Concentration Of Antifreeze Proteins Research Articles

Enhancing Cryopreserved Sperm Quality in Chinese Rare Minnow Gobiocypris rarus: The Impact of Antifreeze Proteins.

The Chinese rare minnow (Gobiocypris rarus), an important model fish in China, faces endangerment in the wild. Sperm cryopreservation facilitates the development of new strains and germplasm conservation, but the quality of its cryopreserved sperm remains low. This study evaluates the protective effects of different concentrations of antifreeze proteins (AFP I and AFP III) on the cryopreservation of Chinese rare minnow sperm. Cryopreserved sperm showed significant declines in progressive motility, curvilinear velocity (VCL), average path velocity (VAP), and lifespan compared to fresh sperm, except for straight-line velocity (VSL). The cryomedium containing 10 μg/mL AFP I improved these parameters to their highest levels. However, no significant difference was found in progressive motility and kinetic parameters between cryopreserved sperm with and without AFPs. Cryopreserved sperm with 10 μg/mL AFP I showed the highest plasma membrane integrity, mitochondrial activity, and DNA integrity, significantly better than without AFPs; importantly, the fertilization rate of cryopreserved sperm with 10 μg/mL AFP I was not significantly different from that of fresh sperm. These results indicate that the addition of 10 μg/mL AFP I to the cryomedium for Chinese rare minnow sperm does not improve kinetic parameters but significantly enhances sperm quality, aiding in its new strain development and germplasm conservation.

Antifreeze protein type I in the vitrification solution improves the cryopreservation of immature cat oocytes

Oocyte cryopreservation is not yet considered a reliable technique since it can reduce the quality and survival of oocytes in several species. This study determined the effect of different concentrations of antifreeze protein I (AFP I) on the vitrification solution of immature cat oocytes. For this, oocytes were randomly distributed in three groups and vitrified with 0 μg/mL (G0, 0 μM); 0.5 μg/mL (G0.5, 0.15 μM), or 1 μg/mL (G1, 0.3 μM) of AFP I. After thawing, oocytes were evaluated for morphological quality, and compared to a fresh group (FG) regarding actin integrity, mitochondrial activity and mass, reactive oxygen species (ROS) and glutathione (GSH) levels, nuclear maturation, expression of GDF9, BMP15, ZAR-1, PRDX1, SIRT1, and SIRT3 genes (normalized by ACTB and YWHAZ genes), and ultrastructure. G0.5 and G1 presented a higher proportion of COCs graded as I and while G0 had a significantly lower quality. G1 had a higher percentage of intact actin in COCs than G0 and G0.5 (P < 0.05). There was no difference (P > 0.05) in the mitochondrial activity between FG and G1 and they were both higher (P < 0.05) than G0 and G0.5. G1 had a significantly lower (P < 0.05) mitochondrial mass than FG and G0, and there was no difference among FG, G0, and G0.5. G1 had higher ROS than all groups (P < 0.05), and there was no difference in GSH levels among the vitrified groups (P > 0.05). For nuclear maturation, there was no difference between G1 and G0.5 (P > 0.05), but these were both higher (P < 0.05) than G0 and lower (P < 0.05) compared to FG. Regarding gene expression, in G0 and G0.5, most genes were downregulated compared to FG, except for SIRT1 and SIRT3 in G0 and SIRT3 in G0.5. In addition, G1 kept the expression more similar to FG. Regardless of concentration, AFP I supplementation in vitrification solution of immature cat oocytes improved maturation rates, morphological quality, and actin integrity and did not impact GSH levels. In the highest concentration tested (1 μg/mL), AFP maintained the mitochondrial activity, reduced mitochondrial mass, increased ROS levels, and had the gene expression more similar to FG. Altogether these data show that AFP supplementation during vitrification seems to mitigate some of the negative impact of cryopreservation improving the integrity and cryosurvival of cat oocytes.

Novel Apoplastic Antifreeze Proteins of Deschampsia antarctica as Enhancer of Common Cell Freezing Media for Cryobanking of Genetic Resources, a Preliminary Study.

