Cooperative Function of Ammonium Polyacrylate with Antifreeze

Activity of antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) is often determined by thermal hysteresis, which is the difference between...
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Cooperative Function of Ammonium Polyacrylate with Antifreeze Protein Type I Kunio Funakoshi,† Takaaki Inada,*,† Hiroshi Kawabata,‡ and Takashi Tomita‡ National Institute of Advanced Industrial Science and Technology (AIST), 1-2-1 Namiki, Tsukuba, Ibaraki 305-8564, Japan, and Fine and Specialty Chemicals Research Center, Nippon Shokubai Company, Ltd., 5-8 Nishi Otabi-cho, Suita, Osaka 564-8512, Japan Received July 4, 2008; Revised Manuscript Received September 23, 2008

Activity of antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) is often determined by thermal hysteresis, which is the difference between the melting temperature and the nonequilibrium freezing temperature of ice in AF(G)P solutions. In this study, we confirmed that thermal hysteresis of AFP type I is significantly enhanced by a cooperative function of ammonium polyacrylate (NH4PA). Thermal hysteresis of mixtures of AFP type I and NH4PA was much larger than the sum of each thermal hysteresis of AFP type I and NH4PA alone. In mixed solutions of AFP type I and NH4PA in the thermal hysteresis region, hexagonal pyramidal-shaped pits densely formed on ice surfaces close to the basal planes. The experimental results suggest that the cooperative function of NH4PA with AFP type I was caused either by the increase in adsorption sites of AFP type I on ice or by the adsorption of AFP type I aggregates on ice.

Introduction In polar and near-polar regions, seawater temperature is about -1.9 °C and air temperature is much lower in winter than the seawater temperature. Some species of marine fish and terrestrial insects, plants, and bacteria, however, can live in such cold environments because they have either antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs).1-5 AF(G)P molecules are known to be adsorbed on ice surfaces.1-5 By the pinning effect of the adsorbed AF(G)P molecules, an ice surface growing in the AF(G)P solution becomes microscopically curved, and thus, the freezing temperature of the curved ice surface is locally depressed in a noncolligative manner due to the Kelvin (Gibbs-Thomson) effect.2,5 Therefore, the nonequilibrium freezing temperature at which ice crystals start to grow in an AF(G)P solution differs from the melting temperature. This temperature difference is called thermal hysteresis.1-5 Because AF(G)Ps suppress growth and recrystallization of ice crystals at temperatures below the melting temperature, they can potentially be used as cryoprotectants for cells, tissues, and organs6 and as additives to frozen foods.6,7 However, AF(G)Ps are currently expensive7 because they are extracted from living organisms. Therefore, nonpeptide compounds that induce thermal hysteresis in themselves8-10 or that enhance thermal hysteresis of AF(G)Ps11-17 need to be developed. Thermal hysteresis of AF(G)Ps is sometimes enhanced by addition of their isoform proteins or other compounds. This cooperative function among AF(G)Ps themselves or between AF(G)Ps and nonpeptide compounds has been reported for various AF(G)Ps. For example, thermal hysteresis of AFGP1-5 is enhanced by AFGP7 and 8.18,19 Thermal hysteresis of AFGP7 and 8 is also enhanced by several nonpeptide compounds.11 Thermal hysteresis of AFGP8 at concentrations higher than 35 mM unexpectedly increases because of the aggregation of

