ArticlePDF AvailableLiterature Review

The multifunctional role of ectoine as a natural cell protectant

Authors:

Abstract and Figures

The protective properties of ectoine, formerly described for only extremophilic microorganisms, can be transferred to human skin. Our present data show that the compatible solute ectoine protects the cellular membrane from damage caused by surfactants. Transepidermal water loss measurements in vivo suggest that the barrier function of the skin is strengthened after the topical application of an oil in water emulsion containing ectoine. Ectoine functions as a superior moisturizer with long-term efficacy. These findings indicating that ectoine is a strong water structure-forming solute are explained in silico by means of molecular dynamic simulations. Spherical clusters containing (1) water, (2) water with ectoine, and (3) water with glycerol are created as model systems. The stronger the water-binding activity of the solute, the greater the quantity of water molecules remaining in the cluster at high temperatures. Water clusters around ectoine molecules remain stable for a long period of time, whereas mixtures of water and glycerol break down and water molecules diffuse out of the spheres. On the basis of these findings, we suggest that the hydrogen bond properties of solutes are not solely responsible for maintaining the water structure form. Moreover, the particular electrostatic potential of ectoine as an amphoteric molecule with zwitterionic character is the major cause for its strong affinity to water. Because of its outstanding water-binding activity, ectoine might be especially useful in preventing water loss in dry atopic skin and in recovering skin viability and preventing skin aging.
Content may be subject to copyright.
The multifunctional role of ectoine as a natural
cell protectant
Ruediger Graf, PhD
a,
, Soheila Anzali, PhD
b
, Joachim Buenger, PhD
a
,
Frank Pfluecker, PhD
a
, Hansjuergen Driller, PhD
a
a
Department of Cosmetics and Food, Merck KGaA, 64293 Darmstadt, Germany
b
R&D New Technology Evaluation, Merck KGaA, 64293 Darmstadt, Germany
Abstract The protective properties of ectoine, formerly described for only extremophilic microorgan-
isms, can be transferred to human skin. Our present data show that the compatible solute ectoine protects
the cellular membrane from damage caused by surfactants. Transepidermal water loss measurements in
vivo suggest that the barrier function of the skin is strengthened after the topical application of an oil in
water emulsion containing ectoine. Ectoine functions as a superior moisturizer with long-term efficacy.
These findings indicating that ectoine is a strong water structure-forming solute are explained in silico
by means of molecular dynamic simulations. Spherical clusters containing (1) water, (2) water with
ectoine, and (3) water with glycerol are created as model systems. The stronger the water-binding
activity of the solute, the greater the quantity of water molecules remaining in the cluster at high
temperatures. Water clusters around ectoine molecules remain stable for a long period of time, whereas
mixtures of water and glycerol break down and water molecules diffuse out of the spheres. On the basis
of these findings, we suggest that the hydrogen bond properties of solutes are not solely responsible for
maintaining the water structure form. Moreover, the particular electrostatic potential of ectoine as an
amphoteric molecule with zwitterionic character is the major cause for its strong affinity to water.
Because of its outstanding water-binding activity, ectoine might be especially useful in preventing water
loss in dry atopic skin and in recovering skin viability and preventing skin aging.
© 2008 Elsevier Inc. All rights reserved.
Introduction
Ectoines, as small organic molecules, occur widely in
aerobic, chemoheterotrophic, and halophilic organisms that
enable them to survive under extreme conditions. These
organisms protect their biopolymers (biomembranes, pro-
teins, enzymes, and nucleic acids) against dehydration
caused by high temperature, salt concentration, and low
water activity by substantial ectoine synthesis and enrich-
ment within the cell.
The organic osmolyte ectoine (Fig. 1) and hydroxyectoine
are amphoteric, water-binding, organic molecules. They are
generally compatible with the cellular metabolism without
adversely affecting the biopolymers or physiologic processes
and are so-called compatible solutes.
1
The protective function of the compatible solutes in a low-
water environment may be explained by the preferential
exclusion model: The solutes are excluded from the
immediate hydration shell of, for example, a protein because
of an unfavorable interaction with the protein surface. The
Corresponding author.
E-mail address: ruediger.graf@merck.de (R. Graf).
0738-081X/$ see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.clindermatol.2008.01.002
Clinics in Dermatology (2008) 26, 326333
consequence is preferential hydration of the protein, thus
promoting its native conformation. Because compatible
solutes do not interact directly with the protein surface, the
catalytic activity remains unaffected.
2,3
Yu and Nagaoka
5
reported interesting results on mole-
cular dynamic simulations performed for water-ectoine
mixture models around chymotrypsin inhibitor 2. According
to their statement, ectoine maintains water at the surface by
slowing down the water diffusion around a protein, where it
is most needed, whereas it does not directly interact with
macromolecules themselves. Thus, ectoine plays an indirect
role in the alteration of the solvent properties and the
modification of the stability of proteins.
4
Ectoine minimizes the denaturation that occurs on the
removal of water molecules by making the unfolding less
favorable.
6
In addition, hydroxyectoine, with its OH group,
can at least partly replace those water molecules lost from the
hydrate shell (replacement hypothesis); in this way, the
native structure of the biopolymers can be further stabilized.
Compatible solutes are amphiphilic in nature and capable of
wettinghydrophobic proteins, thus improving their
hydration capability.
7
The structure-forming and breaking
properties of compatible solutes indirectly influence the
hydration shells and thus the activities of the proteins
involved.
8
In this way, halophilic organisms and other bacteria use
ectoine to protect their cytoplasmic biomolecules against
heat, freezing, dryness, and osmotic stress.
9
Ectoine and
hydroxyectoine can be isolated from halophilic bacteria on a
large scale and thus are available as active ingredients for
skin care.
10
The protective properties of ectoine, formerly described
only for microorganisms, could be transferred to human skin.
Human skin is situated at the interface of the organism and its
environment and therefore is exposed to a variety of
environmental assaults. The stratum corneum in particular
provides a barrier to the evaporation of water from the viable
epidermis. Many factors work to compromise this barrier and
increase the rate of water loss from the skin. Exposure to
extreme environmental conditions, including cold, dry
winter weather, frequent washing with soap and hot water,
or the exposure to surfactants, may cause skin dryness. In
addition to dryness, the cumulative effect of external factors,
such as radiation, wind, and temperature extremes, leads to
accelerated skin aging.
11,12
Various investigations underline the outstanding anti-
aging properties of ectoine. Epidermal dendritic Langerhans
cells are the single most important antigen-presenting cell
population in the skin. The number of Langerhans cells
decreases significantly in aged skin, whereas the decrease in
skin exposed to the sun is greater than that in skin protected
from the sun.
13-15
Topically applied ectoine shows an
immunoprotective potential on the sun-exposed skin of
healthy subjects. The ultraviolet-induced reduction of
Langerhans cells has been prevented by pretreatment with
ectoine before sun exposure.