Antifreeze proteins (AFPs) are natural biomolecules found in cold-adapted organisms that lower the freezing point of water, allowing survival in icy conditions. These proteins have the potential to improve cryopreservation techniques by enhancing the quality of genetic material postthaw. Deschampsia antarctica, a freezing-tolerant plant, possesses AFPs and is a promising candidate for cryopreservation applications. In this study, we investigated the cryoprotective properties of AFPs from D. antarctica extracts on Atlantic salmon spermatozoa. Apoplastic extracts were used to determine ice recrystallization inhibition (IRI), thermal hysteresis (TH) activities and ice crystal morphology. Spermatozoa were cryopreserved using a standard cryoprotectant medium (C+) and three alternative media supplemented with apoplastic extracts. Flow cytometry was employed to measure plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) postthaw. Results showed that a low concentration of AFPs (0.05 mg/mL) provided significant IRI activity. Apoplastic extracts from D. antarctica demonstrated a cryoprotective effect on salmon spermatozoa, with PMI comparable to the standard medium. Moreover, samples treated with apoplastic extracts exhibited a higher percentage of cells with high MMP. These findings represent the first and preliminary report that suggests that AFPs derived from apoplastic extracts of D. antarctica have the potential to serve as cryoprotectants and could allow the development of novel freezing media.

Use of Antifreeze Protein from Tenebrio molitor (TmAFP) in Vitrification of In Vitro-Produced Bovine Embryos: An Ultrastructural Study.

The objective of this study was to evaluate the effects of different concentrations of antifreeze protein (AFP) extracted from the larva of the beetle, Tenebrio molitor (TmAFP), on vitrification of in vitro-produced bovine embryos. In vitro-produced blastocysts were divided into three experimental groups and vitrified using a cryotop. TmAFP was added to the equilibrium solution (ES) and vitrification solution (VS) at a concentration of 0 ng/mL (control), 500 ng/mL (500TmAFP), or 1000 ng/mL (1000TmAFP). Vitrification was carried out by first placing the blastocysts in ES for 2 minutes (7.5% ethylene glycol [EG] and 7.5% dimethyl sulfoxide [DMSO]). The blastocysts were then transferred to VS (15% EG and 15% DMSO) and promptly deposited on a cryotop stem and submerged in liquid nitrogen. Warming was carried out in three steps with decreasing sucrose concentrations. After warming, the blast cells were cultured for 24 hours for subsequent survival analysis and ultrastructural evaluation. There was a significant difference in the survival rate and expansion in the 500TmAFP group compared with the other groups. The ultrastructural analysis revealed intracellular lesions in all vitrified embryos; however, the embryos of the 500TmAFP and 1000TmAFP groups showed fewer cytoplasmic lesions compared with the control group. Taken together, addition of TmAFP can mitigate cellular changes that involve organelles and cellular components essential for proper functioning and improve the viability of warmed and vitrified in vitro-produced bovine embryos.

Molecular Factors of Ice Growth Inhibition for Hyperactive and Globular Antifreeze Proteins: Insights from Molecular Dynamics Simulation.

The molecular mechanism behind the ice growth inhibition by antifreeze proteins (AFPs) is yet to be understood completely. Also, what physical parameters differentiate between the AFP and non-AFP are largely unknown. Thus, to get an atomistic overview of the differential antifreeze activities of different classes of AFPs, we have studied ice growth from different ice surfaces in the presence of a moderately active globular type III AFP and a hyperactive spruce budworm (sbw) AFP. Results are compared with the observations of ice growth simulations in the presence of topologically similar non-AFPs using all-atom molecular dynamics simulations. Simulation data suggest that the ice surface coverage is a critical factor in ice growth inhibition. Due to the presence of an ice binding surface (IBS), AFPs form a high affinity complex with ice, accompanied by a transition of hydration water around the IBS from clathrate-like to ice-like. Several residues around the periphery of the IBS anchor the AFP to the curved ice surface mediated by multiple strong hydrogen bonds, stabilizing the complex immensely. In the high surface coverage regime, the slow unbinding kinetics dominates over the ice growth kinetics and thus facilitates the ice growth inhibition. Due to the non-availability of a proper IBS, non-AFPs form a low-affinity complex with the growing ice surface. As a result, the non-AFPs are continuously repelled by the surface. If the concentration of AFPs is low, then the effective surface coverage is reduced significantly. In this low surface coverage regime, AFPs can also behave like impurities and are engulfed by the growing ice crystal.

