, 19–24]. The
authors of Refs. [19, 20] developed the method of platelet
storage in the presence of xenon. In order to use the xenon in
the storage of biological suspensions and biotechnological
products properly it is necessary to know the molecular
mechanisms of its interactions with cell membrane and cell
components [5, 11, 13, 21]. Nowadays many questions are
still opened.

International Journal of Biochemistry and Biophysics 5(1): 26-36, 2017 27
One important feature of xenon is its ability to create
clathrate hydrates from water molecules at relatively “soft”
conditions when the pressure is still about 0.4–0.7 MPa and
temperature circa 276–278 K [12–14, 21]. When forming
hydrates, lower hydrate formation pressures are desirable in
practice [21, 25, 26]. It is known that crystalline hydrates are
formed by many simple gases that do not interact strongly
with water, and mostly the gas molecules or atoms occupy
“cages” formed by the network of water molecules. The
majority of these gas hydrates adopt one of two possible
cubic cage structures and therefore are called clathrate
hydrates [21, 25, 26]. Although high pressure is often
needed to hold gas atoms inside the water “cages”, xenon
gas is an exception from the rule. Xenon hydrates are the
clathrates of low pressure. They can be stabilized at about
276–280 K [10, 12, 25].
Clathrate hypothesis of anaesthesia of L.Pauling [27, 28]
suggested the existence of clathrate microcrystals formed by
anaesthetic gas and water molecules even at physiological
conditions (at least momentarily): normal atmospheric
pressure and temperature of cells. Some suggestions of
clathrate theory of L.Pauling have been checked later in Refs.
[27–33]. First studies on formation of xenon clathrates in
cellular suspensions of microorganisms have been initiated
in Refs. [9, 13, 14, 34, 35]. Those publications showed how
the methods of magnetic resonance can produce valuable
information about the changes in bacterial medium under gas
pressure. Later publications [21, 36–40] reported more NMR
results on xenon clathrates. The use of paramagnetic
additives expanded the possibilities of NMR relaxation in
studying cell suspensions with gas hydrates [21, 36]. NMR
was also applied for studying clathrate formation in two-
component and complex systems [9, 37–40]. Nevertheless
many questions about the role of xenon and its clathrates in
cell suspensions still remain open.
The most intensively investigated prokaryotic model
organism in biophysics is the Escherichia coli (E.coli), K-12
strain [41, 42]. It is considered as very important
representative of cellular suspensions in biotechnology.
E.coli is a common gram-negative rod-shaped bacterium,
found in normal human (and other animals) bacterial flora. It
is about 2.0 µm in length and its diameter is 0.25–1.0 µm
[41]. These microorganisms are facultative anaerobic
bacteria which can be easily grown in a laboratory.
The current work reports NMR-study of bacterial
suspensions of E.coli under xenon pressure addressing how
the xenon and its hydrates participate in the interactions with
cells and affect the state of intra- and intercellular water. The
biomaterials with crystals of xenon clathrates are
