Sub-zero temperature inactivation of carboxypeptidase Y under high hydrostatic pressure Toshihiko Kinsho1,*, Hiroshi Ueno1,†, Rikimaru Hayashi1, Chieko Hashizume2and Kunio Kimura2,† 1 Di
Trang 1Sub-zero temperature inactivation of carboxypeptidase Y under high hydrostatic pressure
Toshihiko Kinsho1,*, Hiroshi Ueno1,†, Rikimaru Hayashi1, Chieko Hashizume2and
Kunio Kimura2,†
1
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo, Japan;2Meidi-Ya Food Factory Co., Ibaraki, Osaka, Japan
High hydrostatic pressure induced cold inactivation of
carboxypeptidase Y Carboxypeptidase Y was fully active
when exposed to subzero temperature at 0.1 MPa; however,
the enzyme became inactive when high hydrostatic pressure
and subzero temperature were both applied When the
enzyme was treated at pressures higher than 300 MPa and
temperatures lower than)5 °C, it underwent an irreversible
inactivation in which nearly 50% of the a-helical structure
was lost as judged by circular dichroism spectral analysis
When the applied pressure was limited to below 200 MPa,
the cold inactivation process appeared to be reversible In
the presence of reducing agent, this reversible phenomenon,
observed at below 200 MPa, diminished to give an inactive
enzyme; the agent reduces some of disulfide bridge(s) in an
area of the structure that is newly exposed area because of
the cold inactivation Such an area is unavailable if carb-oxypeptidase Y is in its native conformation Because all the disulfide bridges in carboxypeptidase Y locate near the act-ive site cleft, it is suggested that the structural destruction, if any, occurs preferentially in this disulfide rich area A poss-ible mechanism of pressure-dependent cold inactivation of CPY is to destroy the a-helix rich region, which creates an hydrophobic environment This destruction is probably a result of the reallocation of water molecules Experiments carried out in the presence of denaturing agents (SDS, urea, GdnHCl), salts, glycerol, and sucrose led to a conclusion consistent with the idea of water reallocation
Keywords: high hydrostatic pressure; cold inactivation; carboxypeptidase; serine protease; protein denaturation
Carboxypeptidase isolated from Saccharomyces cerevisiae
(CPY) is an exopeptidase specific to C-terminal amino acid
residues of peptide or protein substrates CPY is a
monomeric glycoprotein with 421 amino acid residues in
which four carbohydrate chains are attached via b amide
nitrogen of aspargine residues [1] It has catalytically
essential serine, histidine and aspartate residues, which
make CPY as a member of serine protease family [2] Like
other serine proteases, the enzyme also exhibits an esterase
activity CPY maintains its activity in the presence of
relatively high concentration of SDS, glycerol or salts [3]
High hydrostatic pressure has been used as a denaturant
in study of protein structure Accumulating evidence
suggests that pressure-dependent denaturation of protein
is a reversible process in most cases, where the process of
denaturation by high pressure appears to be significantly
different from that caused by pH, chemical agents or heat
[4,5] Brandts [6], Hawley [7] and others have shown that a
combination of pressure and temperature is highly effective method for the study of protein denaturation Although cold denaturation or inactivation of proteins is a recognized subject matter in protein chemistry [8–11], combined effects
of cold temperature and high pressure on proteins have not been explored extensively
Cold denaturation (in general the terminology cold denaturation is commonly used; however, we use cold inactivation in this study because our emphasis is on the inactivation of enzymic activity) of proteins has been shown previously [12–16], where denaturation was due to subunit dissociation, which is reversible, and sometimes due to aggregation [17,18] In the majority of the cold denaturation experiments, the temperature employed was above freezing point in order to avoid the problems associating with sample being frozen Most proteins, including monomeric ones, are susceptible to freezing and readily become inactive Some attempts have been made to avoid sample freezing
by adding organic solvent [11] Although the presence of organic solvent was successful in lowering the freezing point, down to)100 °C, some of the physical properties of protein
in such solution, i.e thermal denaturation temperature and kinetic data, were altered [8,9,11,19,20] A protein whose activity is preserved, despite prolonged treatment of subzero temperatures in the absence of any antifreezing agent, might
be an ideal candidate for studying high pressure effects on protein structure
In the present study, high hydrostatic pressure and subzero temperature are combined and used as denaturants
on CPY It is also considered the effects of various agents, including those commonly used for protein denaturation and for protein stabilization at neutral pH
Correspondence to R Hayashi, Division of Applied Life Sciences,
Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto
606-8502, Japan.