Effect of antifreeze protein on the quality and microstructure of frozen chicken breasts

The cryo-protective effects of different concentrations of antifreeze protein (AFP, 0 %, 0.10 %, 0.15 %, 0.20 %, 0.25 %, w/v) on chicken frozen at −18 °C for different storage perispeeods were investigated by monitoring water holding capacity (WHC), water distribution, color, protein properties, microstructure and histological changes. The results showed that AFP significantly maintained the quality traits of frozen chicken. Treatment with 0.20 % AFP represented highest cryo-protective effect on the WHC and protein properties during frozen storage. The carbonyl content with 0.20 % AFP was significantly lower than that of without AFP (P < 0.05). Besides, the myofibrils of chicken supplemented with AFP, especially with 0.25 % AFP, had significantly tighter microstructure, smaller ice crystal area and equivalent diameter than those of without AFP treatment (P < 0.05) after 75 d freezing storage. Overall, AFP had a positive effect to improve the quality of frozen chicken, and it was a potential, safe and efficient cryo-protective agent.

Control on moisture distribution and protein changes of Antarctic krill meat by antifreeze protein during multiple freeze-thaw cycles.

Control on the moisture distribution, protein structure changes, and protein degradation of Antarctic krill meat during freeze-thaw (F-T) cycles by presoaking with antifreeze protein (AFP) was investigated. The results from the thawing loss rate and cooking loss rate indicated that 0.1% was the optimal AFP concentration. Magnetic resonance imaging and low-field nuclear magnetic resonance results showed that AFP inhibited the changes in moisture distribution and maintained the moisture in Antarctic krill meat. The contents of nonprotein nitrogen and trichloroacetic acid-soluble peptides indicated that AFP reduced protein degradation. Further, SDS-PAGE showed that AFP reduced the degradation of actin, troponin T, and myosin light chain. The results of fluorescence spectra, circular dichroism, and chemical bond contents indicated that AFP reduced the damage of the protein tertiary and secondary structures of Antarctic krill meat by holding it in a weak polar environment. This study supplied basic theory for the quality control of Antarctic krill meat. PRACTICAL APPLICATION: Protein degradation, moisture distribution, and protein structure changes occurred to Antarctic krill meat during freeze-thaw cycles due to ice crystal growth and recrystallization, which leads to the decrease in quality. Antifreeze protein has been proven to avoid ice crystals' growth and inhibit ice recrystallization. During freeze-thaw cycles, the moisture distribution of Antarctic krill meat treated with antifreeze protein was more uniform, the degree of protein degradation was lower, and the protein structure was protected. This study demonstrated the potential of antifreeze protein as a water and protein protectant of Antarctic krill meat during freeze-thaw cycles.

Antifreeze proteins for low-temperature preservation in reproductive medicine: A systematic review over the last three decades

Antifreeze proteins (AFPs) are synthesized by diverse non-mammalian species, allowing them to survive in severely cold environments. Since the 1990s, the scientific literature reports their use for low-temperature preservation of germplasm. The aim of this systematic review was to compile available scientific evidence regarding the use of AFP for low-temperature preservation of several reproductive specimens. Internet databases were consulted using the terms: “antifreeze protein” OR “AFP” OR “antifreeze glycoprotein” OR “AFGP” OR “ice-binding protein” OR “IBP” OR “thermal hysteresis protein” AND “cryopreservation”. From 56 articles, 87 experiments testing AFPs in low-temperature preservation of gametes, embryos or reproductive tissues/cells were fully analyzed and outcomes were annotated. A positive outcome was considered as a statistically significant improvement on any parameter evaluated after low-temperature preservation with AFP, whereas a negative outcome included worsening of any evaluated parameter, in comparison to untreated groups or groups treated with a lower concentration of AFP. The findings indicated that research on the use of AFP as a cryoprotectant for reproductive specimens has increased markedly over the past decade. Some experiments reported both positive and negative results, which depended, on AFP concentration in the preservation media. Variation in the outcomes associated with species was also observed. Among the 66 experiments conducted in mammals, 77.3% resulted in positive, and 28.8% in negative outcomes after the use of AFP. In fishes, positive and negative outcomes were observed in 71.4% and 33.3% of 21 experiments, respectively. Most positive outcomes included preserving cell post-warming survival. The beneficial effect of AFP supports its use in cryobiological approaches used in human and veterinary medicines and animal protein industry. Moreover, combination of different AFP types, or AFP with antioxidants, or even the use of AFP-biosimilar, comprise some promising approaches to be further explored in cryopreservation.

