- Email: [email protected]
141 Inhibition of ice recrystallization by antifreeze proteins
438
Abstracts / Cryobiology 67 (2013) 398–442
cally relevant cells, will accelerate the progression of cellular therapies from bench to bedside. Source of funding: Loughborough University Graduate School Studentship. Conflict of interest: None declared. Email address: [email protected]
http://dx.doi.org/10.1016/j.cryobiol.2013.09.145
140 The importance of hydrophobic moieties in ice recrystallization inhibitors. Anna K. Balcerzak, Kyle McClymont, Robert N. Ben, Department of Chemistry, University of Ottawa, Ottawa, ON, Canada Ice recrystallization during thawing after cryopreservation results in extensive cellular damage that ultimately leads to cell death and decreased cell viabilities. This is a significant problem particularly with cryopreserved cells utilized in various regenerative medicine therapies. Given the success of these therapies to treat spinal cord injury, cartilage lesions, and cardiac disease, the development of new and improved cryprotectants that minimize cell damage during freeze-thawing and improve cell viability post-cryopreservation are urgently required. Our laboratory has demonstrated that carbohydrate-based hydrogelators can be potent inhibitors of ice recrystallization. While our studies have indicated that a delicate balance between hydrophobic and hydrophilic interactions is crucial for ice recrystallization inhibition (IRI) activity, the essential structural features necessary for potent IRI activity remain unknown. To address this issue, we synthesized and assessed the IRI activity of structurally diverse amino acid-based surfactants/gelators and antiice nucleating using a splat-cooling assay. The results indicate that long alkyl chains and increased hydrophobicity are important for potent IRI activity and that the position of these alkyl chains is essential. Additionally, no correlation was found between IRI activity and critical micelle concentrations, gelation or anti-ice nucleation activity although the counterion of some surfactants did affect IRI activity. The results of our study will facilitate the design of improved ice recrystallization inhibitors for medical, commercial and industrial applications. Source of funding: None declared. Conflict of interest: None declared. Email address: [email protected]
http://dx.doi.org/10.1016/j.cryobiol.2013.09.146
141 Inhibition of ice recrystallization by antifreeze proteins. Shiran Zalis, Maya Bar Dolev, Ido Braslavsky, Institute of Biochemistry, Food Science, and Nutrition Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, Rehovot, Israel Ice recrystallization (IR) is a process in which large ice crystals grow at the expense of smaller ones. It occurs constantly in nature and in industrial processes in conditions of moderate cooling of a partially frozen environment or at accelerated rate due to temperature fluctuations of frozen substances. IR is one of the major causes of smooth texture loss and deterioration of frozen food such as ice cream during storage. In cryopreservation, recrystallization during thawing, damages the cells and tissues due to membrane rapture and cell dehydration, reducing the survival rates. Organisms inhabiting cold environments and prone to IR injuries, such as freeze-tolerating plants and freeze-avoiding organisms like fish and insects have developed antifreeze proteins (AFPs), a subset of the Ice Binding Proteins group which interact with ice for various biological purposes. The AFPs adsorb to ice surfaces and restrict the growth of ice to the areas between bound protein molecules. Such proteins were shown to block IR effectively [Knight and Duman, 1986], and may serve as additives in many applications in which ice recrystallization is an obstacle. Although ice recrystallization inhibition (IRI) activity of a variety of AFPs was studied [Knight et al., 1995], the mechanism by which AFPs inhibit IR is unknown. Particularly, it has been shown that although insect AFPs have considerably higher freezing point depression activity relative to AFPs from fish and plants, their IRI activity is in the same range or lower [Yu et al., 2010]. We intend to study the mechanism of IRI in order to explain the inconsistency between this activity and the freezing point depression activity of insect AFPs. In our study the IRI activity of AFPs from mealworm Tenebriomolitor (TmAFP) was investigated. Sucrose solutions containing various concentration of TmAFP were cooled to 50 °C, then the temperature was elevated and sustained constant for annealing. During annealing, images of recrystallization were acquired using a light microscope equipped with fluorescence illumination and cold-stage. Initial results show an accumulation of TmAFP onto ice crystals and IRI in a range of AFPs concentrations. Our next goals are to develop test methods that will provide reliable, quantitative information on recrystallization and on the accumulation of the protein on the crystals, as well as deple-