16
The exposure of primary human keratinocytes to ultra-
violet A provokes the formation of ceramide by a singlet
oxygen-mediated mechanism. As a consequence of the
increased ceramide level, an intracellular signaling cascade is
activated, leading to expression of the proinflammatory
intercellular adhesion molecule-1. These negative effects are
effectively prevented by ectoine as a result of its singlet
oxygen-quenching properties.
17,18
Because the activity of
antioxidant enzymes and the levels of nonenzymatic
antioxidants decrease with age,
19,20
ectoine could prevent
such oxidative damage in skin.
Skin in particular, which is susceptible to water loss
because of the absence of an optimal skin barrier (eg, the skin
of the elderly, atopic skin, or after surfactant treatment),
shows increased transepidermal water loss (TEWL) and
diminished moisturization.
21
The goal of the present study was to investigate the effect
of ectoine on the moisturization status and barrier function of
the skin after topical application in vivo. Furthermore,
different molecular dynamic simulation systems were
created in silico to compare models of water, water-ectoine,
and water-glycerol. The outstanding activity of ectoine as a
strong water structure former was evaluated against glycerol
as a commonly used humectant in cosmetics.
Materials and methods
Membrane assay
The membrane assay is based on the photometric
quantification of free hemoglobin released from erythrocytes
with a partially damaged membrane provoked by surfactants.
For the different experiments, the erythrocytes are treated as
Fig. 1 Molecular structure of ectoine with the two tautomeric forms (A) and its hydrophilic surface colored according to the corresponding
atomic partial charges (B).
327Multifunctional role of ectoine as a natural cell protectant
follows: (1) Human erythrocytes (2 ×10
8
cells/mL) are
treated for 1 hour with 0%, 0.1%, 0.5%, 1%, and 5% ectoine
to determine the effect of ectoine concentration; and (2) 2 ×
10
8
erythrocytes/mL are treated for 0 (control), 6, 18, and
24 hours with 1% (w/v) ectoine to determine the effect of the
incubation time. Both sets of cells are stressed for 10 minutes
with 0% to 0.04% sodium dodecyl sulfate (SDS) solution,
and the number of cells in lysis is determined spectro-
scopically via the content of free hemoglobin. With two
absorption peaks at 540 and 575 nm, hemoglobin can be
quantified by the determination of absorption at 575 nm,
with the molar absorbance coefficient of 0.125 mmol/L
oxyhemoglobin at A
575nm
= 2.0.
22
The results are shown as
the difference (%) of cells in lysis as a function of the
concentration of ectoine against an untreated control. The
experiment is repeated five times.
Determination of the transepidermal water loss
in vivo
The volar forearm of five volunteers is treated twice daily
for 1 week with an oil in water emulsion (2 mg/cm
2
) con-
taining 0% (placebo), 2%, and 5% ectoine. To achieve a
synthetic increase in TEWL by damaging the skin barrier, the
skin is occlusively treated with 80 μL SDS (2% in water) in
an aluminium chamber for 24 hours. The TEWL is deter-
mined in an acclimatized room at 22°C with an air humidity
of 60% using a TEWAmeter TM210 (Courage + Khazaka,
Koeln, Germany). The TEWL values are visualized before
and after treatment with ectoine-containing emulsion and
after damaging the skin barrier with SDS.
Determination of skin moisture by corneometry
Ectoine treatment and subsequent dehydration
with silica
The skin of the volar forearm of five volunteers is treated
twice daily for 1 week with a cosmetic formulation (2 mg/
cm
2
) containing 0% (placebo), 2%, and 5% ectoine. The
moisture content of the skin is determined with a
Corneometer before application and, after 1 week, 4 hours
after the final application. Silica gel 60 (0.2 g/cm
2
) is applied
under occlusion for 2 hours (dehydration step). On re-
moval of the silica gel, the skin moisture is determined
after 10 minutes, 2 hours, 4 hours, and 24 hours.
Ectoine treatment for long-term hydration
The skin of the volar forearm of five volunteers is treated
twice daily for 12 days with a cosmetic formulation (2 mg/
cm
2
) containing 0% (control), 0.5%, and 1% ectoine. The
skin hydration is determined by corneometry starting at day 8
until day 12. On day 12, the application is stopped for 7 days,
finalizing this experiment on day 19 with a last measurement
of hydration. The measurements are carried out in an
acclimatized room at 22°C with an air humidity of 60%.
Molecular dynamic simulations
The Schrödinger package Impact (Integrated Modelling
Program using Applied Chemical Theory
23
) is used for
molecular dynamic simulations (with OPLS-2005 force
field parameters and partial charges). The OPLS-2005
force field uses experimental data from the liquid state
and quantum mechanical calculations. It is calculated
from the sum of the intramolecular bond, angle, and
torsion motions to set the constituent parameters and the
nonbonded interaction as a van der Waals term together
with an electrostatic term.
Three spheres have been created for: (1) water only; (2) an
ectoine-water mixture; and (3) a glycerol-water mixture. The
creations of spheres are as follows: For each ingredient
(ectoine and glycerol), a 3 ×3×3 matrix is created. For this
purpose, 27 molecules of each ectoine or glycerol are
clustered per sphere. A minimization is performed using the
surface generalized born method with 500 steps of steepest
descent, followed by 500 steps of conjugated gradient.
Ectoine and glycerol are placed in a rectangular box, and
soaking of simple point charge water with a dimension of
70 ×70 ×70 Å is performed.
The spheres are cut out with a radius of 30 Å away from
the centroid atoms. The size of spheres of 30 Å in radius is
sufficient to cover more than one solvation shell for solutes
calculated in spheres. The reason for having so many water
molecules is to ensure that there are at least two shells of
water molecules around the solutes. In addition, we can
examine and compare the indirect effect of solutes on water
molecules on such a large scale.
The shake algorithm is used to constrain the X-H bond,
which allows time steps of 2 fs. Elaborate equilibration runs
of 50 ps at 298.15 K are performed to allow for a careful
accommodation of water structure around the solutes
(ectoine and glycerol). Water oxygen atoms are fixed beyond
25 Å from the defined centroid atoms in each created sphere
in the equilibration. For the dynamic simulations, these
constraints are removed.
The dynamic simulations are performed for water and
water-glycerol for 200 ps and 500 ps at the temperature of
370 K with a temperature relaxation constant value of 0.01
ps. For the water-ectoine mixture, the simulation is
performed for 1 ns to demonstrate the effect of ectoine
with regard to water cluster formation in a long time frame.
The trajectories are recorded every 50 time steps.
Results and discussion
Barrier-improving effects
The membrane of the skin cell can become damaged, for
example, by exposure to surfactants present in washing and
skin-cleansing solutions. Thus, the use of active cleansing
328 R. Graf et al.
surfactants also leads to removal of fat from the skin,
increased TEWL, and dry skin.
For the evaluation of the membrane-protecting properties
of ectoine, the red blood cell (RBC) test was applied. This
assay is a biologic in vitro test for the rapid estimation of
membrane and protein-denaturing properties of surfactants.