“transparent” for radio waves. This allows using the methods
of radio spectroscopy (i.e., NMR) to analyze the structural
and phase state and molecular-dynamic properties of water
in the cell suspensions in the presence of a clathrate phase.
The paper presents how NMR water proton spin-lattice
relaxation times in E.coli suspensions vary under xenon
conditions.
2. Experimental Materials and Methods
2.1. Materials
The cells of E.coli have been grown in flasks with volume
of 0.75 liter using beef-extract broth or mineral liquid
medium with glucose. A sterilized phosphate buffered saline
(PBS) was added. The cells at the stationary phase of growth
were used (age of 14–16 hrs). The samples with cells were
centrifuged at 3000 rpm for 15 min. The precipitate was
washed twice in distilled water, PBS and was used in
subsequent experiments as in Refs. [9, 21, 36]. A
concentration of the biomaterial (cellular suspensions) under
study was expressed in g of dry mass per g H2O and by a
number of cells per 1 ml [21].
2.2. Custom-made Thermostat Chamber with Xenon Kit
We designed and built thermostat chamber with
transparent front door from polymethyl methacrylate. Inside
the chamber a system to transfer the gas to tubes with
samples was built. The gas cylinder (with medical grade
xenon) was connected to the special kit with tubes filled by
suspensions / biomaterials [21, 27, 36]. The tubes with
samples were under gas pressure as described in earlier
publications [21, 37]. Mostly the pressure circa 0.8–1.0 MPa
or less was enough to produce stable clathrates at 278–280 K.
In some experiments “dry” ice (carbon dioxide) was used to
touch the bottom of the tubes for the initiation of clathrate
formation according to described procedure [21, 36, 43].
After saturation of the samples with gas and xenon clathrate
formation the tubes could be detached from the kit in the
thermostat chamber and be used in NMR measurements at
above melting point of common ice but not exceeded 286 K.
2.3. NMR Methods in Studying Water Clathrates of
Xenon
The NMR studies of cell suspensions and suspensions
with clathrate crystals were performed at a proton resonance
frequency of 90 MHz. Some experiments have been carried
out also at 20 MHz. In the single-pulse 1H NMR experiments,
the free induction decay (FID) signals were measured. The
FID consists of a 90° pulse which rotates the magnetization
onto the transverse plane where it decays with characteristic
time. The initial part of the FID is hidden in the dead-time
zone of the spectrometer. The length of the 90° pulse was 8
µs. The FIDs on the samples with clathrates have been
measured according to the procedure of earlier publications
[27, 37, 43]. The signal of total time-domain is the sum of
the signals of the water protons and the protons of clathrate
crystals: S = Swat + Sclath. In the study of water dispersions of
xenon clathrates [21,37] it was shown that spin-spin
relaxation time T2 of the fast relaxing component (protons of
clathrates) did not exceed 25 µs. The structure of clathrate
ice in studied solutions and suspensions at above melting
point of common hexagonal ice was relatively “softer” than
that in common ice with T2 ~ 10 µs. For ice protons the most

