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118. Raman spectroscopic study on the hydrophobic hydration structure in the glassy state
Abstracts / Cryobiology 59 (2009) 370–418 process [1]. It can be considered that these properties will generate a new cryoprotective or lyoprotective effect of the sugar-based surfactants. But it has been unclear how these characteristics could be originated by these sugar surfactants. The purposes of this study are the basic investigation of the freeze-thawing process in an aqueous sugar-based surfactant solution and the study of its frozen state. Octyl-b-D-glucoside (C8Glu) was prepared by a conventional method with little modification as a simple sugar-based surfactant. Sample solutions were prepared by dissolving C8Glu in pure water. Higher concentrations were directly made in an aluminum pan by drying a solution (35 wt%) in the dessicator over phosphorous pentoxide at ambient temperature for differential scanning calorimetry (DSC) measurement. A DSC equipped with a cooling accessory was used to determine the lyotropic phase diagrams, the liquidus curve (Tm), the glass transition temperatures (Tg), and the glass transition temperatures for freeze-concentrated solution (Tg0 ) and its specific heat change (DCp) for this study. To confirm precisely the liquidus curve and the mesophases, a thermal arrest measurement and an optical observation with a polarizing microscope were also conducted. The freeze-thawing behavior of the C8Glu-water binary system was more complicated than that of the simple sugar–water system. When the dilute solutions were frozen, the phase transitions occurred during the thawing process at around 13, 6.2, 1.0 °C, independently plus pure ice melting. The transition peak around 13 °C was observed after the exothermic peak only when a lower scan rate of heating was imposed. Cryomicroscopic observation clearly showed that the phase texture changed from isotropic cubic to anisotropic hexagonal around 6.2 °C, and from hexagonal to isotropic micelle around 1.0 °C during the meling of ice. Thus, the effect of the freeze-concentration of the C8Glu by the formation of ice was clearly found. In the freeze-concentrated state, the unfrozen solutions show the specific heat change in the thawing process from 44 to 40 °C. This phenomenon was considered as the glass transition with freeze-concentration. Its temperature was defined as Tg0 . Two different glass transition temperatures, T1g0 , T2g0 were observed with different thermal hysteresis. On the other hand, the two different Tg curves were also depicted whether the initial phase was lamella (T1g) or gel (T2g). In the supplemented phase diagram, only the coexistence in the magnitude of DCp was recognized at the intersectional glasstransition temperatures at 44 °C in the combination of T2g0 with T1g. Therefore, we concluded that the unfrozen solutions of the C8Glu-water system form the glassy state and it was concluded that the unfrozen solutions in the C8Glu-water system vitrified and the concentration of C8Glu was 91 wt% under maximally freeze-concentrated state. (Conflicts of interest: None declared. Source of funding: None declared.)
403
119. Supercooling and vitrification of aqueous polyethyleneglycol solutions. *K. Kajiwara a, Y. Kitada a, K. Tomizawa b, H. Kanno b, a School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakura Hachioji, Tokyo 192-0982, Japan, b Department of Applied Chemistry, National Defense Academy, Yokosuka, Kanagawa 239-8686, Japan Studies of aqueous solutions at low temperatures are important in cryopreservation of living cells and biotechnology, not to say in basic science. As there is a wide range of molecular weights among water soluble polyethyleneglycols (PEGs), we can study supercooling and vitrification of their aqueous solutions as a function of molecular weight in a systematic way. In this study, we first measured the homogeneous ice nucleation temperatures (THs) of aqueous PEG solutions as a function of PEG concentration and then we tried to vitrify the solutions at the solute concentration at which they give TH of 80, 90 or 100 °C. The average molecular weights of PEGs used in this study are M = 200, 300, 400, 600, 1000, 1540, 2000, and 3000. The emulsification method developed by Rasmussen and MacKenzie [1] was used to measure TH of these aqueous solutions. A simple DTA method was used in the TH measurements. On the other hand, the bulk solutions were used for the measurements of the glass transition temperatures (Tgs). The cooling rate was about 1.8 103 °C/min in the quenching process and the heating rate was about 4 °C in the Tg region. Fig. 1 shows the TH results for the aqueous PEG solutions of M = 1000, 2000 and 3000 and the hypothetical TH curves for the M = 3000 solution by increasing the PEG concentration by 1.5 or 3 times without changing the TH value. Fairly well overlapping of the hypothetical TH curve with the TH curve for the M = 1000 or 2000 clearly demonstrates that additivity rule holds for the supercooling of aqueous PEG solutions. It is interesting to note that Tg of the PEG solution shows systematic variation with the molecular weight (M) of PEG even for the PEG solutions showing the same degree of supercooling. (Conflicts of interest: None declared. Source of funding: None declared.)