Fax: + 81 75 753 6128, Tel.: + 81 75 753 6110.
Abbreviations: ATEE, N-acetyl- L -tyrosine ethyl ester monohydrate;
BTEE, benzoyl- L -tyrosine ethyl ester; CPY, carboxypeptidase Y;
GdnHCl, guanidine hydrochloride.
*Present address: Sanyo Chemical Co., Kyoto, Japan.
Present address: Laboratory of Applied Microbiology and
Biochemistry, Nara Women’s University, Nara 630-8506, Japan.
(Received 19 March 2002, revised 29 June 2002,
accepted 5 August 2002)
Trang 2M A T E R I A L S A N D M E T H O D S
Reagents
Nicotinamide adenine dinucleotide (NADH) and sodium
pyruvate were from Sigma Chemical Co (St Louis, MO,
USA) Benzoyl-L-tyrosine ethyl ester (BTEE),
N-acetyl-L-tyrosine ethyl ester monohydrate (ATEE) and kerosene
were from Nacalai Tesque Co (Kyoto, Japan) CPY was
prepared according to Hayashi et al [3] The other reagents
were highest reagent grade obtained locally
Cold denaturation of CPY under high hydrostatic
pressure
Pressurization apparatus was obtained from Hikari
Kohatsu Instruments Co (model no KP-5B, Hiroshima,
Japan) which generates maximum pressure of 600 MPa
Approximately 200 lL of CPY solution (1–2 mgÆmL)1in
10 mM sodium phosphate buffer, pH 7.0) was placed in
polyethylene pouch and heat-sealed This pouch was
placed in another polyethylene pouch, and distilled water
was used to fill between the two pouches It was
imperative to eliminate any air bubbles from both plastic
bags The double bag was placed inside of the pressure
vessel that was filled with kerosene and pre-equilibrated at
desired temperature The pressure vessel was immersed
into a large refrigerated constant temperature water bath
After the kerosene temperature reached equilibrium,
pressure was applied for 30 min As a control, another
sample was kept at room temperature without
pressuri-zation CPY activity was measured immediately after the
pressure release (ex situ)
CPY activity measurements
Enzymic activity was measured spectrophotometrically on a
Shimadzu UV-210A digital double beam
spectrophoto-meter Both sample and reference cuvettes containing 2 mL
of 50 mM sodium phosphate buffer, pH 7.5 with and
without 1 mM ATEE, respectively, were placed in cell
holders Sample temperature was maintained at 25°C with
a constant temperature water bath As soon as 10 lL of the aliquots of pressure-treated CPY was added into the sample cuvette, a time-dependent linear absorbance change at
237 nm was recorded for 10–15 min The slope of absorb-ance change under 0.1 MPa was used for 100% activity and that in the absence of CPY was 0% activity
Circular dichroism measurements Measurements of CD spectra were performed on a JASCO J-720W spectropolarimeter (JASCO Co., Tokyo, Japan) A quartz cell with 1 mm light path length was used for the native and pressure-treated CPY (2.7 lMin 0.02M potas-sium phosphate buffer, pH 6.0)
Molecular modeling X-ray crystallographic data of carboxypeptidase Y (1ysc) was obtained from the Protein Data Bank (http:// www.pdb.bnl.gov) Molecular modeling was performed
on an IRIS Indigo2-EX Silicon Graphics workstation by using QUANTA 96 software (Molecular Simulations, Inc., California, USA)
R E S U L T S
CPY at subzero temperature When CPY solution was exposed to temperature ranging from 22 to )30 °C at 0.1 MP a (s in Fig 1), nearly full activity was maintained throughout the temperature range examined This suggests that CPY is highly resistant against cold
Effect of high pressure on CPY at low temperature When CPY was exposed to 400 MPa at 10°C, its activity was reduced 50% within 10 min Further treatment did not show any additional changes in the activity (s in Fig 2) This suggests that a part of CPY structure is altered by
Fig 1 Effect of temperature on CPY
inacti-vation under various pressures Enzymic
activ-ities of CPY, which was treated with
atmospheric pressure (0.1 MPa) (s),
100 MPa (d), 200 MPa (h), 300 MPa (j),
and 400 MPa (n) at the indicated
tempera-tures for 30 min, were measured Each data
point represents an average of three separate
measurements Inset shows a water/ice phase
diagram.