28 Xenon-Water Interaction in Bacterial Suspensions as Studied by NMR
part of FID was hidden in the dead-time zone. Only the tail
of the transverse magnetization decay might be observable in
the experiment. Although the part of FID for the clathrates is
a bit longer than the FID for common ice, the situation with
FID in clathrates is comparable with that in ice. To monitor
the water signal only, the contribution of the protons of
clathrate ice should be subtracted. In such a case, normally,
the measurements were carried out at experimental
conditions when transverse magnetization of clathrate
protons decayed very fast to zero (whereas the transverse
magnetization of liquid water has not decayed) and only
protons of water could be measured in the study [37,43]. On
the base of this approach a fraction of water built in clathrate
structures was determined using the difference of the proton
signals of liquid water in suspensions before and after
clathrate formation [21].
The spin-lattice relaxation time T1 was measured by the
inversion recovery (IR) method using the pulse sequence
(180°–t‒90°) at the pulse sequence repetition time ≥ 5T1 [45,
46]. The period of 5T1 was waited between the consecutive
scans of pulse sequence in order to ensure that the fully
recovered signal was acquired. When t is varied then
experimental dependence of magnetization Mt = f (t) can be
obtained and fitted by the law Mt = ∑ M0i·[1‒2·exp(‒t/T1i)],
where i is the number of relaxation component. The analysis
of relaxation data was conducted after transferring the raw
data from spectrometer computer to desk computer using
in-house MatLab® codes. In these NMR studies there was an
aim to model experimental data as a sum of several
relaxation components or distribution of NMR-relaxation
times. To fit the data inverse Laplace transform (ILT) in one
direction was commonly applied [47–50]. The kernel
function in ILT was associated with Mt data obtained from
IR NMR experiments.
3. Results and Discussion
3.1. Spin-lattice Relaxation of Water Protons in
Suspensions of E.coli before and after Xenon
Clathrate Formation
The T1–experiments discovered that the recovery of
longitudinal magnetization of measurable protons in all
studied samples of cell suspensions (concentration range of
0.001–0.24 g dry mass /g H2O) can be fitted by single
exponential curve and characterized by a single value of the
spin-lattice relaxation time T1. However a recovery of
longitudinal magnetization of protons in bacterial
suspensions with clathrates of xenon has bi-exponential
behavior. T1 values of the two exponential components differ
by a factor of 6 to 10. Alternatively, the treatment of the
recovery of longitudinal magnetization using inverse
Laplace transform resulted in a distribution of spin-lattice
relaxation times T1 in the kind of 2 peaks. Fig.1 shows
T1-distributions obtained by one- dimensional ILT applied to
the data sets for original bacterial suspension and E.coli with
xenon clathrates.
The two peaks registered for the suspension of E.coli with
xenon clathrates (Fig.1) had maximums at 0.25 and 1.2 s.
The integral intensities of these peaks corresponded to 66%
and 34 % of measurable protons. The single peak observed in
T1-distribution in suspension of E.coli without clathrates was
at T1 ≈ 0.70 s (Fig.1). This was in line with previously
published NMR relaxation data on cellular suspensions [21,
40] which showed fast exchange between the protons of
intra- and intercellular water, i.e., single exponential T1
-relaxation [51‒53]. The permeability of the cell membrane
to water is quite high (for instance, in red blood cells
exchange time of the order of 10 ms [53]). Water proton
relaxation times in E.coli bacteria showed one exponential
behavior, i.e., two sites (two compartments: intracellular and
extracellular spaces) are in a fast exchange limit. Thus a
researcher needs to reduce one of the relaxation times to less
than exchange time in order to remove this condition and to
generate two exponential behavior [51, 52]. In the presence
of paramagnetic ions of manganese in the extracellular water
shortening the relaxation times outside the cells it was
possible to record two separate NMR signals in the
population of cells with non-damaged cellular membranes
[36, 51, 52].
The relaxation rates of the water molecules in cell
suspensions are governed by several important factors, in
particular, the strength of local magnetic interactions
between water nuclei, the molecular motion and proton
exchange rates. Nuclear magnetic inter- and intramolecular
dipole-dipole coupling describes the interactions between
water nuclei [51‒53]. The magnetic interactions are partially
averaged within the hydration layer by the processes
depending on the interactions of water with
macromolecules/cell surface/clathrates. These are proton
transfer, dynamic orientation and diffusion of water
molecules through regions of different orientations [26, 40,
44, 53]. The NMR relaxation times are sensitive to molecular
motions in the range of 10-8 to 10-12 s [45, 46, 53]. In liquid
solutions water molecules tumble at a rate of about 10-12 s
[45]. This motion is considerably slowed down when water
molecules interact with biological macromolecules/cell
surface. For example, the correlation time is of the order of
10-12 s for the water molecules non-involved in the
association through hydrogen bonds with surface of cells.
The rotational motion of water molecules associated via
hydrogen bonds with polar groups of the macromolecules is
reduced so that their correlation time is even sometimes of
the order of 10-6 s [53]. Under the conditions of rapid
exchange between the hydration and bulk water in
suspensions and macromolecular solutions, single relaxation
rate (1/T1) is observed.
Single exponential relaxation of protons in suspensions of
E.coli confirmed fast exchange between intracellular and
intercellular water on the NMR time scale. Formation of
xenon clathrates affected the exchange rate and resulted in
the measurement of two separate compartments. The
population of fast relaxing T1-component was associated
with the fraction of intracellular water. It depended on the
amount of xenon clathrates.
Fig.2 shows how the amount of clathrate ice, i.e. the
amount of water molecules bounded within the structure of