AFGP8 in water.20 This increase in thermal hysteresis of AFGP8 is also regarded as a cooperative function.20 AFP type III found in notched-fin eelpout has 13 isoforms, and the inactive isoform has a cooperative function with the active isoform.21 Such cooperative function has also been confirmed between the active and the inactive isoforms of insect AFPs.16,22 For insect AFPs, cooperative functions of either their antibodies,12,23 proteins other than AF(G)Ps,12,22 or nonpeptide compounds of low molecular weight13-16 have also been reported. Recent reports have confirmed cooperative functions of many types of salts, such as sodium chloride, with AFGP, AFP type I, and AFP type III.17 In this study, we evaluated ammonium polyacrylate (NH4PA) as a nonpeptide compound that has a cooperative function with AFP type I extracted from the winter flounder, Pleuronectes americanus. Although NH4PA induces thermal hysteresis in itself, its thermal hysteresis is much smaller than that induced by AFP type I; thermal hysteresis of NH4PA is 0.03 °C at 25 mM,9 whereas that of AFP type I is 0.5 °C at 1 mM.24,25 However, due to the difference in their adsorption planes, we expected a cooperative function between AFP type I and NH4PA; AFP type I is adsorbed on the pyramidal planes {202j1} of ice crystals,2-4,26,27 whereas NH4PA is adsorbed on the basal planes {0001}.9 In this study, first, the thermal hysteresis of the mixture of AFP type I and NH4PA was measured. Then the cooperative function between these two compounds was evaluated by comparing their measured thermal hysteresis with the sum of each thermal hysteresis of AFP type I and NH4PA alone. Finally, the role of NH4PA in the cooperative function was also evaluated both from the measured thermal hysteresis and from the morphology of a single crystal of ice grown in mixed solutions of AFP type I and NH4PA.

Experimental Section * To whom correspondence should be addressed. Telephone: +81-29861-7272. Fax: +81-29-851-7523. E-mail: [email protected]. † National Institute of Advanced Industrial Science and Technology (AIST). ‡ Nippon Shokubai Co., Ltd.

Materials. NH4PA was prepared by neutralization of poly(acrylic acid) (PAA) (AQUALIC HL-415, Nippon Shokubai, weight-average molecular weight Mw: 10000) with ammonia aqueous solution (Wako Pure Chemical Industries). Mw of NH4PA was estimated to be about

10.1021/bm800739s CCC: $40.75  2008 American Chemical Society Published on Web 10/11/2008

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Figure 1. Semicylindrical single crystal of ice showing the defined axes and dimensions.

12000 based on that of PAA. AFP type I extracted from the winter flounder, Pleuronectes americanus, was purchased from A/F Protein Canada 2000 Inc. The molecular weight of the winter flounder AFP type I is reportedly about 3300.1 Mixed solutions of AFP type I and NH4PA were prepared with purified deionized water (Milli-Q Jr., Millipore) at an AFP type I (CAFP) concentration controlled within 0 and 0.34 mM and NH4PA (CNHPA) within 0 and 25.0 mM. Preparation of Single Crystal. A single crystal of ice was made from purified deionized water (Milli-Q Jr., Millipore) in a plastic beaker by keeping the water temperature at -0.2 °C in a temperature-controlled room. After spontaneous nucleation of an ice crystal at the water surface, the ice crystal was grown slowly until it was about 7 mm thick. In the single crystal of ice grown using this method, the c-axis was normal to the water surface.28,29 The a-axis was determined by forming negative crystals of hexagonal prismatic morphology in the ice crystal.30 An infrared light was used to grow these negative crystals. Thermal Hysteresis. Details of the apparatus used in this study are described elsewhere.9 Here, 30 mL of a mixed solution of AFP type I and NH4PA was cooled in a jacketed cell (acrylic plastic) by a coolant (FX-3300, 3M) supplied from a thermostat bath (RC 20 CS, Lauda). The solution temperature Ts was measured by a platinum resistance thermometer (Fast Response RTDs 5622-05, CHUB-E4, Hart Scientific). A single crystal of ice was cut into an approximately semicylindrical shape, with the c-axis approximately parallel to the longitudinal axis. The crystal was then mounted on the tip of a stainless steel wire as shown in Figure 1 and finally immersed in the mixed solution. The growth and melting rates of the ice crystal over a 2 h period at different Ts were measured by monitoring the change in crystal size. The size and morphology of the ice crystal were observed by a CCD camera (XC-77R-CE, Sony), recorded using a hard disk drive recorder (VDH8000, Sanyo Electrics), and then analyzed by using image processing software (Image Pro. Plus, Media Cybernetics). The sizes of the single crystal in the a- and c-axes directions were defined by la and lc, respectively, as shown in Figure 1. la and lc in the initial state were between 3 and 5 mm and between 3 and 6 mm, respectively. At first, the ice crystal was immersed in the solution at Ts just above the melting temperature Tm for more than 15 min, so that the ice debris on the ice surface generated during the cutting process would melt. Then Ts was decreased and kept at Tm - 0.1 °C, unless otherwise specified. At this temperature, the ice crystal grew for a while, but finally, crystal growth completely stopped in both the a- and c-axes directions. After the crystal growth stopped, Ts was changed around Tm, and then the growth and melting rates of the ice crystal were measured at different values of Ts.

