抗冻蛋白研究进展(英文)

很多越冬的生物会产生抗冻蛋白,这些抗冻蛋白能够吸附到冰晶的表面改变冰晶形态并抑制冰晶的生长.抗冻蛋白在很多生物体内都被发现,不同的抗冻蛋白结构差异非常大.目前的一些研究揭示了几种抗冻蛋白的结构,并提出了抗冻蛋白与冰晶的结合模型,但是还没有一种机制能解释所有抗冻蛋白的作用机理.抗冻蛋白能被广泛的应用到农业、水产业和低温储藏器官、组织和细胞,利用转基因技术提高植物的抗冻性具有重要应用价值.而抗冻蛋白基因的表达调控则有待进一步阐明.     

昆虫抗冻蛋白的研究

抗冻蛋白是具有热滞活性,能结合并抑制冰晶生长和抑制冰的重结晶的一类蛋白质。近几年来,昆虫抗冻蛋白的研究取得了较快的发展,本文通过分析昆虫抗冻蛋白的结构特点、抗冻活性、作用机制,并讨论了抗冻蛋白在食品工业、医学、基因工程方面的应用。结果表明,昆虫抗冻蛋白虽然结构呈多样性,但有很多关键的残基具有保守性,对维持抗冻蛋白结构和功能的完整性发挥着重要的作用;抗冻蛋白是由多基因家簇编码的。其作用机制主要是吸附一抑制机制,抗冻蛋白依靠氢键吸附到冰晶格,抑制冰晶生长;昆虫抗冻蛋白的应用具有很广阔的前景。     

昆虫抗冻蛋白的研究进展

抗冻蛋白是具有热滞效应,能结合并抑制新的冰晶生长,能抑制冰的重结晶的一类蛋白质。近几年来,昆虫抗冻蛋白的研究取得了较快的发展,本文就昆虫抗冻蛋白的结构,活性的调控,功能与应用做一综述。     

甲虫抗冻蛋白热滞活性的二维吸附结合模型

甲虫抗冻蛋白是一种具有规则结构的昆虫抗冻蛋白。在相同浓度条件下,甲虫抗冻蛋白比鱼类抗冻蛋白有更高的热滞活性,目前已成为人们重点研究的一类抗冻蛋白。根据甲虫抗冻蛋白的结构特点及其在冰晶表面的吸附模式,应用二维吸附结合模型计算分析了具有6￣11个β-螺旋(β-helix)结构片段的甲虫抗冻蛋白变体分子,得到了它们的热滞活性随溶液浓度变化的规律,特别是热滞活性与甲虫抗冻蛋白的β-螺旋结构片段数的关系。结果显示,抗冻蛋白在冰晶表面的覆盖度是一个影响其热滞活性的重要因素。     

昆虫抗冻蛋白的结构与生物学特性研究

抗冻蛋白(antifreeze proteins AFPs)是一类抑制冰晶生长的蛋白质,它能以非依数性形式降低溶液的冰点而对其熔点影响甚微,因而也被称作热滞蛋白。近几年来对于昆虫抗冻蛋白的研究取得了较快的发展,已有20多种昆虫抗冻蛋白被分离纯化。就昆虫抗冻蛋白的结构特征、生物学特性以及在农业、医学和食品工业等方面的应用进行介绍。     

抗冻蛋白溶液的热滞理论

鱼抗冻蛋白溶液有热滞效应,这是一种非ｃｏｌｌｉｇａｔｉｖｅ效应．提出冰晶生长的动力学理论,并根据Ｆｌｏｒｙ－Ｈｕｇｇｉｎ晶格模型计算抗冻蛋白溶液的热滞,结果与实验较好地符合。     

昆虫抗冻蛋白: 规则结构适应功能

抗冻蛋白在环境温度低于体液熔点时能够结合到生物体内的冰核表面,通过限制冰核生长和抑制冰晶重结晶而保护有机体免受结冰引起的伤害。与其他生物抗冻蛋白比较,昆虫抗冻蛋白有很强的活性,结构上具有显著特征,如一级结构规律重复,超二级结构为β-螺旋,可与冰晶发生相互作用,具有TXT基序等。该文综述了近年来关于昆虫抗冻蛋白的结构以及分子生物学等方面研究的新进展,讨论了其结构与功能的关系。     