The standard protocol uses erythrocytes, non-nucleated
blood cells containing hemoglobin. Because hemoglobin is
incapable of crossing the RBC membrane, it is not detectable
outside erythrocytes as long as the RBC membrane is intact.
The assay is based on the photometric quantification of the
hemoglobin released as a consequence of RBC plasma
membrane damage after exposure to surfactants, thus
providing a measure of surfactant aggressiveness.
The stabilization effect on cell membranes pretreated with
ectoine was evaluated. The erythrocytes were incubated for
10 minutes with SDS. SDS destabilizes the membranes of
untreated cells in such a way that lysis occurs in part and cell
components (eg, hemoglobin) are released. The hemoglobin
released serves as an indicator for the spectrophotometric
determination of the degree of cell membrane damage
provoked by SDS. Detecting the released hemoglobin
enabled the number of destroyed erythrocytes to be
determined in our experiments. A modified version of the
RBC test was used to determine the membrane stabilization
achieved by a test substance versus surfactant lysis. This
assay includes the RBC preincubation with a stabilizer
before the addition of surfactant as the lytic agent.
Fig. 2 shows that ectoine protects the cells from damage
caused by SDS treatment. The erythrocytes pretreated with
ectoine are shown to be more resistant to membrane damage
by SDS than those of untreated cells. No stabilizing effect
was observed in cells without ectoine, in which maximum
erythrocyte damage occurred (0% increase of membrane
stability). The higher the ectoine concentration, the greater
the protective effect against membrane damage (Fig. 2A).
Furthermore, the influence of prolonging the incubation
time was investigated. The membrane stability increased to
30% after 6 hours of pretreatment and to approximately 60%
after 24 hours. Thus, the longer the cells are pretreated with
ectoine, the greater the protective effect against membrane
damage by the surfactant SDS (Fig. 2B). The degree of cell
protection that has been linked with the degree of membrane
stabilization depends directly on the ectoine concentration
and the duration of ectoine pretreatment.
Ectoine thus protects the skin barrier against the
damaging effect (water loss) of SDS.
Fig. 3 In vivo determination of TEWL after damage of the skin
barrier by SDS. The forearm skin of the volunteers (n = 5) is treated
twice daily for 1 week with an oil in water emulsion (2 mg/cm
2
)
containing 0% (placebo), 2%, and 5% ectoine. To achieve a
synthetic increase in TEWL by damaging the skin barrier, the skin is
subsequently treated with 2% SDS in water for 24 hours and the
TEWL is determined. The diagram shows the TEWL before and
after treatment with emulsion containing ectoine and after damage
of the skin barrier with SDS.
Fig. 2 Evaluation of the membrane-stabilizing effect of ectoine in
surfactant-stressed cells. Human erythrocytes (2 ×10
8
cells/mL) are
treated (A) for 1 hour with 0%, 0.1%, 0.5%, 1%, and 5% ectoine
and (B) for 0 (control), 6, 18, and 24 hours with 1% ectoine. Both
sets of cells are stressed for 10 minutes with 0% to 0.04% SDS
solution, and the number of cells in lysis is determined spectro-
scopically via the content of free hemoglobin. The diagrams
illustrate the difference (%) of cells in lysis as a function of the
concentration of pretreated ectoine against an untreated control. The
experiment is repeated five times.
329Multifunctional role of ectoine as a natural cell protectant
These data confirm our previous studies of further
cosmetically relevant surfactants in which ectoine showed
a stronger protective effect compared with the well-known
membrane stabilizer phosphatidylcholine.
24
These in vitro findings should also be approved in vivo.
Surfactants have also been used to cause dry skin.
25
For this
reason, after SDS treatment of the skin, the TEWL is
determined as a read-out parameter for the integrity of the
skin barrier. The barrier disruption can be expressed as a
change in TEWL, and the influence of ectoine can be
measured. The study is performed on the lower forearm of
healthy volunteers.
The application of a cosmetic emulsion containing
different amounts of ectoine leads to a remarkable
reduction of TEWL to 40% (Fig. 3). Fig. 3 shows that
skin pretreated with ectoine becomes less susceptible to
damage by the surfactant SDS. The ectoine emulsion thus
protects the skin against surfactant damage and the con-
sequent loss of water.
Protection against dehydration
One of the major goals of cosmetics is still the protection
of the skin against stress factors that lead to dehydration. Dry
air, particularly during periods of freezing or hot weather and
air conditioning, tends to dry out the skin considerably.
To demonstrate the protective effect of ectoine on skin
moisture, two cosmetic formulations with and without
ectoine were topically applied to the lower forearm of
volunteers twice daily for 1 week. The moisture content of
the skin was determined by corneometry, and the results are
shown in Fig. 4.
The diagram illustrates that ectoine in a cosmetic oil in
water emulsion protects the skin against dehydration. In
addition to this protection, ectoine also produces a higher
moisture content than the basic (placebo) formulation that
already contains 3% glycerol. The results also show that
ectoine, even after 24 hours, maintains a considerably greater
degree of skin moisture than untreated or placebo-treated
skin. Ectoine even protects skin against rapid dehydration
after direct application of hygroscopic silica gel. Skin
moisture can be maintained for a longer period of time by
topically applying ectoine.
Low humidity has been shown to stimulate epidermal
DNA synthesis and amplify the hyperproliferative response
to barrier disruption.
26
Stratum corneum morphology is also
Fig. 4 In vivo determination of skin moisture after treatment
with ectoine and subsequent dehydration with silica gel. The
forearm skin of the volunteers (n = 5) is treated twice daily for
1 week with an oil in water emulsion (2 mg/cm
2
) containing 0%
(placebo), 2%, and 5% ectoine. The moisture content of the skin
is determined before application and, after 1 week, 4 hours after
the final application. Silica gel 60 (0.2 g/cm
2
) is applied under
occlusion for 2 hours (dehydration). On removal of the silica
gel, the skin moisture is determined after 10 minutes, 2 hours, 4 hours,
and 24 hours.
Fig. 5 Long-term moisturizing effect with ectoine. The skin of the volar forearm of five volunteers is treated twice daily for 12 days with a
cosmetic formulation (2 mg/cm
2
) containing 0% (control), 0.5%, and 1% ectoine. The skin hydration is determined by corneometry starting at
day 8 until day 12 (A). On this day the application is stopped for 7 days, finalizing this experiment on day 19 with a last measurement of
hydration (B).
330 R. Graf et al.
influenced by a dry environment, and abnormal desquama-
tion is observed under low humidity.
27,28
With respect to our
findings in the silica-dried skin model,formulations
containing ectoine have a prophylactic effect against such
adverse processes in dry skin.