Results Thermal Hysteresis. Figure 2 shows the change in the growth rate of a single crystal of ice in the c-axis direction in a mixed solution of AFP type I (CAFP ) 0.06 mM) and NH4PA (CNHPA ) 6.7 mM) with Ts. Negative values of the growth rate represent melting. The melting rate was relatively proportional to Ts. For this solution, Tm was -0.52 °C, which was determined

Figure 2. Growth rate of a single crystal of ice along the c-axis in a mixed solution of AFP type I (CAFP ) 0.06 mM) and NH4PA (CNHPA ) 6.7 mM) as a function of solution temperature Ts. Data below the nonequilibrium freezing temperature Tf were difficult to determine due to explosive growth of the crystal.

Figure 3. Thermal hysteresis ∆T of mixtures of AFP type I and NH4PA (CNHPA ) 16.7 mM) as a function of AFP concentration CAFP. Open circles represent measured ∆T and broken line represents expected ∆T, which is defined as the sum of each ∆T of AFP type I and NH4PA alone. Solid circle represents measured ∆T of AFP type I alone, and thin solid line represents ∆T extrapolated from reference data.24,25

by the intersection of the regression line of the melting rates and the x-axis. The nonequilibrium freezing temperature Tf was -0.84 °C, which was determined as the temperature at which the ice crystal started to grow explosively from an arbitrary point. Therefore, thermal hysteresis ∆T of the mixture of AFP type I (CAFP ) 0.06 mM) and NH4PA (CNHPA ) 6.7 mM) was 0.32 °C, which is defined as ∆T ) Tm - Tf. Because explosive growth of the ice crystal in the mixed solutions at Tf was always along the c-axis, ∆T in the a-axis direction would be larger than that in the c-axis direction, although ∆T cannot be determined in the a-axis direction due to the explosive growth of the ice crystal in the c-axis direction. Figure 3 shows the measured ∆T of the mixture of AFP type I and NH4PA as a function of CAFP, when CNHPA ) 16.7 mM. The thick solid line is a guide to the eye. ∆T of the mixture increased with increasing CAFP. The measured ∆T of AFP type I alone at 0.06 mM agrees well with ∆T of AFP type I alone extrapolated from reference data obtained (thin solid line) at higher CAFP.24,25 Because ∆T of NH4PA alone at CNHPA ) 16.7 mM was 0.01 °C,9 the expected value of ∆T of the mixture of AFP type I and NH4PA should be the sum of each ∆T of AFP type I and NH4PA alone (broken line). From these results, the measured ∆T of the mixture of AFP type I and NH4PA was

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Figure 4. Thermal hysteresis ∆T of mixtures of AFP type I (CAFP ) 0.06 mM) and NH4PA as a function of NH4PA concentration CNHPA. Open circles represent measured ∆T and broken line represents expected ∆T, which is defined as the sum of each ∆T of AFP type I and NH4PA alone. Solid circles represent measured ∆T of NH4PA alone obtained from reference data.9