M = 3000 M = 1000 M = 2000 M = 3000(1.5N) M = 3000(3N)
-40
Reference
doi:10.1016/j.cryobiol.2009.10.131
TH /°C
-50 [1] Ogawa S, Osanai S. Inhibition effect of sugar-based amphiphiles on eutectic formation in the freezing–thawing process of aqueous NaCl solution. Cryobiology 2007;54:173–80.
-60
-70 118. Raman spectroscopic study on the hydrophobic hydration structure in the glassy state. *Yukihiro Yoshimura, Takahiro Takekiyo, Department of Applied Chemistry, National Defense Academy, 1-10-20 Hashirimizu, Yokosuka, Kanagawa, Japan It is well known that guanidine hydrochloride (GdmHCl) is used to elucidate the protein folding mechanism. According to previous solubility studies by Nozaki et al. [J. Biol. Chem. 238 (1963) 4047; 245 (1970) 1648], they assumed that the addition of GdmHCl to an aqueous protein solution weakens the hydrophobic interaction of the hydrophobic amino acids such as Val, Ile, and Leu in proteins. However, the details of the hydration structure around proteins under the coexistence of GdmHCl are still unclear. Here we focus on the additive effect on the hydration structure of hydrophobic compounds in aqueous solution at 77 K. We selected tetrapropylammonium chloride (n Pr4 NCl) as a model hydrophobic compound, because n-Pr4N+ cation is widely accepted as a suitable substance for investigating hydrophobic interactions in aqueous solutions [J. Solution Chem. 2 (1973) 253]. The structure of the n Pr4 N+ cation resembles that of hydrophobic amino acids such as Val, ILe, and Leu. Besides, the aqueous n Pr4NCl solution becomes a glass upon fast cooling to liquid nitrogen temperature [J. Phys. Chem. 93 (1989) 4981]. We have measured Raman OH stretching spectra of aqueous n Pr4NCl solution at R (=moles of water/moles of n Pr4NCl) = 30 as a function of GdmHCl. At this concentration, n Pr4NCl is considered to form a hydrophobic hydration. The glassy Raman OH stretching spectrum of aqueous n Pr4NCl solution at 77 K has peaks at around 3100 and 3450 cm1. The Raman intensity at around 3100 cm1 I3100 cm1) of the aqueous n Pr4NCl solution decreases with increasing GdmHCl concentration. The I3100 cm1 decreases drastically from 0 to 1 M GdmHCl, and becomes constant from 1 to 3 M. Above 3 M, the I3100 cm1 decreases again. These results mean that GdmHCl clearly induces the change of hydrophobic hydration in the aqueous n Pr4 NCl solution. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2009.10.132
-80
-90
0.2
0.4 0.6 Molality
0.8
Fig. 1. Test of the additivity rule on the TH data for the PEG solutions of M = 1000, 2000, and 3000.
References [1] Rasmussen DH, MacKenzie AP. Water Structure at the Water Polymer Interface. New York: Plenum Press; 1972. pp. 126–145.
doi:10.1016/j.cryobiol.2009.10.133
120. Optical spectroscopic studies on the secondary structure of proteins in the freezing state. *Takahiro Takekiyo a,b, Timothy A. Keiderling a,b, Yukihiro Yoshimura a, a Department of Applied Chemistry, National Defence Academy, 1-10-20, Hashirimizu, Yokosuka, Kanagawa 239-8686, Japan, b Department of Chemistry, University of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607, USA Study of the structural stability of proteins at low temperatures can provide the basic information about the freezing preservation of biomolecules. On the other hand,
403
119. Supercooling and vitrification of aqueous polyethyleneglycol solutions. *K. Kajiwara a, Y. Kitada a, K. Tomizawa b, H. Kanno b, a School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakura Hachioji, Tokyo 192-0982, Japan, b Department of Applied Chemistry, National Defense Academy, Yokosuka, Kanagawa 239-8686, Japan Studies of aqueous solutions at low temperatures are important in cryopreservation of living cells and biotechnology, not to say in basic science. As there is a wide range of molecular weights among water soluble polyethyleneglycols (PEGs), we can study supercooling and vitrification of their aqueous solutions as a function of molecular weight in a systematic way. In this study, we first measured the homogeneous ice nucleation temperatures (THs) of aqueous PEG solutions as a function of PEG concentration and then we tried to vitrify the solutions at the solute concentration at which they give TH of 80, 90 or 100 °C. The average molecular weights of PEGs used in this study are M = 200, 300, 400, 600, 1000, 1540, 2000, and 3000. The emulsification method developed by Rasmussen and MacKenzie [1] was used to measure TH of these aqueous solutions. A simple DTA method was used in the TH measurements. On the other hand, the bulk solutions were used for the measurements of the glass transition temperatures (Tgs). The cooling rate was about 1.8 103 °C/min in the quenching process and the heating rate was about 4 °C in the Tg region. Fig. 1 shows the TH results for the aqueous PEG solutions of M = 1000, 2000 and 3000 and the hypothetical TH curves for the M = 3000 solution by increasing the PEG concentration by 1.5 or 3 times without changing the TH value. Fairly well overlapping of the hypothetical TH curve with the TH curve for the M = 1000 or 2000 clearly demonstrates that additivity rule holds for the supercooling of aqueous PEG solutions. It is interesting to note that Tg of the PEG solution shows systematic variation with the molecular weight (M) of PEG even for the PEG solutions showing the same degree of supercooling. (Conflicts of interest: None declared. Source of funding: None declared.)