Moisture boost with long-term effect
In a further series of experiments, ectoine was evaluated
according to its long-term effect on skin moisture. The test
was carried out on the volar forearm of volunteers. Twice-
daily applications of 0.5% and 1% ectoine were applied for
12 days. The skin hydration was measured with a
Corneometer starting at day 8 until day 12. On day 12, the
application of ectoine was stopped for 7 days, detecting the
skin hydration finally at day 19. The results of this placebo-
controlled study underline the outstanding activity of
ectoine: After 8 days of application, the hydration increased
markedly up to 200% compared with the placebo-treated
skin and remained constant until the end of the testing period
(Fig. 5A). Although the topical application was stopped on
day 12, the actual hydration status was preserved for
approximately 7 days, underlining a significant long-term
moisturizing effect of ectoine (Fig. 5B).
Ectoine retains the power of water
The protein-stabilizing effects of ectoine can be explained
by the preferential exclusion model as a consequence of
entropically favored surface minimization. The ability of
ectoine as a strong water structure-forming solute is further
processed in comparison with glycerol as a commonly used
humectant in cosmetics.
11
After the dynamic simulation time of 200 ps, as well as
1000 ps, the number of water molecules in the water-ectoine
complex remained unexpectedly constant. In contrast, the
performance of the water-glycerol complex: an extreme
corrosion was observed. The total number of water
molecules decreased significantly after 200 ps of dynamic
simulation, and only 2339 water molecules remained in the
sphere (Table 1).
To explain this phenomena, the total potential energy
(E
pot
) was calculated for the spheres containing water, water-
Fig. 6 Evaluation of the E
pot
-value of different water clusters.
During the dynamic simulation at 370 K, water molecules diffuse
out of the spheres and the total amount of water molecules
decreases. To explain this phenomenon, the total potential energy
has been calculated and plotted as the E
pot
-value. In this
experimental setup, the E
pot
-value can be adopted as the stored
energy or the energy of position of each system.
Table 1
t (ps) Water Water-glycerol Water-ectoine
0 3618 3429 3139
200 3026 2339 3138
500 NC 1288 3112
1000 NC NC 3103
The number of water molecules retained in spherical water clusters
during the dynamic simulation time. The simulation is carried out at 370
K, and the water molecules are counted after 0, 200, 500, and 1000 ps.
NC, Not calculated.
Fig. 7 Molecular dynamic simulation of different models
containing (A) water, (B) water and ectoine, and (C) water and
glycerol. The pictures are taken at the beginning of the simulation
(t = 0, A1, B1, C1) and after 200 ps (A2), 1000 ps (B2), and 500 ps
(C2) at a constant temperature of 370 K. Water clusters around
ectoine molecules remain stable for a long period of time, whereas
the cluster of water and glycerol breaks down and water molecules
diffuse out of the spheres. The pictures represent the number of
water molecules counted during the dynamic simulation as shown
in Table 1. The solutes are green.
331Multifunctional role of ectoine as a natural cell protectant
glycerol, and water-ectoine. In this experimental setup, the
E
pot
-value can be adopted as the stored energy or the energy
of position in such a system.
With regard to water and the water-glycerol complexes,
the E
pot
-values decreased dramatically during the simulation
time, whereas the E
pot
-value of the water-ectoine sphere
remained constant even throughout a longer simulation time
(Fig. 6). The E
pot
-value of the water-ectoine sphere remained
constant at the level indicated in the diagram (data not
shown). It is remarkable that the E
pot
-value of regular water
molecules per se was greater than that of the water-ectoine
mixture, indicating the strong organizing and complexing
properties of ectoine.
The dynamic simulation and animations, and the
statistical analysis, demonstrated that the water diffusion
out of the spheres was limited and decreased enormously by
adding ectoine molecules to the sphere (Fig. 7A and B; see
also the stick presentation of water and ectoine atoms in
Fig. 8). Even a 5-fold longer simulation time showed a stable
water structure form attributable to ectoine properties, which
is superior compared with water itself and outstanding
compared with a water-glycerol complex (Fig. 7).
We propose that the hydrogen bond properties of solutes
are not solely responsible for maintaining the water structure
form. Moreover, the particular electrostatic potential of a
compatible solute, such as ectoine, as an amphoteric
molecule with zwitterionic character is the major reason for
its affinity to water.
Conclusions
Our recent studies demonstrate the outstanding role of the
compatible osmolyte ectoine in preventing water loss caused
by surfactant-induced barrier damage. Ectoine functions as a
more potent moisturizer than glycerol and features long-term
moisturizing efficacy. These in vivo findings were explained
in silico by means of molecular dynamic simulations. Water
clusters around ectoine molecules remain stable for a long
period of time, whereas mixtures of water and glycerol are
disintegrated by the diffusion of water molecules out of the
spheres. Because of its strong water-binding activity, ectoine
may be especially useful in the prevention of dehydration in
dry atopic skin and the recovery of skin viability and
prevention of skin aging.
Acknowledgments
We thank Dr Jianxin Duan, of Schrödinger GmbH,
Mannheim, Germany, for the fruitful discussions and
technical support for the dynamic simulations.
References
1. Galinski EA. Compatible solutes of halophilic eubacteria: molecular
principles, water-solute interaction, stress protection. Experienta 1993;
49:487-96.
2. Galinski EA, Stein M, Amendt B, Kinder M. The kosmotropic
(structure-forming) effect of compensatory solutes. Comp Biochem
Physiol 1997;117A:357-65.
3. Kolp S, Pietsch M, Galinski EA, Guetschow M. Compatible solutes as
protectants for zymogens against proteolysis. Biochim Biophys Acta
2006;1764:1234-42.
4. Goeller K, Galinski EA. Protection of a model enzyme (lactate
dehydrogenase) against heat, urea and freeze-thaw treatment by
compatible solute additives. J Mol Catal B Enzym 1999;7:37-45.
5. Yu I, Nagaoka M. Slowdown of water diffusion around protein in
aqueous solution with ectoine. Chem Phys Lett 2004;388:316-21.
6. Crowe JH, Carpenter JF, Crowe LM, Anchordoguy TJ. Are freezing
and dehydration similar stress vectors? A comparison of modes of
interaction of stabilizing solutes with biomolecules. Cryobiology 1990;
27:219-31.
7. Schobert B, Tschesche H. Unusual solution properties of praline and its
interaction with proteins. Biochem Biophys Acta 1978;541:270-7.
Fig. 8 Stick representation of atoms in the ectoine-water sphere. The grey area of the ectoine-water cluster is presented at a higher resolution
to illustrate the molecular composition of the cluster. The small picture corresponds with Fig. 7, B1.
332 R. Graf et al.
8. Wiggins PM. Role of water in some biology processes. Microbiol Rev
1990;54:432-49.
9. Lippert K, Galinski EA. Enzyme stabilization by ectoine-type
compatible solutes: protection against heating, freezing and drying.
Appl Microbiol Biotechnol 1992;37:61-5.
10. Lentzen G, Schwarz T. Extremolytes: natural compounds from
extremophiles for versatile applications. Appl Microbiol Biotechnol
2006;72:623-34.