much larger than the expected ∆T. This ∆T enhancement clearly indicates the cooperative function between AFP type I and NH4PA. Ice crystals of several mm in size were used in this study to measure ∆T, whereas much smaller crystals are generally used to measure ∆T of AF(G)Ps. Although the effect of ice crystal size on ∆T has been pointed out for some fish and insect AFPs,31-33 ∆T of AFP type I measured in our study was almost the same as those reported by others who used an ice crystal of several tens µm,24,25 as shown in Figure 3. Figure 4 shows the measured ∆T of the mixture of AFP type I and NH4PA as a function of CNHPA, when CAFP ) 0.06 mM. Here, ∆T of the mixture increased linearly with increasing CNHPA. Previously obtained values of ∆T of NH4PA9 are also shown in Figure 4. Because ∆T of AFP type I alone at 0.06 mM was 0.09 °C (Figure 3), the expected ∆T of the mixture of AFP type I and NH4PA (broken line in Figure 4) is the sum of each ∆T of AFP type I and NH4PA alone. The measured ∆T of the mixture of AFP type I and NH4PA was much larger than the expected ∆T, indicating the cooperative function between AFP type I and NH4PA. Morphology of Ice Crystal. Figure 5 shows observed changes in morphology of a single crystal of ice in a mixed solution of AFP type I (CAFP ) 0.06 mM) and NH4PA (CNHPA ) 16.7 mM). The initial ice crystal was surrounded by two flat surfaces close to the basal planes {0001}, a flat surface close to the primary prism planes {101j0}, and a rounded surface approximately parallel to the c-axis, as shown in Figure 1. Here, we call the first two flat surfaces the basal planes, and call the second flat surface the primary prism plane, although these are not accurate definitions. When a single crystal of ice was immersed in the solution at Tm - 0.1 °C, the ice crystal grew in both the a- and c-axes directions for a while. During this growth, small pits densely formed on the basal planes (not visible in Figure 5a), whereas the primary prism plane and the rounded surface did not change significantly. When the basal planes were fully covered with pits, the ice crystal completely stopped growing in both the a- and c-axes directions (Figure 5a). Therefore, ∆T of the mixture of AFP type I (CAFP ) 0.06 mM) and NH4PA (CNHPA ) 16.7 mM) was larger than 0.1 °C. When Ts was reduced to Tm - 0.67 °C, the ice crystal started to grow explosively from arbitrary points on the basal planes as a needle-like shape along the c-axis (Figure 5b). Therefore, ∆T of the mixture of AFP type I (CAFP ) 0.06 mM) and NH4PA (CNHPA ) 16.7 mM) was 0.67 °C

Figure 5. Morphology change in a single crystal of ice in a mixed solution of AFP type I (CAFP ) 0.06 mM) and NH4PA (CNHPA ) 16.7 mM). (a) Single crystal of ice at solution temperature Ts of 0.1 °C lower than the melting temperature Tm. The crystal had stopped growing. (b) Same crystal below the nonequilibrium freezing temperature Tf. The crystal started to grow explosively from arbitrary points as a needle-like shape along the c-axis.

Figure 6 shows the morphology changes in the basal plane of a single crystal of ice immersed in mixed solutions of AFP type I (CAFP ) 0.06 mM) and NH4PA (CNHPA ) 6.7 mM and 16.7 mM) at various immersion times at Tm - 0.1 °C. Figures 6a-c show the changes at the lower CNHPA (6.7 mM) at 0, 60, and 180 min after immersion, respectively, and Figures 6d-f show the changes at the higher CNHPA (16.7 mM) at 0, 25, and 60 min after immersion, respectively. For both mixtures, the initial basal planes were relatively flat (0 min, Figures 6a and d). Then, pits gradually formed on the basal planes (Figures 6b and e). When the basal planes were fully covered with pits (Figures 6c and f), the ice crystal completely stopped growing in both the a- and c-axes directions. At the higher CNHPA, the formation rate of the pits (i.e., number of pits formed per minute) was higher and the final size of the pits was smaller. The pits on the basal plane at the lower CNHPA (Figures 6b and c) were hexagonal pyramidal, although the shape of the pits at the higher CNHPA (Figures 6e and f) could not be resolved due to the resolution limit of the microscopy. The hexagonal pit edges were oriented along , rotated by 30° with respect to the a-axis. The pyramidal planes constituting the pits were {112jx}, where the last index x could not be determined because the depth of the pits could not be resolved (Figures 6b and c). Here we call these pyramidal planes the secondary pyramidal planes because their edges lie on the secondary prism planes {112j0}. Figure 7 shows the morphology change in the basal plane of a single crystal of ice immersed in a solution of AFP type I alone (CAFP ) 0.06 mM) at Tm - 0.04 °C. The basal plane was relatively flat just after being immersed in the solution (0 min, Figure 7a). Then, pits gradually formed on the basal plane (Figure 7b). When the basal plane was fully covered with pits (Figure 7c), the ice crystal completely stopped growing in both the a- and c-axes directions. The final size of the pits in the absence of NH4PA (Figure 7c) was larger than that of pits in the presence of NH4PA (Figures 6c and f). The pits (Figures