tion of the protein from the solution. Such methods will enable quantification of the ice recrystallization phenomena and subsequently quantification of the differences between the IRI activities of various types of AFPs. Understanding the mechanism of ice recrystallization and the effects of AFPs on it may improve and expand the use of AFPs in many applications in the medical sector, in cryopreservation, and in the frozen food industry. Source of funding: European Research Council (ERC). Conflict of interest: None declared. Email address: [email protected] http://dx.doi.org/10.1016/j.cryobiol.2013.09.147
142 Ice growth in the presence of an antifreeze protein. Maddalena Bayer-Giraldi, Ilka Weikusat, Cornelia Isert, Sepp Kipfstuhl. Alfred-Wegener Institute for Polar and Marine Research, Bremerhaven, Germany Antifreeze proteins (AFPs) have evolved in cold-adapted organisms to control ice crystal growth when exposed to sub-zero temperature conditions. It has been suggested that the effect of the proteins results in small ice crystal size, thus avoiding the mechanical damage to frozen tissues and cells caused by large ice grains. The polar diatom Fragilariopsis cylindrus, a dominant species within sea-ice assemblages, produces AFPs. We expressed in E. coli a recombinant form of this protein and isolated it by affinity chromatography. We studied its effect on ice grain size after shock-freezing and subsequent annealing, and under slow freezing conditions. Shock-freezing ( 40 °C) produced small sized crystals, and during annealing at 4 °C AFPs successfully inhibited recrystallization already at low concentrations (1.2 lM), as observed at light microscopy and using the Otago optical recrystallometer. However, slow ice growth at 5 °C, more likely to resemble natural freezing conditions, surprisingly resulted in the formation of larger crystals in the presence of AFPs than in the negative controls. Further characteristic microstructural features, such as gradual c-axis transition within individual grains and sublimation etching patterns, were observed under crossed polarizers and at light microscopy. These features are possibly due to the incorporation of proteins into the ice lattice during growth, causing local defects. Our observations remain to be clarified, but should be taken into account when considering the biological role of AFPs as well as potential industrial applications of the proteins. Source of funding: Public funding by German AiF/IGF. Conflict of interest: None declared. Email address: [email protected] http://dx.doi.org/10.1016/j.cryobiol.2013.09.148
143 Ice shaping in solutions of ice-binding proteins – Melting vs growing morphologies. Maya Bar Dolev a, J.J. Liu b, Yangzong Qin b, Yeliz Celik b, Ran Drori a, John Wettlaufer c, Peter L. Davies d, Ido Braslavsky a,b, a Faculty of Agriculture, Food & Environmental Sciences, The Hebrew University of Jerusalem, Rehovot, Israel, b Department of Physics and Astronomy, Ohio University, Athens, OH, USA, c Department of Physics, and Department of Geology and Geophysics, Yale University, New Haven, CT, USA, d Department of Biochemistry, Queen’s University, Kingston, ON, Canada A fundamental strategy evolved by organisms in cold habitats, obliged to cope with ice and freeze injuries, is ice-binding proteins (IBP). These proteins directly interact with ice surfaces and serve to depress the freezing point of the body fluids, to inhibit ice recrystallization, or to promote ice nucleation. The delicate control over ice growth make these proteins applicable in any field that requires control over ice growth, namely, in cryopreservation of cells, tissues and organs, in cryosurgery and in agriculture, in the frozen food industry and in complex material engineering. A variety of IBPs have been studied in the past decade, differing in sequences, structures, and specific activities. Most IBPs are grossly classified to moderate and hyperactive according to their thermal hysteresis activity, which is the gap between the equilibrium melting point and the freezing point of an ice crystal grown in IBP solution. We have studied the ability of IBPs to shape ice crystals in distinguishable forms characteristic of the particular protein type. IBPs with moderate thermal hysteresis activities induce elongated bipyramidal crystals – often with well-defined facets – while hyperactive IBPs produce more varied crystal shapes, such as the ‘‘lemon-like” crystals typically observed with Tenebrio molitor IBP. These unique morphologies are frequently considered to be growth shapes. We conducted a systematic study of ice shaping in solutions containing a wide range of IBPs. We found that although ice crystals in solutions of moderate IBPs do indeed grow into their faceted shapes, in the presence of most hyperactive IBPs, ice melts into its final shape. These melting shapes result from the affinity of the hyperactive