Trang 3high-pressure treatment, but the rest appears to be resistant
when the treatment was carried out at 10°C
The apparent inactivation of CPY was evident when the
enzyme was exposed to lower temperatures (0 and)10 °C)
and at 400 MPa (d and h in Fig 2) The time course of
CPY inactivation at 0°C appeared to be a biphasic, and at
)10 °C it became monophasic, indicating that the cold
inactivation at)10 °C underwent a two-phase transition
mechanism Thus, it is clear that the cold inactivation of
CPY is either induced or accelerated when high hydrostatic
pressure is applied
As summarized in Table 1, pressure treatment of CPY
was carried out under two different conditions to investigate
a possible effect of ice formation during the experiments
In one condition, high pressure was applied after thermal equilibrium at the desired temperature was achieved During this equilibrium period, ice could form at tempera-tures below 10°C In the other condition, both pressuriza-tion and depressurizapressuriza-tion steps were performed at room temperature to avoid any ice formation Because there were
no significant differences on the degree of enzymic inacti-vation between the two conditions, the idea of ice formation being a contributing factor for the cold inactivation of CPY was eliminated
Circular dichroism analysis of cold inactivated CPY Far-ultraviolet CD spectra (190–250 nm range) of both the native and cold inactivated CPY (treated under 400 MPa at )5 °C for 30 min) are shown in Fig 3 Native CPY exhibits
a typical a/b mixed-type CD spectrum as having a negative peak within 210–220 nm The CD spectrum of the cold inactivated CPY showed reduction of peaks, the positive one at 194 nm and the negative one at 210–220 nm The results indicate a significant alteration in its secondary structure of the cold inactivated CPY As we performed a quantitative analysis of the secondary structure according to the method of Yang et al [21] an approximately 50% loss of the helical structure was estimated
Combined effects of low temperature and high pressure on CPY
As Fig 1 exhibits smooth transition curves for pressure treatment at 0.1–400 MPa at a temperature ranging from 22
to)30 °C, the effect of freezing was excluded as the cause of inactivation Curves also indicate that the loss of enzymic activity is a pressure-dependent phenomenon It is evident that pressure-dependent inactivation becomes the predomi-nant event at the temperature below)10 °C
Reversibility of low temperature inactivation of CPY under high pressure
Time-dependent recovery of CPY activity was observed after the release of pressure (Fig 4) When CPY activity was
Fig 2 Kinetics of the pressure inactivation of CPY at 400 MPa under
different temperatures Enzymic activities of CPY at different time
points were measured while CPY was pressurized under 400 MPa at
10 °C (s), 0 °C (d), and )10 °C (h).
Fig 3 Circular dichroism spectra of the native and pressure-treated CPY Native CPY spectrum is shown as a solid line and the pressure-treated CPY is shown as a broken line CPY was pressure-pressure-treated under 400 MPa at )5 °C for 30 min.
Table 1 Effect of ice formation on the pressure- and
temperature-dependent CPY inactivation In method 1, CPY activity was measured
immediately following the depressurization of the sample treated by
the pressure as described under methods, where the pressurization
process was initiated after the inside pressure vessel reached the desired
temperature In method 2, the pressure vessel was pressurized at room
temperature before it was incubated at the indicated temperature for
60 min Before the pressure release, the vessel was placed in the 25 °C
water bath for 10 min in order to prevent the icing during the
depressurization period.