Cooperative Function with Antifreeze Protein Type I

Figure 6. Morphology changes in the basal plane of a single crystal of ice in mixed solutions of AFP type I and NH4PA at solution temperature Ts of 0.1 °C lower than the melting temperature Tm. (a-c) AFP type I concentration CAFP ) 0.06 mM and NH4PA concentration CNHPA ) 6.7 mM: (a) the basal plane immediately after immersion (0 min), (b) after 60 min, and (c) after 180 min. (d-f) CAFP ) 0.06 mM and CNHPA ) 16.7 mM: (d) the basal plane immediately after immersion (0 min), (e) after 25 min, and (f) after 60 min.

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Figure 7. Morphology change in the basal plane of a single crystal of ice in a solution of AFP type I (0.06 mM) at solution temperature Ts of 0.04 °C lower than the melting temperature Tm: (a) the basal plane immediately after immersion (0 min), (b) after 330 min, and (c) after 420 min.

7b and c) had hexagonal pyramidal shape, and the pyramidal planes constituting the pits were the secondary pyramidal planes {112jx}. Figure 8 shows the relationship between thermal hysteresis ∆T of mixtures of AFP type I (CAFP between 0 and 0.34 mM) and NH4PA (CNHPA between 0 and 25.0 mM) and the average size of pits formed on the basal planes of a single crystal of ice. The size of each pit was measured along the a1-axis in Figures 6 and 7. Independent of the solution composition, the trend was the smaller the average size of the pits, the larger ∆T.

Discussion The cooperative function between AFP type I and NH4PA was confirmed (Figures 3 and 4); the measured ∆T of a mixture of AFP type I and NH4PA was much larger than the expected ∆T (defined as the sum of each ∆T of AFP type I and NH4PA alone). The maximal ∆T of 1.0 °C was achieved when CAFP ) 0.06 mM and CNHPA ) 25.0 mM in the mixed solution (Figure 4). If additional AFP type I was added to this mixed solution, then AFP type I was separated from the mixed solution probably due to the solubility limit. If additional NH4PA was added to this mixed solution, it was difficult to determine ∆T because

Figure 8. Relationship between thermal hysteresis ∆T of mixtures of AFP type I and NH4PA and average size of pits on the basal planes of a single crystal of ice.

the growth rate of an ice crystal before stopping became too slow to form pits on the basal plane due to the slower mass transfer rate of water molecules in the mixed solution.9 At this maximum ∆T, the cooperative function yielded a ∆T enhancement ratio (the ratio of the measured ∆T to the expected ∆T)