11. Orth DS, Appa Y. Glycerine: a natural ingredient for moisturizing skin.
In: Loden M, Maibach HI, editors. Dry skin and moisturizers. London:
CRC Press; 2000. p. 213-28.
12. Rabe JH, Mamelak AJ, McElgunn PJS, Morison WL, Sauder DN.
Photoaging: mechanisms and repair. J Am Acad Dermatol 2006;55:1-19.
13. Toyoda M, Bhawan J. Ultrastructural evidence for the participation of
Langerhans cells in cutaneous photoaging process: a quantitative
comparative study. J Dermatol Sci 1997;14:87-100.
14. Grewe M. Chronological aging and photoageing of dendritic cells. Clin
Exp Dermatol 2001;26:608-12.
15. Bushan M, Cumberbatch M, Dearman RJ, Andrew SM, Kimber I,
Griffiths CE. Tumor necrosis factor-alpha-induced migration of human
Langerhans cells: the influence of aging. Br J Dermatol 2002;146:
32-40.
16. Pfluecker F, Buenger J, Hitzel S, et al. Complete photo protection
going beyond visible endpoints. SÖFW J 2005;131:20-30.
17. Buenger J, Driller HJ. Ectoine: an effective natural substance to prevent
UVA-induced premature photoaging. Skin Pharmacol Physiol 2004;17:
232-7.
18. Grether-Beck S, Timmer A, Felsner I, Brenden H, Brammertz D,
Krutmann J. Ultraviolet A-induced signaling involves a ceramide-
mediated autocrine loop leading to ceramide de novo synthesis. J Invest
Dermatol 2005;125:545-53.
19. Yasui H, Sakurai H. Age-dependent generation of reactive oxygen
species in the skin of live hairless rats exposed to UVA light. Exp
Dermatol 2003;12:298-300.
20. Tolmasoff JM, Ono T, Cutler RG. Superoxide dismutase: correlation
with life-span and specific metabolic rate in primate species. Proc Natl
Acad Sci U S A 1980;77:2777-81.
21. Loden M. The skin barrier and use of moisturizers in atopic dermatitis.
Clin Dermatol 2003;21:145-57.
22. Pape WJ, Hoppe U. Standardization of an in vitro red blood cell test for
evaluating the acute cytotoxic potential of tensides. Arzneimittel-
forschung 1990;40:498-502.
23. Impact, version 4.0. New York, NY: Schrödinger, LLC; 2005.
24. Buenger J, Degwert J, Driller HJ. The protective function of compatible
solute ectoine on the skin cells and its biomolecules with respect to UV-
radiation, immunosuppression and membrane damage. IFSCC Mag
2001;4:1-6.
25. VanderValk PGM, Stam-Westerveld EB, Paye M. A model to study the
drying potential of detergent formulations on the skin. In: Maibach HI,
editor. Dermatologic research techniques. Boca Raton (Fla): CRC
Press; 1996. p. 195-205.
26. Denda M, Sato J, Tsuchiya T, Elias PM, Feingold KR. Low
humidity stimulates epidermal DNA synthesis and amplifies the
hyperproliferative response to barrier disruption: implication of
exacerbations of inflammatory dermatoses. J Invest Dermatol 1998;
111:873-8.
27. Denda M, Sato J, Masuda Y, et al. Exposure to dry environment
enhances epidermal permeability barrier function. J Invest Dermatol
1998;111:858-63.
28. Sato J, Denda M, Nakanihi J, Koyama J. Dry conditions affect
desquamation of stratum corneum in vivo. J Dermatol Sci 1998;18:
163-9.
333Multifunctional role of ectoine as a natural cell protectant
... Thus the name extremolyte is a coinage: organic osmolytes which are synthesized by extremophilic microorganisms. The presence of these molecules allows microorganisms to resist extreme living conditions like drastic temperature variations and high salinity [1,2]. Interestingly, these solutes are biologically inert and accumulate at high concentration in the cytoplasm without interfering with the overall cellular functions; hence they are called compatible solute [3]. ...
... Long used hygroscopic molecules like urea have been systematically replaced by extremolytes in a stepwise manner [10]. Specifically ectoine and hydroxyectoine have been used as cell protectant in skin care [2,10] due to their acces-sibility of large scale production [1,[4][5][6]. Further biological importance is given by the stabilization of proteins in presence of compatible solutes [11,12]. ...
... In addition we discuss the hygroscopic properties of the different molecules in atomistic detail. Our results allow to interpret the numerical findings of a recent publication [21] and validate the experimental consensus that extremolytes are more appropriate for water binding than other compatible solutes [1,2,10,25]. We will show that the length scale of solvent perturbation is identical to the distance between a protein and a compatible solute in agreement to Ref. [21]. ...
Preprint
We have performed Molecular Dynamics simulations of ectoine, hydroxyectoine and urea in explicit solvent. Special attention has been spent on the local surrounding structure of water molecules. Our results indicate that ectoine and hydroxyectoine are able to accumulate more water molecules than urea by a pronounced ordering due to hydrogen bonds. We have validated that the charging of the molecules is of main importance resulting in a well defined hydration sphere. The influence of a varying salt concentration is also investigated. Finally we present experimental results of a DPPC monolayer phase transition that validate our numerical findings.
... Ectoine is a marine bacteria-and algaederived aminoacid generated in harsh marine environmental conditions, specifically in osmotic stress conditions [91,92] (Fig. 7). Reported for its antioxidant potential, ectoine possesses high ROS scavenger activity, especially toward hydroxyl radicals [93], and is a long-lasting moisturizer that avoids epidermal dehydration [93,94]. It also lowers skin irritation and it has been researched for the treatment of mild atopic dermatitis [95]. ...
... Ectoine is a marine bacteria-and algaederived aminoacid generated in harsh marine environmental conditions, specifically in osmotic stress conditions [91,92] (Fig. 7). Reported for its antioxidant potential, ectoine possesses high ROS scavenger activity, especially toward hydroxyl radicals [93], and is a long-lasting moisturizer that avoids epidermal dehydration [93,94]. It also lowers skin irritation and it has been researched for the treatment of mild atopic dermatitis [95]. ...