Improvement of sperm cryo-survival of cynomolgus macaque (Macaca fascicularis) by commercial egg-yolk–free freezing medium with type III antifreeze protein

When nonhuman primate sperm undergoes cryopreservation in an egg yolk medium there is an increased risk that the egg yolk might adversely affect the sperm due to containing of avian pathogens. Although commercial egg-yolk-free medium for human sperm cryopreservation has been used for macaque sperm, the cryo-survival remains less than optimal. The present study, therefore, was conducted to determine the optimal concentration of antifreeze protein (AFP) III supplemented in a commercial egg-yolk-free medium for cynomolgus macaque (Macaca fascicularis) sperm cryo-survival. The function of frozen-thawed sperm was evaluated by post-thaw sperm motility, acrosome integrity, and mitochondrial function. Results indicate that the sperm motilities were greater when 0.1, 1, and 10 μg/ml of AFP III were supplemented into the sperm freezing medium (P < 0.05). In addition, the mitochondrial membrane potential was greater in the sperm cryopreserved with the medium that was supplemented with 0.1 μg/ml of AFP III (P < 0.05). The addition of AFP III at any of the concentrations, however, did not have any cryoprotection effect on the sperm acrosome, and the greatest concentrations of AFP III at 100 and 200 μg/ml had detrimental effects on acrosomal integrity (P < 0.05). Results of the present study indicated the methods used are effective for the cryopreservation of cynomolgus monkey sperm while reducing associated health risks due to avian pathogens being present in egg yolk-based extenders.

Ice-binding proteins and the applicability and limitations of the kinetic pinning model.

Ice-binding proteins (IBPs) are unique molecules that bind to and are active on the interface between two phases of water: ice and liquid water. This property allows them to affect ice growth in multiple ways: shaping ice crystals, suppressing the freezing point, inhibiting recrystallization and promoting nucleation. Advances in the protein's production technologies make these proteins promising agents for medical applications among others. Here, we focus on a special class of IBPs that suppress freezing by causing thermal hysteresis (TH): antifreeze proteins (AFPs). The kinetic pinning model describes the dynamics of a growing ice face with proteins binding to it, which eventually slow it down to a halt. We use the kinetic pinning model, with some adjustments made, to study the TH dependence on the solution's concentration of AFPs by fitting the model to published experimental data. We find this model describes the activity of (moderate) type III AFPs well, but is inadequate for the (hyperactive) Tenebrio molitor AFPs. We also find the engulfment resistance to be a key parameter, which depends on the protein's size. Finally, we explain intuitively how TH depends on the seeding time of the ice crystal in the protein solution. Using this insight, we explain the discrepancy in TH measurements between different assays. This article is part of the theme issue 'The physics and chemistry of ice: scaffolding across scales, from the viability of life to the formation of planets'.

Effect of Herring Antifreeze Protein Combined with Chitosan Magnetic Nanoparticles on Quality Attributes in Red Sea Bream (Pagrosomus major)

The effects on the quality of frozen red sea bream which were pretreated by soaking in solutions containing trehalose, chitosan magnetic nanoparticles (CS@Fe3O4 nanoparticles), and glycerin or different concentration herring antifreeze proteins (AFPs) were investigated in this study. The DSC, dynamic rheology, shear force, and TPA were conducted to analyze the physical characteristics. Raman and intrinsic fluorescence spectra were used to measure the protein secondary and tertiary structures. So-ANS and zeta potential were carried out to explore the degree of protein aggregation. Low-field NMR was used to test the water migration, and light microscope was performed to observe the fiber microstructure. Results showed that prior to freezing, the pretreatment of red sea bream samples with soak solutions could minimize drip loss, preserve the meat tenderness and texture, and improve the fish fillet thermal stability and viscoelasticity, and the free water content was decreased while the immobilized water content was increased; the longitudinal and transverse fiber structures were clear and less destructive. Compared with the control, the protein secondary and tertiary structures of samples, among the tested soaking solutions, tended to stability, and the degree of protein oxidation and aggregation was decreased. Therefore, this methodology was proved to be an effective method to inhibit the ice crystal growth and modify ice crystal form in order to improve the quality of the final product, and these effects were amplified with the concentration of AFPs increasingly.

Multivalent Display of Antifreeze Proteins by Fusion to Self-Assembling Protein Cages Enhances Ice-Binding Activities.