抗冻蛋白应用于水稻悬浮细胞超低温保存的研究

本文将鱼类抗冻蛋白应用于植物细胞的超低温保存。结果表明,在水稻悬浮细胞的两步法保存中,浓度为0.01mg/ml的抗冻蛋白具有特别的负作用,相对高浓度的抗冻蛋白则能减小细胞存活率的波动性。在玻璃化法保存中,浓度为0.2mg/ml的抗冻蛋白能改善保存效果,更高浓度的抗冻蛋白(>5mg/ml)反而会降低保存效果。环境冰晶量、抗冻蛋白浓度、低温保护剂浓度和细胞膜组成等是影响抗冻蛋白使用效果的几大因素。作者在机理分析中认为,一方面,抗冻蛋白能和冰晶作用,抑制重冰晶,防止去玻璃化;另一方面,抗冻蛋白也能和细胞膜作用,诱发膜表面冰晶形成。     

昆虫抗冻蛋白的研究进展

抗冻蛋白是一类与冰晶有亲合力,能够与冰晶结合并抑制冰晶生长的蛋白或糖蛋白。自20世纪60年代以来,研究人员已经分别从鱼类、昆虫、植物、真菌和细菌中发现多种抗冻蛋白。其中已知鱼类抗冻蛋白有5种,也是研究最详细的。但是,近几年来发现昆虫抗冻蛋白活性普遍比较高,因此受到研究人员重视,研究取得了较快的发展。主要讨论昆虫抗冻蛋白的结构特点、抗冻活性、作用机制和应用,并分析目前的研究现状提出一些待解决的问题,以期望对昆虫抗冻蛋白的研究进行比较系统化的整理。     

抗冻蛋白与细胞的低温和超低温保存

抗冻蛋白(AFP,Antifreeze,Proteins)最早发现于极地海洋鱼类,它能非连续地降低体液冰点,并通过吸附于冰晶的特殊表面有效阻止和改变冰晶生长。现已证明,一些陆生节肢动物、维管植物、非维管植物、真菌和细菌等,也存在抗冻蛋白。鱼类抗冻蛋白一般分成四     

The mechanism by which fish antifreeze proteins cause thermal hysteresis

Antifreeze proteins are characterised by their ability to prevent ice from growing upon cooling below the bulk melting point. This displacement of the freezing temperature of ice is limited and at a sufficiently low temperature a rapid ice growth takes place. The separation of the melting and freezing temperature is usually referred to as thermal hysteresis, and the temperature of ice growth is referred to as the hysteresis freezing point. The hysteresis is supposed to be the result of an adsorption of antifreeze proteins to the crystal surface. This causes the ice to grow as convex surface regions between adjacent adsorbed antifreeze proteins, thus lowering the temperature at which the crystal can visibly expand. The model requires that the antifreeze proteins are irreversibly adsorbed onto the ice surface within the hysteresis gap. This presupposition is apparently in conflict with several characteristic features of the phenomenon; the absence of superheating of ice in the presence of antifreeze proteins, the dependence of the hysteresis activity on the concentration of antifreeze proteins and the different capacities of different types of antifreeze proteins to cause thermal hysteresis at equimolar concentrations. In addition, there are structural obstacles that apparently would preclude irreversible adsorption of the antifreeze proteins to the ice surface; the bond strength necessary for irreversible adsorption and the absence of a clearly defined surface to which the antifreeze proteins may adsorb. This article deals with these apparent conflicts between the prevailing theory and the empirical observations. We first review the mechanism of thermal hysteresis with some modifications: we explain the hysteresis as a result of vapour pressure equilibrium between the ice surface and the ambient fluid fraction within the hysteresis gap due to a pressure build-up within the convex growth zones, and the ice growth as the result of an ice surface nucleation event at the hysteresis freezing point. We then go on to summarise the empirical data to show that the dependence of the hysteresis on the concentration of antifreeze proteins arises from an equilibrium exchange of antifreeze proteins between ice and solution at the melting point. This reversible association between antifreeze proteins and the ice is followed by an irreversible adsorption of the antifreeze proteins onto a newly formed crystal plane when the temperature is lowered below the melting point. The formation of the crystal plane is due to a solidification of the interfacial region, and the necessary bond strength is provided by the protein "freezing" to the surface. In essence: the antifreeze proteins are "melted off" the ice at the bulk melting point and "freeze" to the ice as the temperature is reduced to subfreezing temperatures. We explain the different hysteresis activities caused by different types of antifreeze proteins at equimolar concentrations as a consequence of their solubility features during the phase of reversible association between the proteins and the ice, i.e., at the melting point; a low water solubility results in a large fraction of the proteins being associated with the ice at the melting point. This leads to a greater density of irreversibly adsorbed antifreeze proteins at the ice surface when the temperature drops, and thus to a greater hysteresis activity. Reference is also made to observations on insect antifreeze proteins to emphasise the general validity of this approach.     