Article
Full-text available
Excessive exposure to sunlight can contribute for skin photo-damage, such as sunburn, dryness, wrinkles, hyperpigmentation, immunosuppressive events and skin sensitization reactions. The use of aftersun products is an effective strategy to reduce the visible signs and symptoms of acute photodamage in the skin. Aiming to unveil the active ingredients able to offset acute sun damage, this work focuses on the characterization of the aftersun products market. A total of 84 after-sun formulations from 41 international brands currently marketed in Portugal were analyzed concerning the composition described on the product label, identifying natural and synthetic/semi-synthetic ingredients with the ability to mitigate solar-induced effects. The majority of aftersun formulations contained ingredients derived from terrestrial and marine sources (> 80%). An in-depth examination of these compounds is also offered, revealing the top of the most used natural and synthetic/semi-synthetic ingredients present in aftersun products, as well as their mechanism of action. A critical appraisal of the scientific data was made aiming to highlight the scientific evidence of ingredients able to mitigate skin photodamage. Amino acids and peptides, and A. barbadensis extract were tested for their in vivo efficacy. Nevertheless, all the ingredients were analyzed with in vitro studies as preliminary screening before in vivo, ex vivo and/or clinical studies. In summary, this study provides an overview of the use of active ingredients in commercial aftersun products to understand better the benefits associated with their use in cosmetic formulations and identify opportunities for innovation. Graphical abstract
... Additionally, the DEJ index showed significant improvement following the eight-week application period. This, combined with increased skin elasticity and firmness, as well as a reduction in the area and length of wrinkles in the lower eyelids and lateral orbital regions, suggests that the anti-aging active ingredients within the eye cream, such as Tabebuia Impetiginous bark extract [26], acetyl hexapeptide-8 [27], dipeptide diaminobutyroyl benzylamide diacetate [28], arginine/lysine polypeptide [29], niacinamide [30], ergothioneine [31], and ectoine [32], may mitigate the undulation of the DEJ. However, no significant improvement was observed in the SAAID, for which two primary reasons are postulated. ...
Article
Full-text available
Background: Skin aging is a multifactorial process influenced by genetic and environmental factors, manifesting prominently on the face. Variations in aging effects across facial areas necessitate advanced imaging for detailed assessment. Materials and Methods: We utilized a portable handheld two-photon microscope (TPM) for in vivo exploration of skin aging alterations in the periocular and cheek areas of a Chinese female population. This study included 107 healthy volunteers, aged between 18 to 60 years, categorized into four age intervals. Our research extended to a clinical trial to evaluate the efficacy of a novel anti-aging eye cream rich in plant extracts, peptides, and antioxidants. This formulation was assessed for its potential in enhancing skin hydration, elasticity, and reducing wrinkles. Participants applied the eye cream twice daily for 8 weeks, with skin parameters measured at baseline and at intervals throughout the study period. Results: Our findings reveal significant variations in epidermal thickness (ET) and dermal-epidermal junction (DEJ) indices with age, particularly noting a decrease in ET and flattening of the DEJ in older age groups. The anti-aging eye cream demonstrated marked improvements in skin hydration levels, elasticity, and a reduction in under-eye wrinkles and crow's feet, underscoring the formula's anti-aging benefits. Conclusion: This study highlights the value of two-photon microscopy in providing detailed insights into the structural changes associated with skin aging, particularly in facial regions. The clinical evaluation of the anti-aging eye cream further confirms its efficacy in addressing key signs of aging, paving the way for personalized skincare treatments. Our findings underscore the therapeutic promise of targeted anti-aging formulations in mitigating the visible effects of skin aging.
... Deepsane is an exopolysaccharide that is extracted from the marine bacteria Alteromonas macleodii and is used to protect sensitive skin from UVB, mechanical, and chemical stress (Balkrishna et al. 2018). An osmoprotectant ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) generated by various bacterial species under osmotic stress, was first isolated from Ectothiorhodospira kalochloris (Graf et al. 2008). Additionally, other halophilic bacteria, such as Actinobacteridae, alpha-and gamma-proteobacteria, also synthesise ectoine in highly saline conditions. ...
Chapter
The need for physiologically active ingredients is being pushed by the rising demand from consumers for natural cosmetics. Because natural biopolymers and novel bioactive compounds have so many benefits over synthetic ones, customers are looking for cosmetics made of these materials more and more often. As a result, cosmetics made of chemical extracts or biomass derived from plants and marine life are being introduced. Because of their activity as antibacterial, antifungal, anti-microalgal, and antioxidant qualities, seaweeds have drawn the interest of researchers. Compared to terrestrial species, marine microorganisms have different metabolic routes and adaption mechanisms, which lead to their distinctive composition, great variety, and significant biological activity. The high concentration of physiologically active compounds and biodiversity can be found in marine ecosystems, which also hold untapped medical, nutraceutical, and cosmetic potential. Marine organisms supply tiny compounds like trichodin A to prevent microbial contamination and ectoine to moisturise skin, in addition to bulk materials like agar and carrageenan that gel and thicken cosmetic formulas. This book chapter focuses on compounds originating from marine environments, including novel chemical entities that have the potential to be used as cosmeceuticals, their modes of action, and the health advantages they offer.
... The analysis conducted using AntiMASH (26) revealed Marinobacter sp. MMG032 has gene clusters for ectoine, a putative thermoprotectant (27). Flagellimonas sp. ...
Article
Full-text available
Here, we report the draft genome sequences of Flagellimonas sp. MMG031 and Marinobacter sp. MMG032, isolated from coral-associated dinoflagellate Symbiodinium pilosum, assembled and analyzed by undergraduate students participating in a Marine Microbial Genomics (MMG) course. A genomic comparison suggests MMG031 and MMG032 are novel species and a resource for restoration and biotechnology.
... This BGC had up to nine genes encoding ectoine biosynthesis (Fig. S2). Ectoine is commonly used in the moisturizing and anti-ageing creams to enhance the skin resistance against the skin cleansers [32]. Table 1 Putative secondary metabolites coding cluster of K. schroeteri based on genome analysis by antiSMASH. ...
... The unique ecological niches provided by extreme environments make microbial resources from these habitats an important source of novel microbial pigments (de Menezes et al. 2023). Additionally, in other high-value industries, ectoine, an amino acid derivative discovered in microorganisms from desert salt lakes, helps microbial cells cope with high osmotic pressure and thermal stress by regulating cellular osmotic balance (Graf et al. 2008). Following its development, ectoine has made a strong entry into the cosmetics industry, with skincare and anti-aging products based on its properties continuously emerging in the market, creating substantial commercial value (Liu et al. 2021). ...
Article
Full-text available
Extreme environments such as hyperarid, hypersaline, hyperthermal environments, and the deep sea harbor diverse microbial communities, which are specially adapted to extreme conditions and are known as extremophiles. These extremophilic organisms have developed unique survival strategies, making them ideal models for studying microbial diversity, evolution, and adaptation to adversity. They also play critical roles in biogeochemical cycles. Additionally, extremophiles often produce novel bioactive compounds in response to corresponding challenging environments. Recent advances in technologies, including genomic sequencing and untargeted metabolomic analysis, have significantly enhanced our understanding of microbial diversity, ecology, evolution, and the genetic and physiological characteristics in extremophiles. The integration of advanced multi-omics technologies into culture-dependent research has notably improved the efficiency, providing valuable insights into the physiological functions and biosynthetic capacities of extremophiles. The vast untapped microbial resources in extreme environments present substantial opportunities for discovering novel natural products and advancing our knowledge of microbial ecology and evolution. This review highlights the current research status on extremophilic microbiomes, focusing on microbial diversity, ecological roles, isolation and cultivation strategies, and the exploration of their biosynthetic potential. Moreover, we emphasize the importance and potential of discovering more strain resources and metabolites, which would be boosted greatly by harnessing the power of multi-omics data.