International Journal of Biochemistry and Biophysics 5(1): 26-36, 2017 29
xenon clathrates, depends on the concentration of cells in
suspension. The fraction of clathrates has been calculated
according to the procedure [37‒40] which compares the
measurements of the FID in original bacterial suspension
and suspension with xenon clathrates. The transition of
liquid water into solid phase of clathrates resulted in a
change of spin-spin relaxation times T2 of protons. T2 ~ 1‒2 s
for free mobile water whereas T2 ~ 10 µs for protons of solid
phase. Such a difference in T2-values gives an opportunity to
separate proton signals of liquid and solid phases much
better. The results of Fig.2 are in line with the previously
published data on the xenon clathrates formation in solutions
of biomacromolecules [12, 27, 31, 32, 34]. It was shown [31,
32, 34] that a water fraction bound in clathrate structures was
due to abundance of hydrophobic groups on the surface of
proteins. Probably some groups on the cellular surfaces also
participate in the formation of xenon hydrates stabilizing
clathrate lattice from water molecules [21,28,31]. As a result
the amount of stable clathrate structures was increasing with
cell concentration in studied range. One observed that the
amplitude of fast relaxing T1–component (relatively to total
water content in cell suspension) increased nonlinearly with
an increase in the initial cell concentration. Apparently this is
due to the advent of solid phase of water (clathrates) and
subsequent displacement of the microorganisms in the free
volume of the suspension.
Fig.3 shows calculated fraction of intracellular water
(open circles) and the dependence of the population of the
fast relaxing T1-component on cell concentration
recalculated to the signal of liquid water. This recalculation
shows the true concentration of the cells remaining in the
liquid phase of the suspension after the formation of
clathrates. The dependence of the population of this
T1-component looks almost linear, and this assumes that the
fast component of T1 is due to the protons of intracellular
water. The slow T1-component can be attributed then to the
extracellular water protons. This assignment is further
supported by a decrease of the population of the slow
component with increasing concentration of cells. A fraction
of the extracellular water protons which are not in clathrate
structures decreased. According to this analysis, the cell
concentration increased relatively to volume of liquid phase
after clathrate formation. A fraction of intracellular water
(Fig. 3, open circles) was calculated on the base of summary
volume of cells and had a linear dependence on
concentration of cells.
0.1 1 10
0.000
0.005
0.010
0.015
T1 (s)
Intensity (a.u.)
Figure 1. T1-distribution of 1H obtained by one-dimensional ILT on the recovery of longitudinal magnetization data set measured in the suspension of
E.coli with concentration of 0.07 g dry mass/g H2O (open circles) and in the same sample after xenon clathrate formation at 1.2 MPa (solid line). T=280 K.
Frequency is 90 MHz.
The integral intensities of 2 peaks in the suspension of E.coli with xenon clathrates (from left to right) are 66% and 34 % of measurable protons. The signals
in each T1-experiment have been normalized per maximal signal registered after total recovery of longitudinal magnetization.

