Sub-zero temperature inactivation of carboxypeptidase Y under high hydrostatic pressure Toshihiko Kinsho 1, *, Hiroshi Ueno 1, †, Rikimaru Hayashi 1 , Chieko Hashizume 2 and Kunio Kimura 2, † 1 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo, Japan; 2 Meidi-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 Ôcold denaturationÕ is commonly used; however, we use Ôcold inactivationÕ 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) Eur. J. Biochem. 269, 4666–4674 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03167.x MATERIALS AND METHODS 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 )1 in 10 m M 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 m M sodium phosphate buffer, pH 7.5 with and without 1 m M ATEE, respectively, were placed in cell holders. Sample temperature was maintained at 25 °Cwith a constant temperature water bath. As soon as 10 lLofthe 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 l M in 0.02 M 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). RESULTS CPY at subzero temperature When CPY solution was exposed to temperature ranging from 22 to )30 °Cat0.1MPa(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)attheindicatedtempera- tures for 30 min, were measured. Each data point represents an average of three separate measurements. Inset shows a water/ice phase diagram. Ó FEBS 2002 Cold inactivation of CPY under high pressure (Eur. J. Biochem. 269) 4667 high-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 depressurization 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-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 2 200 25 90 93 10 90 88 07783 )10 67 73 )20 54 46 300 25 77 77 10 68 62 05754 )53548 –15 19 29 4668 T. Kinsho et al. (Eur. J. Biochem. 269) Ó FEBS 2002 measured immediately after the treatment at 10 °Candat 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 °Cis 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 °Cand )22 °C in the presence or absence of 0.1 M 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 6 M urea, 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 1 M urea 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 1 M or 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 m M 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 m M . 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 )2ME +2ME 22 0.01 100 100 0 100 92 89 3.3 200 90 86 4.4 300 77 70 9.1 400 63 59 6.4 )22 0.01 94 90 4.3 100 78 4.5 94 200 56 3.9 93 Fig. 4. Change in CPY activity following the release of pressure. Enzymic activities of CPY, which was exposed to 400 MPa pressure at 10 °Cfor60min(s), at 0 °Cfor90min(d), and at )10 °Cfor 30 min (h), were measured. Table 3. Effect of urea and guanidine HCl on CPY inactivation at low temperature. Experiments were performed at )10 °Cand200MPafor 30 min as described under methods. Denaturant Concentration ( M ) Activity remaining (%) Urea 0 79 127 221 311 Guanidine HCl 0 79 1 9.0 2 0.0 Ó FEBS 2002 Cold inactivation of CPY under high pressure (Eur. J. Biochem. 269) 4669 Effect 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 2 M ) for glycerol and 1.0 M for 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 3 M (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 + , NH 4 + ,K + ,andNa + , 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 °Cfor 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). 4670 T. Kinsho et al. (Eur. J. Biochem. 269) Ó FEBS 2002 The 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. DISCUSSION 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. Ó FEBS 2002 Cold inactivation of CPY under high pressure (Eur. J. Biochem. 269) 4671 pressure-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 normalwater.BecauseLi + 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 NH 4 + 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. NH 4 + 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. 4672 T. Kinsho et al. (Eur. J. Biochem. 269) Ó FEBS 2002 suggests 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 2 M urea instead of 8 M 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]). ACKNOWLEDGEMENTS 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. REFERENCES 1. Shimizu, H., Ueno, H. & Hayashi, R. 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