Pressure
(MPa)
Temperature
(°C)
Activity (%) Method 1 Method 2
Trang 4measured immediately after the treatment at 10°C and at
400 MPa for 30 min, it reduced to around 45% On
standing at 4°C, this sample showed a gradual activity
recovery over 50 min This suggests that the process of
structural alteration occurred under 400 MPa at 10°C is
reversible On the other hand, when CPY was treated at 0 or
)10 °C under 400 MPa, no significant activity recovery was
observed The results indicate that low temperature
treat-ment of CPY under 400 MPa exhibits a cold inactivation of
the enzyme, in which an irreversible damage is evident
Effect of 2-mercaptoethanol upon cold inactivation
of CPY under high pressure
Table 2 summarizes the effect of 2-mercaptoethanol on
CPY, which is exposed to high pressure at 22°C and
)22 °C in the presence or absence of 0.1M
2-mercaptoeth-anol for 30 min At 22°C, the effect of 2-mercaptoethanol
was insignificant in the pressure range we examined At
)22 °C, near complete inactivation of CPY was observed
for 100 and 200 MPa treatments in the presence of
2-mercaptoethanol The results indicated that disulfide
bond(s) became accessible to reducing agent at or above
100 MPa below subzero temperature
Effects of denaturing agents upon cold inactivation of CPY under high pressure
CPY maintains 80% of its activity after 1 h incubation with
6Murea, in neutral pH buffers, at 25°C under atmospheric pressure (A Yamazaki, H Ueno & R Hayashi, unpub-lished results) This suggests that CPY is a highly resistant protein against urea denaturation The cold inactivation of CPY was observed readily when either urea or guanidine HCl was present (Table 3) A concentration of 1Murea was sufficient to achieve more than 70% inactivation at)10 °C under 200 MPa for 30 min Higher concentrations of urea lead to nearly 90% inactivation under the identical condi-tions Guanidine HCl is a stronger denaturant than urea; near complete inactivation is achieved at guanidine HCl concentration of 1Mor above
Effect of SDS upon cold inactivation of CPY under high pressure
CPY was incubated at)10 °C for 30 min under 400 MPa
in the presence of various concentrations of SDS (Fig 5) SDS concentration at 1 mM or less (< 0.03% w/w) exhibited a maximum (30%) protection from the inactiva-tion This protective action of SDS disappeared at the concentration higher than 2 mM The amount of SDS used
in the experiments was less than SDS/PAGE analysis
Table 2 Effect of 2-mercaptoethanolrcaptoethanol upon CPY activity treated with high hydrostatic pressures at 22 and )22 °C CPY activity was measured immediately after the pressure treatment as described under methods, where the pressurization process was initiated after the inside pressure vessel reached the desired temperature Concentration of 2-mercaptoethanol was 0.1 M 2ME, 2-mercaptoethanol.
Temperature
(°C)
Pressure (MPa)
Activity remaining (%)
% Loss of activity upon addition of 2ME
Fig 4 Change in CPY activity following the release of pressure.
Enzymic activities of CPY, which was exposed to 400 MPa pressure at
10 °C for 60 min (s), at 0 °C for 90 min (d), and at )10 °C for
30 min (h), were measured.
Table 3 Effect of urea and guanidine HCl on CPY inactivation at low temperature Experiments were performed at )10 °C and 200 MP a for
30 min as described under methods.
Denaturant Concentration ( M ) Activity remaining (%)
Guanidine HCl 0 79
Trang 5Effect of glycerol and sucrose upon cold inactivation
of CPY under high pressure
CPY was incubated at)10 °C for 30 min under 400 MPa
in the presence of either glycerol or sucrose This condition
was chosen because both polyols showed a protective effect
against cold inactivation The effects were linearly increased
up to 20% (about 2M) for glycerol and 1.0Mfor sucrose
(Fig 6)
Effect of inorganic salt and cations upon cold
inactivation of CPY under high pressure
Sodium chloride acted in a manner similar to glycerol; its
protective effect increased linearly as the concentration was
raised up to 3M(Fig 7)
Effects of monovalent cations were also examined, where
protective roles of monovalent cations were demonstrated
Except for Li+ ion, the examined cations, Cs+, Rb+,
NH4+, K+, and Na+, fit into a linear line when CPY
activity was plotted against 1/ionic radii (Fig 8) The results
indicate that smaller cationic ions have higher protective
effects than larger ions
Structural summary of CPY
Fig 9 represents a structural feature of CPY based upon
X-ray crystallographic data of Endrizzi et al [22] where five
pairs of disulfide bonds and the catalytic triad,
Ser146-His397-Asp338, are colored in green Peptide backbone was
also colored gradually according to the temperature factor
Three principal features of CPY structure are shown: (a)
spatial orientation of a-helices; (b) location of disulfide
bridges; and (c) distribution of movable (flexible) residues
Active site cleft of CPY is constructed with five a-helices,
these locate as if they surround the catalytic triad In addition, five disulfide bridges (Cys56–298, Cys198–207, Cys217–240, Cys224–233 and Cys262–268) also locate close
to this active center Three of them (Cys198–207, Cys217–
240, and Cys224–233, where the latter two are called disulfide zipper) are on the a-helices Cys56–298 locates at very end of the short a-helix (300–313) Although the role of the disulfide bridges in CPY is still undefined, they seem to
be important in maintaining the active structure because the loss of disulfide bridge(s) results in the inactive enzyme [3]
Fig 5 Effect of SDS on the pressure-induced inactivation of CPY under
the subzero temperature Enzymic activities of CPY were measured
after the treatment of CPY with 400 MPa pressure at )10 °C for
30 min in the presence of indicated concentration of SDS.