30 Xenon-Water Interaction in Bacterial Suspensions as Studied by NMR
0 2 4 6 8
0
20
40
60
80
100
C (x1010 cell/ml)
water in clathrates (%)
Figure 2. The dependence of the amount of H2O molecules bounded within the clathrate structures (i.e., protons of solid phase with T2 ~ 10 µ s) on the
concentration of cells in the suspensions of E.coli with xenon clathrates. The results were obtained from NMR (FID) measurements taking into account the
procedure of Refs. [37, 38]. T = 280 K. Pressure is 1.2 MPa. Frequency is 20 MHz.
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35
0
20
40
60
80
100
C (g dry mass / g H2O)
Intensity (%)
Figure 3. The relative amplitude of fast relaxing T1-component (vertical line segments) and calculated fraction of intracellular water (open circles) in the
liquid phase of E.coli suspensions with xenon clathrates as a function of cell concentration (C). Solid line is the linear fit to the data (open circles) calculated
on the base of summary volume of cells.
T = 280 K. Pressure is 1.2 MPa. Frequency is 90 MHz.

International Journal of Biochemistry and Biophysics 5(1): 26-36, 2017 31
It is worth to emphasize that the dependence of
intracellular water amount on the concentration of cells in
suspension (Fig.3, open circles) ideally should be a straight
line coming through the origin of the coordinates and the
meaning which can be obtained at tightly packed cells, i.e.,
when extracellular water would be completely removed. The
experimental curve for the population of the fast relaxing
T1-component is positioned lower than that based on the total
volume of the cells (Fig.3). This could be explained by the
osmotic compression of the cell due to an increase in the
concentration of solutes in extracellular space upon xenon
clathrate formation and a decrease in the amount of
remaining liquid water. This is in line with the published data
on paramagnetic doping (when the Mn2+ ions are introduced
into the extracellular water to separate the proton signals of
intracellular and extracellular compartments) in studying the
xenon clathrate formation in the extracellular compartments
of bacterial suspensions [21, 36].
The behavior of T1-components in E.coli suspensions with
xenon hydrates depended on the amount of clathrate
microcrystals. This suggests that the proton exchange
between the extracellular water and the protons of solid
clathrate lattice could also affect T1-component of the
extracellular water. This is in line with some published
NMR-data [37, 39, 43, 44, 51]. Ref. [44] showed that the T1
of protons of ice was increasing with decreasing the
temperature from 273 K to 213 K. For example, at T=273 K
the T1 of protons of ice was 0.5 s increasing to 3 s at 250 K. In
contrast to ice the T1 of protons of liquid water decreased
with decreasing the temperature of water [43, 44, 51].
According to the published data on dispersions of xenon
clathrates in pure water [37, 39], the temperature dependence
of T1 for water protons showed a minimum. A proton
exchange between liquid water and solid clathrate phase
resulted in relaxation behavior of the protons of water
molecules displaced among clathrate microcrystals in
dispersion. Upon this influence dependence of T1 on
temperature for the protons of water remaining in liquid state
became similar to that for the protons of ice. Thus the
protons of liquid water near the crystals of clathrate ice could
experience spin-lattice relaxation with increased values of T1
exceeded typical values for liquid water. However an
interpretation of longitudinal relaxation in E.coli suspension
with xenon hydrates could be much harder than it was for
clathrate dispersions of xenon in pure water because of
additional relaxation sinks and hydration of cell surfaces. For
phase of clathrate crystals at the temperatures above 273 K
T1 is probably of the order of few seconds (the estimate is
based on the mobility of water molecules in ice at 213‒273 K
and in clathrate crystals at 275‒280 K [25, 26, 30, 38, 44,
54]). Then for the protons of liquid water remaining in
extracellular compartment and dispersed between hydrate
crystals and bacterial cells we can expect T1 to be of the order
of 1 s. The amount of water molecules participating in the
exchange with clathrate phase was increasing with growth of
the amount of hydrate crystals in suspensions as surface of
boundaries of water-clathrate ice was growing up. This
resulted in changing spin-lattice relaxation time T1 for slow
component of longitudinal magnetization recovery. In one
particular example for E.coli suspension with xenon
hydrates, T1 doubled compared to the initial spin-lattice
relaxation constant of T1=0.54 s at a cell concentration of
0.23 g dry mass / g H2O.
According to the data of Ref. [44], the proton spin-lattice
relaxation times in carefully purified ice have been measured
for the temperature range of 213‒273 K. The authors of this
work considered a relationship of T1 with rotational jump
time τj as follows: T1=2.14×105×τj. The dependence of T1
against inverse temperature has been characterized by single
apparent activation energy of Ea ≈59 kJ/mol [44] (or Ea ≈56.3
kJ/mol according to the data published in Ref. [40]) and T1 in
ice was frequently dependent as ~ω02 [44]. So the
measurements of T1 at different resonance frequencies could
also clarify some details in frequently connecting motion of
protons in suspensions with xenon hydrates. From the data of
Refs. [37, 43] the temperature dependence of T1 (an
Arrhenius plot [21,40] in the range of 278‒286 K) for the
protons of water molecules displaced among clathrate
microcrystals in dispersion gave the apparent activation
energy of Ea =26.4±3.7 kJ/mol. NMR data of Ref. [55]
presented apparent activation energy for xenon hydrates as
Ea =24.7±3.3 kJ/mol.
In order to estimate correlation times τc (as characteristics
of molecular motions based on NMR relaxation time
measurements) in E.coli suspensions with xenon clathrates
we applied the approach of earlier works [45, 46, 51, 53, 56]
assuming the motions involved in intramolecular relaxation
and using the expressions reported for 1/T1 in terms of the
correlation times:
)},2(4),({
3
21 00
1cc JJC
T