Article
Streptomyces rochei is a species of Streptomyces with a diverse range of biological activities. S. rochei strain A144 was isolated from desert soils and exhibits antagonistic activity against several plant pathogenic fungi. The genome of S. rochei A144 was sequenced and revealed the presence of one linear chromosome and one plasmid. The chromosome length was found to be 8,085,429 bp, with a GC content of 72.62%, while the Plas1 length was 177,399 bp, with a GC content of 69.08%. Comparative genomics was employed to analyse the S. rochei group. There is a high degree of collinearity between the genomes of S. rochei strains. Based on pan-genome analysis, S. rochei has 10,315 gene families, including 4051 core and 2322 unique genes. AntiSMASH was used to identify the gene clusters for secondary metabolites, identifying 33 secondary metabolite genes on the A144 genome. Among them, 18 clusters were found to be >70% identical to known biosynthetic gene clusters (BGCs), indicating that A144 has the potential to synthesize secondary metabolites. The majority of the BGCs were found to be conserved within the S. rochei group, including those encoding polyketide synthases (PKS), terpenes, non-ribosomal peptide synthetases (NRPS), other ribosomally synthesised and post-translationally modified peptides (RiPP), nicotianamine-iron transporters, lanthipeptides, and a few other types. The S. rochei group can be a potential genetic source of useful secondary metabolites with applications in medicine and biotechnology.
Article
As the global population continues to grow, so does the demand for longer, healthier lives and environmentally responsible choices. Consumers are increasingly drawn to naturally sourced products with proven health and wellbeing benefits. The marine environment presents a promising yet underexplored resource for the cosmetics industry, offering bioactive compounds with the potential for safe and biocompatible ingredients. This manuscript provides a comprehensive overview of the potential of marine organisms for cosmetics production, highlighting marine-derived compounds and their applications in skin/hair/oral-care products, cosmeceuticals and more. It also lays down critical safety considerations and addresses the methodologies for sourcing marine compounds, including harvesting, the biorefinery concept, use of systems biology for enhanced product development, and the relevant regulatory landscape. The review is enriched by three case studies: design of macroalgal skincare products in Iceland, establishment of a microalgal cosmetics spin-off in Italy, and the utilization of marine proteins for cosmeceutical applications.
Article
Full-text available
Evidence is reviewed that freezing and dehydration are fundamentally different stress vectors: (a) Proteins, membranes, phospholipids, and living cells and organisms all contain about 0.25 g nonfreezable H2O/g dry weight. By definition, this H2O is not removed by freezing. (b) Dehydration, by contrast with freezing, can remove the nonfreezable H2O. Removing this H2O results in profound changes in the physical properties of biomolecules, particularly phospholipids and proteins, (c) The mechanisms of preservation of proteins during freezing and drying are completely different. The specificity for solute requirements for stabilization of proteins during freezing is low; any solute that is preferentially excluded from the hydration shell of a protein is also a cryoprotectant. (d) By contrast, stabilization of proteins during drying requires direct interaction between the stabilizing molecule and the protein, probably involving hydrogen bonding between the stabilizer and polar residues in the protein. The specificity is very high in this case; only carbohydrates are effective, and of those that have been tested trehalose is the most effective, (e) Less is understood about the mechanism of stabilization of phospholipid bilayers during freezing, but it is clear that while many solutes will preserve liposomes during freezing, only a few (of which trehalose is the most effective) will preserve them during drying. Stabilization of bilayers during drying requires direct interaction between the sugar and polar head groups of the phospholipids.
Article
The state of intracellular water has been a matter of controversy for a long time for two reasons. First, experiments have often given conflicting results. Second, hitherto, there have been no plausible grounds for assuming that intracellular water should be significantly different from bulk water. A collective behavior of water molecules is suggested here as a thermodynamically inevitable mechanism for generation of appreciable zones of abnormal water. At a highly charged surface, water molecules move together, generating a zone of water perhaps 6 nm thick, which is weakly hydrogen bonded, fluid, and reactive and selectively accumulates small cations, multivalent anions, and hydrophobic solutes. At a hydrophobic surface, molecules move apart and local water becomes strongly bonded, inert, and viscous and accumulates large cations, univalent anions, and compatible solutes. Proteins and many other biopolymers have patchy surfaces which therefore induce, by the two mechanisms described, patchy interfacial water structures, which extended appreciable distances from the surface. The reason for many conflicting experimental results now becomes apparent. Average values of properties of water measured in gels, cells, or solutions of proteins are often not very different from the same properties of normal water, giving no indication that they are averages of extreme values. To detect the operation of this phenomenon, it is necessary to probe selectively a single abnormal population. Examples of such experiments are given. It is shown that this collective behavior of water molecules amounts to a considerable biological force, which can be equivalent to a pressure of 1,000 atm (1.013 x 10(5) kPa). It is suggested that cells selectively accumulate K+ ions and compatible solutes to avoid extremes of water structure in their aqueous compartments, but that cation pumps and other enzymes exploit the different solvent properties and reactivities of water to perform work of transport or synthesis.
Article
Aging involves the whole organism including the immune system. Age-dependent alterations of immune functions are located in both, its adaptive and its innate part. The most important cell type of the innate immune system are the dendritic cells, because their capacity to induce primary immune responses via professional antigen presentation is indispensible for the initiation of the adaptive immune response. Evidence exists, that dendritic cells of the systemic immunity, as represented by lymph node and blood derived dendritic cells, as well as of local immunity, represented by Langerhans cells of the skin, participate in aging processes. In animal models of older mice, dendritic cells of lymph nodes show degenerative characteristics with decreased adhesion molecule expression, less dendrite formation, and reduced antigen trapping capacity, together implying disturbed functional activity. In contrast, dendritic cells generated from peripheral blood of elderly people were not impaired in their capacity to induce T-cell responses. Together, these findings indicate that in old individuals in vivo, dendritic cells of the systemic immunity are reduced in their functional capacity to stimulate immune responses, whereas in vitro generated dendritic cells are fully functional, and therefore, might be used for therapeutic approaches to treat age-associated malfunctions of the immune system. Thus far, only morphological descriptions exist about age associated changes of dendritic cells of the skin, in particular the Langerhans cells. In the skin, effects of natural occuring aging have to be differentiated from UVradiation-induced aging processes. The hallmark of Langerhans cell changes in natural as well as UVinduced skin aging is their reduction in cell number within the epidermis. In addition, they show an atrophic morphology with less dendrites, and less Birbeck granules. It is assumed, that these morphological changes are associated with loss of dendritic cell functions, and that this contributes to age-associated development of skin cancer. Therapeutic strategies against natural and UV-induced skin aging should include improvement of these changes of Langerhans cells in order to strengthen the immunological functions of the body's outer surface.