Antifreeze proteins (AFPs) are small monomeric proteins that adsorb to the surface of ice to inhibit ice crystal growth and impart freeze resistance to the organisms producing them. Previously, monomeric AFPs have been conjugated to the termini of branched polymers to increase their activity through the simultaneous binding of more than one AFP to ice. Here, we describe a superior approach to increasing AFP activity through oligomerization that eliminates the need for conjugation reactions with varying levels of efficiency. A moderately active AFP from a fish and a hyperactive AFP from an Antarctic bacterium were genetically fused to the C-termini of one component of the 24-subunit protein cage T33-21, resulting in protein nanoparticles that multivalently display exactly 12 AFPs. The resulting nanoparticles exhibited freezing point depression >50-fold greater than that seen with the same concentration of monomeric AFP and a similar increase in the level of ice-recrystallization inhibition. These results support the anchored clathrate mechanism of binding of AFP to ice. The enhanced freezing point depression could be due to the difficulty of overgrowing a larger AFP on the ice surface and the improved ice-recrystallization inhibition to the ability of the nanoparticle to simultaneously bind multiple ice grains. Oligomerization of these proteins using self-assembling protein cages will be useful in a variety of biotechnology and cryobiology applications.

Thermal hysteresis of antifreeze proteins considering Fragilariopsis cylindrus

The diatom species Fragilariopsis cylindrus produces antifreeze proteins (AFPs) of moderate thermal hysteresis. Two concepts are often used in order to describe a thermal hysteresis, on the one hand the irreversible nucleation growth described by the Gibbs-Thomson relation and on the other hand a nonlinear adsorption kinetics of the ice-binding proteins. We refer to the Landau's phase transition theory and predict a saturation concentration of AFPs for the maximal freezing depression. The derived functional relation between temperature and AFP concentration is more complex than a simple power law as suggested by thermal hysteresis experiments. With the parameter set obtained for Fragilariopsis cylindrus the modelled curve is comparable with the often used square root law.

The effect of antifreeze protein on the cryopreservation of chrysanthemums

To our knowledge, this is the first study to show the application of antifreeze protein (AFP) in the cryopreservation of ornamental plants. We studied the effect of AFP on the cryopreservation of Chrysanthemum grandiflorum (Ramat.) Kitam ‘Borami’, ‘Secret Pink’, and ‘Yellow Cap’. For all cultivars, survival and regrowth rates were higher in shoot tips vitrified with plant vitrification solution 3 (PVS3) containing various concentrations of AFP than in those vitrified with PVS3 alone. The optimal AFP concentration was genotype-dependent. Scanning electron microscopy revealed that shoot tips vitrified with PVS3 containing AFP had less freezing injuries than those treated with PVS3 alone. Analysis of the enthalpy in each sample by differential scanning calorimetry supported the finding that AFP is a potent cryoprotectant that can reduce the freezing point of water in plant tissues. We expect that our results will facilitate the successful application of AFPs in the cryopreservation of rare and commercially important plant germplasm.

Effects of three different types of antifreeze proteins on mouse ovarian tissue cryopreservation and transplantation.