Abstracts / Cryobiology 67 (2013) 398–442
cally relevant cells, will accelerate the progression of cellular therapies from bench to bedside. Source of funding: Loughborough University Graduate School Studentship. Conflict of interest: None declared. Email address: [email protected]
http://dx.doi.org/10.1016/j.cryobiol.2013.09.145
140 The importance of hydrophobic moieties in ice recrystallization inhibitors. Anna K. Balcerzak, Kyle McClymont, Robert N. Ben, Department of Chemistry, University of Ottawa, Ottawa, ON, Canada Ice recrystallization during thawing after cryopreservation results in extensive cellular damage that ultimately leads to cell death and decreased cell viabilities. This is a significant problem particularly with cryopreserved cells utilized in various regenerative medicine therapies. Given the success of these therapies to treat spinal cord injury, cartilage lesions, and cardiac disease, the development of new and improved cryprotectants that minimize cell damage during freeze-thawing and improve cell viability post-cryopreservation are urgently required. Our laboratory has demonstrated that carbohydrate-based hydrogelators can be potent inhibitors of ice recrystallization. While our studies have indicated that a delicate balance between hydrophobic and hydrophilic interactions is crucial for ice recrystallization inhibition (IRI) activity, the essential structural features necessary for potent IRI activity remain unknown. To address this issue, we synthesized and assessed the IRI activity of structurally diverse amino acid-based surfactants/gelators and antiice nucleating using a splat-cooling assay. The results indicate that long alkyl chains and increased hydrophobicity are important for potent IRI activity and that the position of these alkyl chains is essential. Additionally, no correlation was found between IRI activity and critical micelle concentrations, gelation or anti-ice nucleation activity although the counterion of some surfactants did affect IRI activity. The results of our study will facilitate the design of improved ice recrystallization inhibitors for medical, commercial and industrial applications. Source of funding: None declared. Conflict of interest: None declared. Email address: [email protected]
http://dx.doi.org/10.1016/j.cryobiol.2013.09.146
141 Inhibition of ice recrystallization by antifreeze proteins. Shiran Zalis, Maya Bar Dolev, Ido Braslavsky, Institute of Biochemistry, Food Science, and Nutrition Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, Rehovot, Israel Ice recrystallization (IR) is a process in which large ice crystals grow at the expense of smaller ones. It occurs constantly in nature and in industrial processes in conditions of moderate cooling of a partially frozen environment or at accelerated rate due to temperature fluctuations of frozen substances. IR is one of the major causes of smooth texture loss and deterioration of frozen food such as ice cream during storage. In cryopreservation, recrystallization during thawing, damages the cells and tissues due to membrane rapture and cell dehydration, reducing the survival rates. Organisms inhabiting cold environments and prone to IR injuries, such as freeze-tolerating plants and freeze-avoiding organisms like fish and insects have developed antifreeze proteins (AFPs), a subset of the Ice Binding Proteins group which interact with ice for various biological purposes. The AFPs adsorb to ice surfaces and restrict the growth of ice to the areas between bound protein molecules. Such proteins were shown to block IR effectively [Knight and Duman, 1986], and may serve as additives in many applications in which ice recrystallization is an obstacle. Although ice recrystallization inhibition (IRI) activity of a variety of AFPs was studied [Knight et al., 1995], the mechanism by which AFPs inhibit IR is unknown. Particularly, it has been shown that although insect AFPs have considerably higher freezing point depression activity relative to AFPs from fish and plants, their IRI activity is in the same range or lower [Yu et al., 2010]. We intend to study the mechanism of IRI in order to explain the inconsistency between this activity and the freezing point depression activity of insect AFPs. In our study the IRI activity of AFPs from mealworm Tenebriomolitor (TmAFP) was investigated. Sucrose solutions containing various concentration of TmAFP were cooled to 50 °C, then the temperature was elevated and sustained constant for annealing. During annealing, images of recrystallization were acquired using a light microscope equipped with fluorescence illumination and cold-stage. Initial results show an accumulation of TmAFP onto ice crystals and IRI in a range of AFPs concentrations. Our next goals are to develop test methods that will provide reliable, quantitative information on recrystallization and on the accumulation of the protein on the crystals, as well as deple-