Fig 6 Protective action of glycerol and sucrose against pressure-induced inactivation of carboxypeptidase Y under subzero temperature Enzymic activities of CPY, which was treated with 400 MPa pressure
at )10 °C for 30 min in the presence of indicated amount of glycerol (d) or sucrose (s), were measured.
Fig 7 Protective action of sodium chloride against pressure-induced inactivation of carboxypeptidase Y under subzero temperature Enzymic activities of CPY were measured after it was treated with 400 MPa pressure at )10 °C for 30 min in the presence of indicated amount of NaCl (d).
Trang 6The red colored region in Fig 9 represents highly flexible
area in CPY structure and is likely to be destroyed during
the heat denaturation It is of interest to point out that the
central cleft rich in a-helix (colored blue) is a rather
immobile region CPY is considered relatively stable enzyme
against various denaturants, i.e chemical agents [23], pH
change [3], salts [24,25], and freezing [23] The stability of
CPY may originate from the above-described three
princi-pal features
D I S C U S S I O N
Unique properties of CPY CPY exhibits a typical pressure-dependent cold inactivation phenomenon When both subzero temperature and pressure above 300 MPa are applied, the enzyme activity is strongly inhibited However, the enzyme remains fully active after the exposure to subzero temperature under atmospheric pressure Other enzymes we have examined, like chymo-trypsinogen A, pancreatic ribonuclease A, and lactate dehydrogenase, showed a significant loss (10–20%) of enzymic activity just by standing at subzero temperature for
30 min without extra pressure applied (T Kinsho, H Ueno
& R Hayashi, unpublished results.) To this extent, CPY has ideal properties for the study of the pressure-dependent cold inactivation process Previously, our own [26] and other [27] studies have demonstrated the phenomenon of pressure-induced cold inactivation of CPY In this study, detailed molecular events in the pressure-induced cold inactivation of CPY are described
Action of reducing agent which reveals site(s) of cold inactivation
The action of reducing agent was drastic: only when two denaturation elements, high pressure and subzero tempera-ture, were combined, did the reducing agent successfully inactivate the enzyme (Table 2) Because CPY is highly resistant to reduction under native or pressurized conditions
at ambient temperature, the observed inactivation must associate with some conformational changes occurring at the sites near disulfide bond(s) Such conformational changes enable the penetration of the reducing agent, as well as solvents
Figure 9 indicates the location of disulfide bonds in CPY
It is apparent that both the disulfide bonds and a-helix rich region locate at or near the central cleft as if they surround the catalytic triad Our present results with reducing agent suggest that the structural alterations under the
Fig 8 The correlation between the size of monovalent cationic ions and
their effects on activity of CPY treated under high pressure at subzero
temperature Enzymic activities of CPY were measured after the
treatment with 400 MPa pressure at )10 °C for 30 min in the presence
of 1 M of chloride salt of Li + (s), Na + (d), K + (h), Rb + (j), NH 4+
(n), and Cs + (m) Dotted line indicates activity level for the absence of
salts.
Fig 9 Spatial orientation of disulfide bonds
and temperature factor in computer generated
CPY model CPY structure was visualized on
a SGI Indigo2-EX computer where location
of disulfide bonds is labeled Temperature
factor listed in 1ysc PDB file was used for
coloring Red is designated for 100 and blue
for 0.