BackgroundOvarian tissue (OT) cryopreservation is effective in preserving fertility in cancer patients who have concerns about fertility loss due to cancer treatment. However, the damage incurred at different steps during the cryopreservation procedure may cause follicular depletion; hence, preventing chilling injury would help maintain ovarian function.ObjectiveThis study was designed to investigate the beneficial effects of different antifreeze proteins (AFPs) on mouse ovarian tissue cryopreservation and transplantation.MethodologyOvaries were obtained from 5-week-old B6D2F1 mice, and each ovary was cryopreserved using two-step vitrification and four-step warming procedures. In Experiment I, ovaries were randomly allocated into fresh, vitrification control, and nine experimental groups according to the AFP type (FfIBP, LeIBP, type III) and concentration (0.1, 1, 10 mg/mL) used. After vitrification and warming, 5,790 ovarian follicles were evaluated using histology and TUNEL assays, and immunofluorescence for τH2AX and Rad51 was used to detect DNA double-strand breaks (DSBs) and repair (DDR), respectively. In Experiment II, 20 mice were randomly divided into two groups: one where the vitrification and warming media were supplemented with 10 mg/mL LeIBP, and the other where media alone were used (control). Ovaries were then autotransplanted under both kidney capsules 7 days after vitrification together with the addition of 10 mg/mL LeIBP in the vitrification-warming media. After transplantation, the ovarian follicles, the percentage of apoptotic follicles, the extent of the CD31-positive area, and the serum FSH levels of the transplanted groups were compared.Principal FindingsIn Experiment I, the percentage of total grade 1 follicles was significantly higher in the 10 mg/mL LeIBP group than in the vitrification control, while all AFP-treated groups had significantly improved grade 1 primordial follicle numbers compared with those of the vitrification control. The number of apoptotic (TUNEL-positive) follicles was significantly decreased in the groups treated with 1 and 10 mg/mL LeIBP. The proportion of τH2AX-positive follicles was significantly reduced in all AFP-treated groups, while the proportion of Rad51-positive follicles was significantly decreased in only the FfIBP- and LeIBP-treated groups. In Experiment II, after autotransplantation of OT vitrified with 10 mg/mL of LeIBP, the percentage of total grade 1 and primordial grade 1 follicles, and the extent of the CD31-positive area, were increased significantly. Moreover, the levels of serum FSH and the percentage of TUNEL-positive follicles were significantly lower in the LeIBP-treated than in the control group.ConclusionA supplementation with high concentrations of AFPs had protective effects on follicle preservation during OT vitrification-warming procedures. The group treated with LeIBP was protected most effectively. The beneficial effects of LeIBP were also observed after autotransplantation of vitrified-warmed OT. Further studies are necessary to determine the exact mechanism of these protective effects.

The influence of adsorption orientation on the statistical mechanics model of type I antifreeze protein: The coverage rate

Based on the statistical mechanics model, the coverage rate of the type I antifreeze protein (AFP) on the surface of ice-crystal in a protein solution is studied by including the adsorption orientation, as well as both the AFP–ice and AFP–water hydrophobic interactions. As an example, the computed results for HPLC-6 are given and discussed. It is shown that the coverage rate increases with the AFP concentration for the dilute protein solutions. The effect of the adsorption orientation of AFP molecules on the coverage rate cannot be ignored. Including the adsorption orientation enhances the coverage rate when the AFP solution is very dilute, but reduces it for the slightly thick solution. The stronger the AFP–ice and AFP–water hydrophobic interactions are, the larger the coverage rate is.

Dynamical mechanism of antifreeze proteins to prevent ice growth.

The fascinating ability of algae, insects, and fishes to survive at temperatures below normal freezing is realized by antifreeze proteins (AFPs). These are surface-active molecules and interact with the diffusive water-ice interface thus preventing complete solidification. We propose a dynamical mechanism on how these proteins inhibit the freezing of water. We apply a Ginzburg-Landau-type approach to describe the phase separation in the two-component system (ice, AFP). The free-energy density involves two fields: one for the ice phase with a low AFP concentration and one for liquid water with a high AFP concentration. The time evolution of the ice reveals microstructures resulting from phase separation in the presence of AFPs. We observed a faster clustering of pre-ice structure connected to a locking of grain size by the action of AFP, which is an essentially dynamical process. The adsorption of additional water molecules is inhibited and the further growth of ice grains stopped. The interfacial energy between ice and water is lowered allowing the AFPs to form smaller critical ice nuclei. Similar to a hysteresis in magnetic materials we observe a thermodynamic hysteresis leading to a nonlinear density dependence of the freezing point depression in agreement with the experiments.

064 The dynamic nature of antifreeze protein activity

Ice-binding proteins include antifreeze proteins (AFPs), ice nucleating proteins, ice adhesins, and ice recrystallization inhibition proteins. These proteins have great potential for controlling ice growth in various cryobiological and industrial applications. AFPs have been investigated for several decades. However, due to the unstable nature of ice crystals near their melting temperature, few experimental projects had addressed the kinetics of the AFP – ice interaction. When AFPs adsorb to ice they produce a thermal hysteresis (TH), which is the temperature gap between melting temperature of the ice in the solution of AFP, and the lowered temperature at which the crystal starts to grow. The basis for the relationship between TH and the concentration of AFP in solution is not known. We have used temperature-controlled microfluidic devices and fluorescently labeled AFPs to follow on the surface concentration of AFP on ice crystals. We find that the binding of the proteins to the ice surfaces is irreversible (”Superheating of ice crystals in antifreeze protein solutions” PNAS 2010 and ”Microfluidic experiments reveal that antifreeze proteins bound to ice crystals suffice to prevent their growth” PNAS 2013). Furthermore, we confirm that TH increases with the time that an ice crystal is expose to solutions that contain AFPs. We also find a direct connection between the surface concentrations of AFP and the measured TH. These experimental findings allow for testing different theoretical models regarding the mechanism of AFP’s ability to inhibit ice growth. Understanding the dynamic nature of AFP action will allow for rational design of their use in cryobiology applications. Source of funding: This research was funded by the Europe Research Council (ERC), the Israel Science Foundation (ISF), and the Canadian Institutes of Health Research (CIHR). Conflict of interest: None declared. ido.braslavsky@mail.huji.ac.il