The mechanism by which fish antifreeze proteins cause thermal hysteresis

Antifreeze proteins are characterised by their ability to prevent ice from growing upon cooling below the bulk melting point. This displacement of the freezing temperature of ice is limited and at a sufficiently low temperature a rapid ice growth takes place. The separation of the melting and freezing temperature is usually referred to as thermal hysteresis, and the temperature of ice growth is referred to as the hysteresis freezing point. The hysteresis is supposed to be the result of an adsorption of antifreeze proteins to the crystal surface. This causes the ice to grow as convex surface regions between adjacent adsorbed antifreeze proteins, thus lowering the temperature at which the crystal can visibly expand. The model requires that the antifreeze proteins are irreversibly adsorbed onto the ice surface within the hysteresis gap. This presupposition is apparently in conflict with several characteristic features of the phenomenon; the absence of superheating of ice in the presence of antifreeze proteins, the dependence of the hysteresis activity on the concentration of antifreeze proteins and the different capacities of different types of antifreeze proteins to cause thermal hysteresis at equimolar concentrations. In addition, there are structural obstacles that apparently would preclude irreversible adsorption of the antifreeze proteins to the ice surface; the bond strength necessary for irreversible adsorption and the absence of a clearly defined surface to which the antifreeze proteins may adsorb. This article deals with these apparent conflicts between the prevailing theory and the empirical observations. We first review the mechanism of thermal hysteresis with some modifications: we explain the hysteresis as a result of vapour pressure equilibrium between the ice surface and the ambient fluid fraction within the hysteresis gap due to a pressure build-up within the convex growth zones, and the ice growth as the result of an ice surface nucleation event at the hysteresis freezing point. We then go on to summarise the empirical data to show that the dependence of the hysteresis on the concentration of antifreeze proteins arises from an equilibrium exchange of antifreeze proteins between ice and solution at the melting point. This reversible association between antifreeze proteins and the ice is followed by an irreversible adsorption of the antifreeze proteins onto a newly formed crystal plane when the temperature is lowered below the melting point. The formation of the crystal plane is due to a solidification of the interfacial region, and the necessary bond strength is provided by the protein “freezing” to the surface. In essence: the antifreeze proteins are “melted off” the ice at the bulk melting point and “freeze” to the ice as the temperature is reduced to subfreezing temperatures. We explain the different hysteresis activities caused by different types of antifreeze proteins at equimolar concentrations as a consequence of their solubility features during the phase of reversible association between the proteins and the ice, i.e., at the melting point; a low water solubility results in a large fraction of the proteins being associated with the ice at the melting point. This leads to a greater density of irreversibly adsorbed antifreeze proteins at the ice surface when the temperature drops, and thus to a greater hysteresis activity. Reference is also made to observations on insect antifreeze proteins to emphasise the general validity of this approach.     