Article
The production and/or accumulation of organic osmolytes, which serve to compensate for osmotic pressure and low cytoplasmic water activity, are the typical properties of many halophilic microorganisms. These so-called compatible/compensatory solutes not only maintain osmotic equilibrium but also protect and stabilize cytoplasmic components against a variety of stress factors. A molecular basis for this is seen in the kosmotropic nature of these solutes, referring to the structure-forming ability in water. Using a gel filtration method and near-infrared spectroscopy, we were able to demonstrate that nature's prime compensatory solutes (betaine, ectoines, proline, N-acetylated diamino acids) strongly influence surrounding water molecules. The hydration numbers observed (three to five molecules of water per molecule of solute) are comparable with those of the “unfreezable water” recently reported for trehalose and are markedly higher than those of disturbing (chaotropic) salts. In addition, a Gaussian analysis of hydration spectra revealed vibration bands similar to those observed in frozen water, indicating that strong hydrogen bonds are induced by the presence of compensatory solutes.
Article
Ectoine is one of the most common compatible solutes found in halophilic bacteria, and has an effect to introduce a tolerance to high salt concentration or high temperature. By analyzing 1 ns molecular dynamics simulations at 370 K, we have shown that, in the ectoine aqueous solution, the water diffusion slows down around a protein (chymotrypsin inhibitor 2 (CI2)), keeping the protein hydration structure essentially unchanged. It is concluded that the slowdown of water diffusion around the backbone amide protons must be one of the decisive factors in reducing the exchange rate of the backbone amide protons, whose reduction is experimentally believed closely related to the tolerance effect.
Article
Compatible solutes are best described as organic osmolytes responsible for osmotic balance and at the same time compatible with the cells' metabolism. A comprehensive survey (using HPLC and NMR methods) on halophilic/halotolerant eubacteria has revealed the full diversity of compatible solutes employed in nature. Molecular principles derived from the spectrum of compounds found in the bacterial world may be summarized as follows. Compatible solutes are polar, highly soluble molecules and uncharged at physiological pH. With the exception of proline (a proteinogenic amino acid) they are characterized as amino acid derivatives of the following types: betaines, ectoines, N-acetylated diamino acids and N-derivatized carboxamides of glutamine. Using nearinfrared spectroscopy we have also been able to demonstrate that compatible solutes are strong water-structure formers and as such probably excluded from the hydration shell of proteins. This preferential exclusion probably explains their function as effective stabilizers of the hydration shell of native proteins (protection against heating freezing and drying). Hence these typical products of halophilic eubacteria have a considerable potential as stabilizing/protecting agents on both molecular and whole-cell level. Thorough understanding of common structural principles and fundamental water-solute interactions will ultimately enable us to design novel highly efficient stress protectants and stabilizers of biomolecules.
Article
The aim of this study was to elucidate the protective effect of the new compatible solutes, ectoine and hydroxyectoine, on two sensitive enzymes (lactic dehydrogenase, phosphofructokinase). The solutes tested also included (for reasons of comparison) other compatible solutes such as glycine betaine and a number of disaccharides (sucrose, trehalose, maltose). All compatible solutes under investigation displayed remarkable stabilizing capabilities. However, the degree of protection depended on both the type of solute chosen and the enzyme used as a test system. The most prominent protectants were trehalose, ectoine and hydroxyectoine, which are very often found in nature (singly or in combinationn) as part of the compatible solute cocktail of moderately halophilic eubacteria.
Article
In this study on M4-lactate dehydrogenase (LDH) we were able to show that the addition of compatible solutes (glycine betaine, hydroxyectoine) shifts the enzyme's activity curve towards higher temperature. This increase in temperature stability is gained at the expense of a slightly reduced maximal activity and is also reflected in an increase in activation energy. In addition, tryptophan fluorescence spectroscopy has been used to monitor structural changes of the enzyme under conditions of freeze-thawing and urea treatment in the presence of a number of organic and inorganic solutes. As the data revealed that changes in fluorescence intensity are directly related to changes in enzyme activity, we were able to evolve a method for rapid assessment of enzyme stabilisation on the basis of fluorescence measurements. All organic solutes under investigation displayed remarkable stabilising properties, although the degree of stabilisation depended on both the type of solute and the stress factor chosen. It has to be noted that ammonium sulphate also performed very well as a stabiliser against heat and urea treatment, whereas the addition of inorganic salts during freeze-thawing apparently destabilises protein structure, at least under the test conditions employed.
Article
Proline in aqueous solution shows several properties which are unusual for low molecular weight substances. Investigations of solubility, density and viscosity revealed behaviour which is characteristic for hydrophilic colloids. 1H-NMR studies indicated a strong hydrogen bonding of water in proline solutions, especially at high concentrations of the solute. From these results it was concluded that proline forms aggregates by stepwise stacking and hydrophobic interaction of the pyrrolidine ring. Thus, the proposed multimer contans a hydrophobic backbone and hydrophilic groups on the surface, exposed to water. Proline solutions are able to increase the solubility of sparingly soluble proteins. The enhancement effect depends on the nature of the protein and on the proline concentration. It is postulated that by a hydrophobic interaction of proline with hydrophobic surface residues of proteins their hydrophilic area is increased. The presence of proline in solutions of the well soluble protein bovine albumin reduces the precipitation of this protein by ethanol and (NH4)2SO4, presumably by an increased water-binding capacity of the proline-protein solution.
Article
The state of intracellular water has been a matter of controversy for a long time for two reasons. First, experiments have often given conflicting results. Second, hitherto, there have been no plausible grounds for assuming that intracellular water should be significantly different from bulk water. A collective behavior of water molecules is suggested here as a thermodynamically inevitable mechanism for generation of appreciable zones of abnormal water. At a highly charged surface, water molecules move together, generating a zone of water perhaps 6 nm thick, which is weakly hydrogen bonded, fluid, and reactive and selectively accumulates small cations, multivalent anions, and hydrophobic solutes. At a hydrophobic surface, molecules move apart and local water becomes strongly bonded, inert, and viscous and accumulates large cations, univalent anions, and compatible solutes. Proteins and many other biopolymers have patchy surfaces which therefore induce, by the two mechanisms described, patchy interfacial water structures, which extended appreciable distances from the surface. The reason for many conflicting experimental results now becomes apparent. Average values of properties of water measured in gels, cells, or solutions of proteins are often not very different from the same properties of normal water, giving no indication that they are averages of extreme values. To detect the operation of this phenomenon, it is necessary to probe selectively a single abnormal population. Examples of such experiments are given. It is shown that this collective behavior of water molecules amounts to a considerable biological force, which can be equivalent to a pressure of 1,000 atm (1.013 x 10(5) kPa). It is suggested that cells selectively accumulate K+ ions and compatible solutes to avoid extremes of water structure in their aqueous compartments, but that cation pumps and other enzymes exploit the different solvent properties and reactivities of water to perform work of transport or synthesis.