with
22
( , ) 1c
cc
J

 

(1)
where ω0 is the Larmor angular frequency in a constant
magnetic field B0. It is related to the resonance frequency ν0
by the relation ω0=2πν0. C = 2.51010 s–2 is the constant of
dipole binding of water molecules.
The use of Eq.(1) can give first estimation of the
relaxation behavior of the protons of remaining water
molecules displaced among clathrate microcrystals in
bacterial suspensions assuming some values of correlation
times. According to published data [56‒58] T1 values for the
unfrozen water in some biomaterials (e.g., solutions of
agarose or lysozyme) indicate that mobility of water
remaining in liquid state can be described by a distribution of
correlation times with single peak centered at τc ~ 10-9 s. For
the conditions (temperature, pressure) when xenon still did
not form clathrates in suspension each water molecule
spends part of its time as isotropic water with single
correlation time. The correlation between motion of protons
from the water molecules and from macromolecules in
principle gives rise to the process of cross-relaxation or
spin-diffusion. The observed relaxation rates of water
protons can be influenced by cross-relaxation rates.
Spin-diffusion occurs by way of mutual exchange of

32 Xenon-Water Interaction in Bacterial Suspensions as Studied by NMR
spin-magnetization between water protons of the hydration
water and protons on macromolecules / cell surface. The
contribution of cross-relaxation can become very important
under the conditions of slow molecular diffusion.
The mobility of water remaining in suspensions after
xenon hydrate formation might be comparable with that for
unfrozen water in the solutions of biomolecules. This allows
a calculation of T1 at two experimental resonance
frequencies (90 and 20 MHz) at the value of τc, for instance,
of 2.010-9 s. The results show reasonable difference (3‒4
times) in ratio of T1-values for 90 and 20 MHz although the
value T1≈28 ms (at 90 MHz) looks very small in comparison
with experimental T1-values for extracellular water (0.5‒1.2
s) in bacterial suspension with xenon clathrates. For changed
value of correlation time (one order) to τc=2.010-10 s the
calculation of T1 according to Eq.(1) would result in
T1=60‒63 ms (these simulated values are still small in
comparison with T1 from experiments) showing very small
difference between simulated T1 values for 90 and 20 MHz.
The estimation for next change to τc=2.010-11 s was also
unsuccessful to match the experimental meanings of T1 for
two resonance frequencies by the calculated values. This
suggests that the motions with correlation times τc ~10-9 s (or
less for one-two orders) are not likely to be main input in
bacterial suspensions with xenon clathrates. So the approach
with one correlation time based on literature data should be
definitely changed to distribution of τc to fit the experiments
in E.coli suspension with clathrates of xenon.
3.2. Factors Defining Mobility of Water among Xenon
Clathrates in Bacterial Suspension
The approach of Ref. [56] which studied bound water in
frozen erythrocytes by NMR could be probed on bacterial
cells adopting the usual formulations of the relationship
between relaxation times and correlation times and assuming
as well a log-normal distribution of correlation times with
distribution function:
dZ
Z
dP ])(exp[
1
)( 2