062 Antifreeze proteins from Japanese organisms: Functional analyses for their general use

Antifreeze protein (AFP) binds to the surface of an ice crystal to inhibit its growth below 0 °C, and also to the membrane surface of a cell to improve its viability above 0 °C. To clarify the mechanism of these two extraordinary functions and to realize a new AFP-utilizing technique, we have been performed exploration of AFPs from Japanese organisms, development of a new mass-preparation method of fish AFP utilizing their muscle homogenate, and structure-function analyses of the AFPs by employing various techniques including X-ray and NMR. The following three topics are presented in this symposium. (1) Mass-purification of fish AFP: In Cryo2009 we reported a simple method to purify an industrial amount (∼kg) of crude AFPI ∼ III and AFGP8 from fish muscle homogenates. In this symposium, we show a development of this method to prepare AFPI of 90% purity from the crude sample, whose production efficiency was 5 g/week in our lab. The biochemical characterization of this purified AFPI will also be presented. (2) Structural determination of fungal AFP: We solved an irregularly ordered solenoid structure of a 223-residue fungal AFP having a triangular cross-section, and determined which of its surfaces forms an ice-binding site (IBS). This IBS is capable of binding to both prism and basal planes, although it has irregularly arrayed surface-bound waters differently to the known hyperactive AFPs. (4) Prolonging cell life-time at 4 °C with AFP: AFP-induced effect on the survival rate was examined for human hepatoma cell (HepG2), rat insulinoma cell (RIN-5F), and bovine blastcyst under hypothermic (+4 °C) temperature. A dramatic increase of the rate was observed at the AFP concentration of 10 mg/ml. The hatching rate of bovine blastcyst after 10-day preservation was improved from 3% to 20% by addition of a recombinant protein of AFPIII. The pregnancy was verified for two recipient heifers so far, for each the 10-day preserved blastcyst with AFP was transferred. The present obtained results will help understanding of the AFP functions and will provide an example of AFP-utilizing technique. Source of Funding: This work was supported by a Grant-in-Aid for scientific research from the Japan Society for the Promotion of Science (JSPS) (No. 23310171) and from Japan Bio-oriented Technology Research Advancement Institution (BRAIN). Conflict of Interest: None declared. s.tsuda@aist.go.jp

Antifreeze protein detection using Rhodamine B as photoluminescence label in porous silicon

A novel method is demonstrated to detect Antifreeze proteins (AFPs) based on photoluminescence (PL) using porous silicon (PS) coated with silver as a substrate. Ag/PS substrate is obtained through immersion of PS in silver nitrate (AgNO3) solutions and is incubated with Rhodamine B (RB) as PL label. This substrate is easy to be fabricated and the pore size of PS is large enough for biological molecules to infiltrate, which is an ideal platform for biological molecule detection. Through functionalization used glutaraldehyde (GTA) and 4-(N-Maleimidomethyl) cyclohexane-1-carboxylicacid (Sulfo-SMCC) as cross-linkers separately, we test the role of the AFPs antibodies in selective capturing the AFPs antigen and explain the reason of the enhancement of PL intensity. The result shows a significant enhancement of the PL intensity of RB at around 590 nm due to the interaction of antibody–antigen competitive binding with AFPs. Therefore, the PL corresponding to RB was selected to detect the target AFPs and the PL intensity of RB proportional to the AFPs concentration. The detection limit was found to be 1.65 μg/ml for AFPs when GTA was used as cross-linker, and the detection limit was 16.5 ng/ml with Sulfo-SMCC as cross-linker.