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of about 8. This ∆T enhancement ratio is larger than that previously reported for AFP type I, which is estimated to be at most 2.17 For practical applications of AF(G)Ps, the cooperative function can reduce the amount of AF(G)Ps required for a given ∆T. For example, only 0.06 mM of AFP type I is needed for ∆T of 1.0 °C by addition of 25.0 mM of NH4PA (Figure 4), whereas 3.7 mM of AFP type I is required for the same ∆T without addition of NH4PA.24,25 In mixed solutions of AFP type I and NH4PA, at temperatures in the thermal hysteresis region (Tf < Ts < Tm), hexagonal pyramidal-shaped pits consisting of the secondary pyramidal planes {112jx} densely formed on the basal planes of a single crystal of ice, whereas the basal planes that had originally existed in the initial shape of the ice crystal disappeared concurrently during the pit formation (Figure 6). Similar hexagonal pyramidal pits were observed on the basal planes of a single crystal of ice also in a solution of AFP type I alone when Tf < Ts < Tm (Figure 7). In contrast, in solutions of NH4PA alone, the basal planes with no detectable pits appear when Tf < Ts < Tm.9 Therefore, the hexagonal pyramidal pits that formed in the mixed solutions of AFP type I and NH4PA in the current study were caused not by NH4PA, but by AFP type I. Similar hexagonal pyramidal pits consisting of the secondary pyramidal planes have been observed on the basal planes of a single crystal of ice also in solutions of various types of fish AF(G)Ps,34 indicating that various fish AF(G)Ps affect the secondary pyramidal planes of ice. The size of the pits in mixed solutions of AFP type I and NH4PA (Figure 6) was smaller than that in a solution of AFP type I alone (Figure 7) and also smaller than that previously reported for other fish AF(G)P solutions,34 although the shape of the pits was similar for all these solutions. Therefore, NH4PA in a mixed solution affects the size and number of pits and, thus, affects the cooperative function (Figure 8). The pits that form on the basal planes of ice consisted of the secondary pyramidal planes {112jx} in a solution of fish AF(G)Ps34 or in a mixed solution of AFP type I and NH4PA (Figures 6b and c), whereas the external shape of a single crystal of ice in a fish AF(G)P solution generally consists of {101jx} planes,2-4,26,27 which we call here the primary pyramidal planes. Simultaneous existence of such two different crystallographic planes of ice rotated by 30° with respect to each other has been reported.35,36 At a pressure as high as 2000 bar, a single crystal of ice in water without any additives exhibits the primary prism planes {101j0} during growth, whereas it exhibits the secondary prism planes {112j0} during melting.35 This asymmetry of the crystal shape can be explained from the growth and melting kinetics of ice near the roughening transition temperature of the prism plane,35,36 which is similar to the melting temperature of ice at 2000 bar.37 Generally, observing such asymmetry in water at ambient pressure is difficult because the primary prism planes become molecularly rough near the melting temperature at ambient pressure and thus disappear. Nevertheless, the same asymmetry of crystal shape can be observed in a solution of spruce budworm AFP (sbwAFP) even at ambient pressure, because sbwAFP stabilizes the primary prism planes against roughening transition.36 The secondary pyramidal planes of pits that formed in mixed solutions of AFP type I and NH4PA (Figures 6 and 7) or in solutions of fish AF(G)Ps34 are essentially different from the above-mentioned secondary prism planes observed during melting,35,36 because the secondary pyramidal planes of pits are

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Figure 9. Various adsorption models (b-e) of cooperative function between AFP type I and NH4PA, compared with normal adsorption model (a) of AFP type I in the absence of NH4PA. (b) Complexes of AFP type I and NH4PA are formed in solution and then adsorbed on ice surface. Number of adsorption site is not affected by NH4PA. (c) AFP type I molecules are adsorbed on ice surface. Number of adsorption site is increased by the existence of NH4PA. (d) Aggregates of AFP type I molecules are formed in solution and then adsorbed on ice surface. Number of adsorption site is not affected by NH4PA. (e) AFP type I and NH4PA molecules are adsorbed separately on different ice surfaces.

formed not by the melting but by the growth of the basal planes of ice, although the formation mechanism of these pits is still unknown.36 Enhancement of ∆T by the cooperative function has been explained by a few models as follows. In the first model (Figure 9b), an AF(G)P molecule binds to another molecule such as NH4PA and, thus, forms a complex that covers a larger area of ice by being adsorbed on a specific ice surface, compared with the adsorption area when only AF(G)P molecules are present (Figure 9a).12,15,18,22,23 Consequently, the area of the bare ice surface with no adsorbed molecules is reduced by the complex adsorption, even if the number density of the adsorption sites on the ice surface is the same. Thus, from the Kelvin (Gibbs-Thomson) effect, ∆T will increase with decreasing area