Raman spectra of a solid antifreeze glycoprotein and its liquid and frozen aqueous solutions.

Raman spectroscopy was used to study the anomalous decrease in the freezing temperature of water produced by an antifreeze glycoprotein obtained from the sera of an Antarctic fish. An active fraction of this glycoprotein has a molecular weight of approximately 18,000 by equilibrium sedimentation compared to an apparent weight of 20 by freezing temperature depression. The Raman spectra of water present in a 1% antifreeze glycoprotein solution and of ice frozen from this solution were indistinguishable from the spectra of pure water and ice, respectively. These results indicate that the bulk properties of water and ice are unaffected by the presence of the antifreeze glycoprotein. Raman measurements on ice grown slowly, using as seed an oriented single crystal of ice in contact with 1% glycoprotein solutions, showed that the active glycoprotein was not excluded from the ice phase. On the other hand, we found that a smaller, inactive glycoprotein was excluded. Comparison of the Raman spectra of active and inactive glycoprotein components as solids, in 5% solutions, and rapidly frozen 5% solutions, showed that the two components differ in conformation and possibly in the environment of their carbohydrate hydroxyls. These observations suggest that hydrogen bonding of the carbohydrate hydroxyls of the active glycoprotein at the ice-solution interface may physically prevent growth of the ice lattice.     

Salt-induced enhancement of antifreeze protein activity: a salting-out effect

Antifreeze proteins are a structurally diverse group of proteins characterized by their unique ability to cause a separation of the melting- and growth-temperatures of ice. These proteins have evolved independently in different kinds of cold-adapted ectothermic animals, including insects and fish, where they protect against lethal freezing of the body fluids. There is a great variability in the capacity of different kinds of antifreeze proteins to evoke the antifreeze effect, but the basis of these differences is not well understood. This study reports on salt-induced enhancement of the antifreeze activity of an antifreeze protein from the longhorn beetle Rhagium inquisitor (L.). The results imply that antifreeze activity is predetermined by a steady-state distribution of the antifreeze protein between the solution and the ice surface region. The observed salt-induced enhancement of the antifreeze activity compares qualitatively and quantitatively with salt-induced lowering of protein solubility. Thus, salts apparently enhance antifreeze activity by evoking a solubility-induced shift in the distribution pattern of the antifreeze proteins in favour of the ice. These results indicate that the solubility of antifreeze proteins in the solution surrounding the ice crystal is a fundamental physiochemical property in relation to their antifreeze potency.     

差示扫描量热法直接测定沙冬青抗冻蛋白的热滞效应

用差示扫描量热法直接测定了从沙冬青中提取的一种抗冻蛋白（ＡＦＰ）组分的低温热行为。结果表明,该组分的低温热行为远较文献报道的各种抗冻蛋白复杂。在降、升温过程中,在低和高温侧都给出两个放或吸热峰,两个峰表现出相互独立而又相互依存的热滞行为。低温峰的热滞活性远高于高温岭。我们认为,这种ＡＦＰ分子对水及冰晶很可能有两种不同的相互作用和影响。     

Antifreeze glycoproteins: influence of polymer length and ice crystal habit on activity

The effect of the ice crystalline habit and the length of the polymer on the ability of the antifreeze glycoproteins (AFGP) from polar fish to depress the freezing temperature of water was investigated. The low-molecular-weight components of the glycoproteins, AFGP- 6–8, are inactive when a solution of such a sample is nucleated at ?6°C. A solution of large AFGP (1–4) is fully functional under the same conditions. The low-molecular-weight components differ from the height-molecular-weight components in that they contain some proline replacing the alanine in the Ala-Ala-Thr · disaccharide polymer unit. In the present experiments, antifreeze activity was examined in the presence of two different forms of ice crystal growth habits, and homodimders of AFGP 6 and 8 were prepared to investigate the function of polymer length and the on antifreeze activity at different degrees of supercooling. The results indicate that the ice crystal growth habit and the introduction of proline into the polymer unit may be responsible for the loss of activity at deep supercooling (?6°C) of AFGP 6–8. The loss in the ability of AFGP to depress the freezing temperature of water at deep supercooling is not solely due to polymer length, as carbodiimide-linked dimers of AFGP 6 do not function under these freezing conditions. A Model of antifreezing action based on Langmuirian adsorption of AFGP on the ice surface and direct competition between water and AFGP molecules for the incorporation sites in the ice crystal lattice is presented.     