(2)
Here Z=ln(τ/τ*) and τ* is the median of the correlation
time distribution and β is the distribution width. The authors
of Ref. [56] assumed that β is independent of temperature
and τ* is dependent of temperature following to Arrhenius
expression with activation energy for motion corresponding
to the correlation times. The spin-lattice relaxation rate 1/T1
is then [56]:






02
0
02
01
]
)2(1 )(2
2
)(1 )(
[
3
21




dPdP
C
T
(3)
For the evaluation of the relaxation rates it is necessary to
estimate the integrals in Eq.(3). In Ref. [56] this was done
numerically using the relation [57]:




dX
dP 12
02
0
0)])[cosh(exp(
2
1
)(1 )( 





(4)
for a log-normal distribution where X =ln(ω0·τ*).
The approach to fit relaxation data in bacterial
suspensions with xenon clathrates is based on certain
assumptions (water molecules are undergoing to
translational and rotational motions governed by a
distribution of correlation times) which allow several
adjustable parameters for each correlation time: dipole
constant C, β, τ* and activation energy stemming from
temperature dependence of τ*. In principle these parameters
could be determined by a computer fit of the data using an
iterative method. In reality, the dipole constant of water, in
other words, the rigid-lattice second moment is in the range
from 1.51010 s–2 for isolated molecule of water to 2.61010
s–2 for crystals of ice [56]. There is indefinite choice for the
constant of dipole binding as the relaxation data on T1 were
obtained for the remaining water in E.coli suspensions after
clathrate formation experiencing an exchange with solid
phase of clathrate ice.
Fitting the data to the model with one log-normal
distribution of correlation times gives the values for the most
of the parameters as widely varied. The authors of Ref. [56]
for the remaining liquid water in frozen red blood cell (RBC)
suspension proposed a model which describes NMR
relaxation data by a distribution with two peaks. Additional
parameters for the fraction of water molecules under first
peak and for the fraction of water molecules under second
peak were introduced in order to fit the distribution of τc.
Analysing the published model for RBC suspensions we
suppose that for clathrates of xenon in cell suspensions more
consistent model could be developed further. Some
additional experiments should involve relaxation of xenon
atoms and introduction of parameters responsible for the
presence of xenon in suspensions. An idea to explain a cell
metabolism during/after xenon clathrate formation could be
developed to add the details to total pattern of cell changes.
An increase of salt concentration in extracellular water at
xenon clathrate formation causes shrinking the cells by loss
of free water. When the cells compressed the anion and
cation flux across the membrane can be controlled and
coupled to the cell metabolism involving a regulation of
water structure. The remaining intracellular water can be
more highly structured resulting in decreased relaxation
times (i.e., increased rates R1=1/T1) with cell concentration
(Fig.4, Fig.5).
For the future modelling the suspensions with xenon it is
necessary also to take into account the changes produced by
xenon dissolved in cell suspension. It is possible to consider
extracellular water as saline with a concentration 0.9% by
weight. According to the data [59] the solubility of xenon in
saline water is low, in particular, Ostwald coefficient is equal
to 0.0926 (at standard temperature and 0.101 MPa of gas
pressure). However the T1 value of xenon is quite long (66 s,
when measured at magnetic field B0 =9.4 T) due to dipolar
couplings with proton spins [59].

International Journal of Biochemistry and Biophysics 5(1): 26-36, 2017 33
0 1 2 3 4 5 6 7 8
0
1
2
3
4
C (x1010 cell/ml)
R1, slow (s-1)
Figure 4. Spin-lattice relaxation rate R1=1/T1 of slow relaxing component of longitudinal magnetization of water protons in liquid phase of E.coli
suspensions with xenon clathrates as a function of cell concentration. T = 280 K. Pressure is 1.2 MPa. Frequency is 20 MHz
0 1 2 3 4 5 6 7 8
0
5
10
15
20
25
30
35
40
C (x1010 cell/ml)
R1, fast (s-1)
Figure 5. Spin-lattice relaxation rate R1=1/T1 of fast relaxing component of longitudinal magnetization of water protons in liquid phase of E.coli suspensions
with xenon clathrates as a function of cell concentration. T = 280 K. Pressure is 1.2 MPa. Frequency is 20 MHz