M = 3000 M = 1000 M = 2000 M = 3000(1.5N) M = 3000(3N)
-40
Reference
doi:10.1016/j.cryobiol.2009.10.131
TH /°C
-50 [1] Ogawa S, Osanai S. Inhibition effect of sugar-based amphiphiles on eutectic formation in the freezing–thawing process of aqueous NaCl solution. Cryobiology 2007;54:173–80.
-60
-70 118. Raman spectroscopic study on the hydrophobic hydration structure in the glassy state. *Yukihiro Yoshimura, Takahiro Takekiyo, Department of Applied Chemistry, National Defense Academy, 1-10-20 Hashirimizu, Yokosuka, Kanagawa, Japan It is well known that guanidine hydrochloride (GdmHCl) is used to elucidate the protein folding mechanism. According to previous solubility studies by Nozaki et al. [J. Biol. Chem. 238 (1963) 4047; 245 (1970) 1648], they assumed that the addition of GdmHCl to an aqueous protein solution weakens the hydrophobic interaction of the hydrophobic amino acids such as Val, Ile, and Leu in proteins. However, the details of the hydration structure around proteins under the coexistence of GdmHCl are still unclear. Here we focus on the additive effect on the hydration structure of hydrophobic compounds in aqueous solution at 77 K. We selected tetrapropylammonium chloride (n Pr4 NCl) as a model hydrophobic compound, because n-Pr4N+ cation is widely accepted as a suitable substance for investigating hydrophobic interactions in aqueous solutions [J. Solution Chem. 2 (1973) 253]. The structure of the n Pr4 N+ cation resembles that of hydrophobic amino acids such as Val, ILe, and Leu. Besides, the aqueous n Pr4NCl solution becomes a glass upon fast cooling to liquid nitrogen temperature [J. Phys. Chem. 93 (1989) 4981]. We have measured Raman OH stretching spectra of aqueous n Pr4NCl solution at R (=moles of water/moles of n Pr4NCl) = 30 as a function of GdmHCl. At this concentration, n Pr4NCl is considered to form a hydrophobic hydration. The glassy Raman OH stretching spectrum of aqueous n Pr4NCl solution at 77 K has peaks at around 3100 and 3450 cm1. The Raman intensity at around 3100 cm1 I3100 cm1) of the aqueous n Pr4NCl solution decreases with increasing GdmHCl concentration. The I3100 cm1 decreases drastically from 0 to 1 M GdmHCl, and becomes constant from 1 to 3 M. Above 3 M, the I3100 cm1 decreases again. These results mean that GdmHCl clearly induces the change of hydrophobic hydration in the aqueous n Pr4 NCl solution. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2009.10.132
-80
-90
0.2
0.4 0.6 Molality
0.8
Fig. 1. Test of the additivity rule on the TH data for the PEG solutions of M = 1000, 2000, and 3000.
References [1] Rasmussen DH, MacKenzie AP. Water Structure at the Water Polymer Interface. New York: Plenum Press; 1972. pp. 126–145.
doi:10.1016/j.cryobiol.2009.10.133
120. Optical spectroscopic studies on the secondary structure of proteins in the freezing state. *Takahiro Takekiyo a,b, Timothy A. Keiderling a,b, Yukihiro Yoshimura a, a Department of Applied Chemistry, National Defence Academy, 1-10-20, Hashirimizu, Yokosuka, Kanagawa 239-8686, Japan, b Department of Chemistry, University of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607, USA Study of the structural stability of proteins at low temperatures can provide the basic information about the freezing preservation of biomolecules. On the other hand,