抗冻糖蛋白溶液中冰晶生长速率的研究

在分析了溶液中抗冻糖蛋白与冰晶表面的相互作用的基础上,提出了在抗冻糖蛋白溶液中冰晶沿c轴方向生长的理论。给出了冰晶在抗冻糖蛋白溶液中生长速率的定量计算,而且理论值与实验结果有较好的符合,解释了冰晶在抗冻糖蛋白溶液中生长速度和生长习性的各向异性。     

Zero-sized effect of nano-particles and inverse homogeneous nucleation. Principles of freezing and antifreeze

It was found that freezing of water in terms of homogeneous nucleation of ice never occurs even in ultra-clean micro-sized water droplets under normal conditions. More surprisingly, at sufficiently low supercoolings, foreign nano-particles exert no effect on the nucleation barrier of ice; it is as if they physically "vanished." This effect, called hereafter the "zero-sized" effect of foreign particles (or nucleators), leads to the entry of a so-called inverse homogeneous-like nucleation domain, in which nucleation is effectively suppressed. The freezing temperature of water corresponds to the transition temperature from the inverse homogeneous-like nucleation regime to foreign particle-mediated heterogeneous nucleation. The freezing temperature of water is mainly determined by (i) the surface roughness of nucleators at large supercoolings, (ii) the interaction and structural match between nucleating ice and the substrate, and (iii) the size of the effective surface of nucleators at low supercoolings. Our experiments showed that the temperature of -40 degrees C, commonly regarded as the temperature of homogeneous nucleation-mediated freezing, is actually the transition temperature from the inverse homogeneous-like nucleation regime to foreign particle-mediated heterogeneous nucleation in ultra-clean water. Taking advantage of inverse homogeneous-like nucleation, the interfacial tensions between water and ice in very pure water and antifreeze aqueous solutions were measured at a very high precision for the first time. The principles of freezing promotion and antifreeze and the selection for the biological ice nucleation and antifreeze proteins are obtained. The results provide completely new insights into freezing and antifreeze phenomena and bear generic implications for all crystallization systems.     

Biochemistry of fish antifreeze proteins

Four distinct macromolecular antifreezes have been isolated and characterized from different marine fish. These include the glycoprotein antifreezes (Mr 2.5-33 K), which are made up of a repeating tripeptide (Ala-Ala-Thr)n with a disaccharide attached to the threonyl residues, and three antifreeze protein (AFP) types. Type I is an alanine-rich, amphiphilic, alpha-helix (Mr 3-5 K); type II is a larger protein (Mr 14 K) with a high content of reverse turns and five disulfide bridges; and type III is intermediate in size (Mr 6-7 K) with no distinguishing features of secondary structure or amino acid composition. Despite their marked structural differences, all four antifreeze types appear to function in the same way by binding to the prism faces of ice crystals and inhibiting growth along the a-axes. It is suggested that type I AFP binds preferentially to the prism faces as a result of interactions between the helix macrodipole and the dipoles on the water molecules in the ice lattice. Binding is stabilized by hydrogen bonding, and the amphiphilic character of the helix results in the hydrophobic phase of the helix being exposed to the solvent. When the solution temperature is lowered further, ice crystal growth occurs primarily on the uncoated, unordered basal plane resulting in bipyramidal-shaped crystals. The structural features of type I AFP that could contribute to this mechanism of action are reviewed. Current challenges lie in solving the other antifreeze structures and interpreting them in light of what appears to be a common mechanism of action.