34 Xenon-Water Interaction in Bacterial Suspensions as Studied by NMR
An increase of the cell concentration in E.coli suspensions
with xenon clathrates resulted in decrease of slow (and fast)
components of longitudinal relaxation time T1 (Figs.4, 5).
This could be associated with increasing viscosity because of
the concentration of solutes (inside and outside cells)
increased. This is also could be considered as a result of
increasing exchange between the protons of liquid water and
the protons of macromolecular surface structures of cell in
the case of spin-diffusion. Last suggestion is rather unlikely
as published data indicated that spin-diffusion affects the
relaxation of protons at high concentration of proteins and
macromolecules [45, 60‒62]. A temperature dependence of
slow T1 component in the range of 277‒289 K (Arrhenius
plot) resulted in apparent activation energy Ea=27.2±2.0
kJ/mol which exceeds Ea-value for pure water (~21 kJ/mol
[40]). Probably, a mobility of water molecules in liquid
phase of cell suspensions at xenon clathrate formation
decreased not only due to increasing cell concentration and
compress of cytoplasm but significantly due to dissolution of
xenon gas in cells in the amount enough to create separate
clathrate cages but not enough to build up a space network of
completed clathrate structures. According to the NMR data
on 129Xe dissolved in physiological saline at 0.1 MPa and
injected into RBC suspension gas-exchange processes
between extra- and intracellular compartments occur on the
time scale of a few tens milliseconds [59,63]. The mixing of
xenon between physiological saline and RBCs resulted in
accumulation of 129Xe NMR signals inside cells with
intensity comparable to that in extracellular compartment for
just the period of ~1 s [59]. However an affinity of xenon to
build up the clathrate structures in extra- and intracellular
spaces at the temperatures above melting point of ice can be
strongly depending on increased gas pressure and other
conditions in cells and outside cells including changed
metabolism (flux of water and ions via membrane),
availability of free liquid water and accumulation of xenon in
hydrophobic part of membrane.
4. Conclusions
Structural-dynamic changes in bacterial cell properties
induced by xenon have been studied by proton spin-lattice
relaxation measurements. The inert gas xenon is able to form
clathrate hydrates from water molecules at pressure circa
0.4–0.7 MPa and temperature about 276–278 K. An amount
of water bound in solid phase of xenon clathrates can be
estimated from NMR data. The results clarified how NMR
method can monitor a process of clathrate formation and
how the xenon atoms and its hydrates interact with cells. The
formation of xenon hydrates affected exchange dynamics of
water between two compartments and resulted in apparent
two T1-components.The results of T1-measurements showed
that the mobility of intra- and intercellular water can be
changed at above melting point of common ice with the aid
of xenon hydrates. The ability of xenon hydrates to lower
water proton relaxation time inside the cells may reflect their
propensity to structure and influence on exchange between
extracellular and intracellular water.
Detailed interpretation of correlation times as the
characteristics of molecular motions in compartments of
bacterial suspension in the presence of clathrate ice is not
easy because of the lack of suitable mathematical models.
However, the results showed that the surface of cells
participates in clathrate formation. It affects the dynamics of
the water molecules and determines their correlation times.
The changes in mobility of extracellular water at xenon
hydrate formation result in changes in cell properties.
Molecular mechanisms of these changes (affecting
membrane permeability and activity of proteins and lipids)
must be further explored involving different NMR facilities.
There are many NMR methods for probing the
compartments of heterogeneous, in particular, cellular
systems. NMR has a useful role in characterizing the
molecular properties of separate spaces of cellular
suspensions giving a possibility to probe water, solutes and
surfaces non-invasively. This allows keeping the
biomaterials under xenon conditions further without
distortions.
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