Trang 7pressure-dependent cold inactivation occurs in this a-helix
rich region
Figure 9 also represents flexibility of CPY based upon
crystallographically determined temperature factor It
pro-vides an important clue concerning the structural aspects of
the pressure-dependent cold inactivation It is evident that
the above-mentioned a-helix rich region is found in the blue
colored area, suggesting that the a-helix rich region is less
inert towards heat but can be sensitive to the
pressure-dependent cold inactivation as described above in the study
with the reducing agent It is also suggested that there are
separate denaturation pathways in CPY towards heat and
pressure-dependent cold inactivation (Scheme 1) A similar
conclusion was previously obtained by others in the study of
subtilisin inhibitor, where the thermal denaturation process
was different from the cold denaturation process as judged
by the spectroscopic means [28]
The use of reducing agent can be useful for probing
protein conformation [29] Whether the reduction of
disulfide bond(s) would be the sequential event and which
disulfide bond(s) would be reduced are important questions
that remained to be investigated Nevertheless,
conforma-tional change around the sulfhydryl groups or disulfide
bonds must be associated with an increased solvent
accessibility leading to a more efficient reaction of SH and
SS groups This lets us focus on how water molecules affect
the CPY molecule, as discussed in the following sections
Effects of polyols
The presence of glycerol or sucrose, compounds known to
increase hydration to free water molecules, protects CPY
from pressure-dependent inactivation at subzero
tempera-ture, e.g 40% glycerol shows a maximum protection
against 400 MPa pressure at)10 °C The protective role
of polyols, including some mono- or di-saccharides, for the
stability of native structure of proteins against heat or cold
denaturation has been reported [30–37] The strong
inter-action between polyols and free water molecules may help
to reduce the action of those free water molecules; thus, the
ionic and hydrophobic amino acid residues critically
involved in the construction of the stable protein
confor-mation are ineffective in participating in binding with water
molecules [38] It is feasible that free water molecules in the
active site cleft participate in the destruction of the CPY structure, in which the presence of polyalcohols can prevent penetration of water molecules to the active site to give the protective effect
Effects of cations The protective power of small cationic ions (Fig 8) can
be explained, although speculatively, by their high surface charge density, which enhances hydration by interacting with more water molecules, via electrostatic interactions
Frank and Wen proposed an ion–solvent interaction model to explain the roles of ions in water structure [39] Ions such as Li+ and Na+ are surrounded by three concentric regions of the water: the innermost (region A) water is highly immobilized, the second (region B) in which the water is less ice-like, and the third (region C) contains normal water Because Li+and Na+ions form a strong dipole interaction with water molecules in region A, they are named as structure-making ions On the other hand, ions such as K+, Rb+, Cs+and NH4+are surrounded by the water; thus, these ions are called structure-breaking ions Based upon Frank and Wen’s model, the high protective effect of Na+ ion can be explained by having region A water structure around Na+ion, which resists making ice-like ordered water clusters NH4+ion takes a tetrahedral conformation, which fits well into ice-forming water struc-ture [40]; thus, there is no protective effect Li+ion, despite
of having region A water structure, does not show any protective effect The property of the Li+ion is unique in making a tetrahedral conformation whose structure is indistinguishable from regions B and C water under the pressurized condition at subzero temperature [41] Our results are consistent with the Frank and Wen model
Effects of chaotropic agents The presence of urea or GdnHCl accelerates CPY inacti-vation at subzero temperature under high pressure Kauzmann described similar observations on ovalbumin [42], where ovalbumin treated with urea at 0°C under
100 MPa accelerated the denaturation rate compared to the rate at 40°C under 100 MPa This acceleration strongly
Scheme 1 Hypothetical denaturation pathway of heat denaturation versus pressure-dependent cold inactivation.
Trang 8suggests the combined application of urea or GdnHCl with
subzero temperature and high pressure for protein
chem-istry field For example, SH modification protocol described
by Crestfield et al [43] can be performed with only 1 or 2M
urea instead of 8M if one uses low temperature and high
pressure This should make the remaining step of removing
excess urea from the samples, subjected to classical
carbo-xymethylation, less problematic [44]
The protective action of SDS may be explained by its
ability in disturbing the highly structured ice formation in
protein molecule; thus, preserving the hydrophobic
interac-tions, which maintains the native-like structure Meanwhile,
a further understanding of SDS properties under the high
pressure conditions, i.e changes in solubility and critical
micelle concentration (CMC), is desired
Effects of subzero temperature and high pressure
on water molecules
It is suggested that water molecules tend to form highly
ordered clusters with enhanced hydrogen bond interactions
under cold temperature [19] Formation of such water
clusters causes weaker hydrophobic interactions and
enhances the hydrogen bond interaction within water
molecules locating inside the cluster or in the surface water
molecules
High hydrostatic pressure contributes to further
weak-ening the hydrophobic interactions by creating a hydration
environment in the hydrophobic area of the protein
molecule Therefore, the irreversible inactivation of CPY
under 300 or 400 MPa at subzero temperatures is likely
caused by the weakened hydrophobic interactions Our
present results are consistent with currently accepted
interpretation of pressure effects on proteins (for example
[45,46])
A C K N O W L E D G E M E N T S
Authors indebted to Ms Asako Yamazaki for her help in obtaining
CD spectra Thanks are due to Professor Michael M Cox for his
critical reading of the manuscript.
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