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of the bare ice surface.2,5,38 This complex formation model suggests that ∆T increases as the number of the complex that can be adsorbed on ice increases. Therefore, the linear relationship between ∆T and CNHPA in Figure 4 suggests that the number of the complex continuously increased as CNHPA increased up to 25 mM. However, the complex formation must have been saturated, and thus, ∆T must have reached the upper limit because CNHPA was more than two orders in magnitude higher than CAFP on the molar basis, whereas the molecular weight of NH4PA was in the same order as that of AFP type I. This contradiction indicates that the complex formation model can be excluded in this study. In the second model (Figure 9c), the number of adsorption sites of AF(G)P molecules on the ice surface is increased by the addition of other molecules such as NH4PA, and the distance between the adsorbed AF(G)P molecules is reduced,11 although it is not clear what kind of interaction affects the number of adsorption sites. Also, in this case, from the Kelvin (GibbsThomson) effect, ∆T will increase with decreasing distance of the adsorbed molecules. In the third model (Figure 9d), the addition of other molecules such as NH4PA to AF(G)P solutions causes salting-in or saltingout, which affects the solubility of AF(G)P in water.17 In this study, salting-out was likely to occur, because the solubility of AFP type I decreased by the addition of NH4PA. If AFP type I forms aggregates in the solution by salting-out, these aggregates cover a larger area of ice compared with the adsorption area covered by a single molecule of AFP type I.20 This increase in the adsorption area will increase ∆T in the same manner as the above-mentioned complex formation model. In addition to these models, another model can be considered for the cooperative function between AFP type I and NH4PA (Figure 9e), in which AFP type I and NH4PA molecules separately affect ice surfaces in a mixed solution of AFP type I and NH4PA, because they are adsorbed on different crystallographic ice planes in each solution; AFP type I is adsorbed on the pyramidal planes {202j1},2-4,26,27 whereas NH4PA is adsorbed on the basal planes {0001}.9 In Figure 6, however, the basal planes disappeared when the crystal growth of ice stopped in the thermal hysteresis region in the mixed solutions of AFP type I and NH4PA. The disappearance of the basal planes indicates that NH4PA in itself does not directly affect ice surfaces in the mixed solutions but rather plays an auxiliary role in the cooperative function, as it does in the abovementioned three models. Consequently, the second and third models (Figures 9c and d) seem to adequately explain the cooperative function between AFP type I and NH4PA, although which of the two is more appropriate is unknown. NH4PA is known to be partly dissociated in water into PAAand NH4+ ions.39 However, in our study here, PAA or NH3 either had no cooperative function with AFP type I; ∆T of AFP type I was not enhanced by adding PAA or NH3. Therefore, the role of NH4PA in the cooperative function would not be caused by PAA- and NH4+ ions alone, but rather PAA- and NH4+ ions might together affect the adsorption of AFP type I on ice.

Conclusion A cooperative function of ammonium polyacrylate (NH4PA) with antifreeze protein (AFP) type I was confirmed. Thermal hysteresis of mixtures of AFP type I and NH4PA was much larger than the sum of each thermal hysteresis of AFP type I

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and NH4PA alone. The enhancement ratio of thermal hysteresis by the cooperative function was about 8 when the thermal hysteresis had a maximum. When a single crystal of ice in mixed solutions of AFP type I and NH4PA stopped growing in the thermal hysteresis region, a high concentration of hexagonal pyramidal-shaped pits were observed on ice surfaces close to the basal planes. The pits consisted of the secondary pyramidal planes {112jx}, although the last index x could not be determined. Thermal hysteresis of the mixtures of AFP type I and NH4PA had a relationship with the size of the pits, irrespective of the composition of the mixed solutions. Four models of the cooperative function of NH4PA with AFP type I were discussed, and then we concluded, based on the experimental results, that the cooperative function was caused either by the increase in adsorption sites of AFP type I on ice or by the adsorption of AFP type I aggregates on ice The cooperative function of NH4PA with AFP type I confirmed in this study would be useful in practical applications of AFP type I, because the amount of AFP type I required for a given thermal hysteresis can be significantly reduced. Acknowledgment. This study was supported by the Industrial Technology Research Grant Program from New Energy and Industrial Technology Development Organization (NEDO) of Japan.

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