tion of the protein from the solution. Such methods will enable quantification of the ice recrystallization phenomena and subsequently quantification of the differences between the IRI activities of various types of AFPs. Understanding the mechanism of ice recrystallization and the effects of AFPs on it may improve and expand the use of AFPs in many applications in the medical sector, in cryopreservation, and in the frozen food industry. Source of funding: European Research Council (ERC). Conflict of interest: None declared. Email address: [email protected] http://dx.doi.org/10.1016/j.cryobiol.2013.09.147
142 Ice growth in the presence of an antifreeze protein. Maddalena Bayer-Giraldi, Ilka Weikusat, Cornelia Isert, Sepp Kipfstuhl. Alfred-Wegener Institute for Polar and Marine Research, Bremerhaven, Germany Antifreeze proteins (AFPs) have evolved in cold-adapted organisms to control ice crystal growth when exposed to sub-zero temperature conditions. It has been suggested that the effect of the proteins results in small ice crystal size, thus avoiding the mechanical damage to frozen tissues and cells caused by large ice grains. The polar diatom Fragilariopsis cylindrus, a dominant species within sea-ice assemblages, produces AFPs. We expressed in E. coli a recombinant form of this protein and isolated it by affinity chromatography. We studied its effect on ice grain size after shock-freezing and subsequent annealing, and under slow freezing conditions. Shock-freezing ( 40 °C) produced small sized crystals, and during annealing at 4 °C AFPs successfully inhibited recrystallization already at low concentrations (1.2 lM), as observed at light microscopy and using the Otago optical recrystallometer. However, slow ice growth at 5 °C, more likely to resemble natural freezing conditions, surprisingly resulted in the formation of larger crystals in the presence of AFPs than in the negative controls. Further characteristic microstructural features, such as gradual c-axis transition within individual grains and sublimation etching patterns, were observed under crossed polarizers and at light microscopy. These features are possibly due to the incorporation of proteins into the ice lattice during growth, causing local defects. Our observations remain to be clarified, but should be taken into account when considering the biological role of AFPs as well as potential industrial applications of the proteins. Source of funding: Public funding by German AiF/IGF. Conflict of interest: None declared. Email address: [email protected] http://dx.doi.org/10.1016/j.cryobiol.2013.09.148
143 Ice shaping in solutions of ice-binding proteins – Melting vs growing morphologies. Maya Bar Dolev a, J.J. Liu b, Yangzong Qin b, Yeliz Celik b, Ran Drori a, John Wettlaufer c, Peter L. Davies d, Ido Braslavsky a,b, a Faculty of Agriculture, Food & Environmental Sciences, The Hebrew University of Jerusalem, Rehovot, Israel, b Department of Physics and Astronomy, Ohio University, Athens, OH, USA, c Department of Physics, and Department of Geology and Geophysics, Yale University, New Haven, CT, USA, d Department of Biochemistry, Queen’s University, Kingston, ON, Canada A fundamental strategy evolved by organisms in cold habitats, obliged to cope with ice and freeze injuries, is ice-binding proteins (IBP). These proteins directly interact with ice surfaces and serve to depress the freezing point of the body fluids, to inhibit ice recrystallization, or to promote ice nucleation. The delicate control over ice growth make these proteins applicable in any field that requires control over ice growth, namely, in cryopreservation of cells, tissues and organs, in cryosurgery and in agriculture, in the frozen food industry and in complex material engineering. A variety of IBPs have been studied in the past decade, differing in sequences, structures, and specific activities. Most IBPs are grossly classified to moderate and hyperactive according to their thermal hysteresis activity, which is the gap between the equilibrium melting point and the freezing point of an ice crystal grown in IBP solution. We have studied the ability of IBPs to shape ice crystals in distinguishable forms characteristic of the particular protein type. IBPs with moderate thermal hysteresis activities induce elongated bipyramidal crystals – often with well-defined facets – while hyperactive IBPs produce more varied crystal shapes, such as the ‘‘lemon-like” crystals typically observed with Tenebrio molitor IBP. These unique morphologies are frequently considered to be growth shapes. We conducted a systematic study of ice shaping in solutions containing a wide range of IBPs. We found that although ice crystals in solutions of moderate IBPs do indeed grow into their faceted shapes, in the presence of most hyperactive IBPs, ice melts into its final shape. These melting shapes result from the affinity of the hyperactive