Antifreeze proteins (AFPs) are a structurally diverse class of macromolecules that interact with water molecules located in the surface of an ice crystal at temperatures below the melting point of the solution [1]. Interaction of a substantial number of AFPs with the ice surface modifies the shape of the ice crystal, resulting in unique morphologies, such as a hexagonal bipyramid or hexagonal trapezohedron [2]. AFPs inhibit ice growth by adsorbing onto the ice surface (adsorption-inhibition model) [3,4] as the temperature is lowered, and such inhibition becomes insufficient below a certain temperature that is favourable for the initiation of crystal growth. For AFP solutions, this 'ice-growth initiation temperature' (T ini ) is different from the melting point (T m ) of the ice crystal, and the T ini is referred to as the nonequilibrium freezing point (nonequilibrium T f ). The difference between T m and the nonequilibrium T f is defined as thermal hysteresis (TH), and is generally used as a measure of the growth inhibition ability of an AFP [5]. Therefore, determination of the T ini and T m of a seed ice crystal is an essential procedure to evaluate the TH activity of AFP. However, the mechanism of ice binding that alters the TH value remains unclear [6].The TH value of AFPs has been evaluated using a nanolitre osmometer and the 'capillary technique' [5,7]. In the former, submicrolitre volumes of an AFP solution are introduced into an oil droplet, the temperature of which is controlled by a Peltier device. The TH value is determined by observing the growth of an Antifreeze proteins (AFPs) possess a unique ability to bind to a seed ice crystal to inhibit its growth. The strength of this binding has been evaluated by thermal hysteresis (TH). In this study, we examined the dependence of TH on experimental parameters, including cooling rate, annealing time, annealing temperature and the size of the seed ice crystal for an isoform of type III AFP from notched-fin eelpout (nfeAFP8). TH of nfeAFP8 dramatically decreased when using a fast cooling rate (0.20°CAEmin )1 ). It also decreased with increasing seed crystal size under a slow cooling rate (0.01°CAEmin), but such dependence was not detected under the fast cooling rate. TH was enhanced 1.4-and 2.5-fold when ice crystals were annealed for 3 h at 0.05 and 0.25°C below T m , respectively. After annealing for 2 h at 0.25°C below T m , TH activity showed marked dependence on the size of ice crystals. These results suggest that annealing of an ice crystal for 2-3 h significantly increased the TH value of type III AFP. Based on a proposed adsorption-inhibition model, we assume that type III AFP undergoes additional ice binding to the convex ice front over a 2-3 h time scale, which results in the TH dependence on the annealing time.Abbreviations AFGP, antifreeze glycoprotein; AFP, antifreeze protein; nfeAFP8, an isoform of type III AFP from Notched-fin eelpout; TH, thermal hysteresis.

We found that Notched‐fin eelpout, which lives off the north east coast of Japan, expresses an antifreeze protein (AFP). The liver of this fish contains DNAs that encode at least 13 type III AFP isoforms (denoted nfeAFPs). The primary sequences of the nfeAFP isoforms were categorized into SP‐ and QAE‐sephadex binding groups, and the latter were further divided into two subgroups, QAE1 and QAE2 groups. Ice crystals observed in HPLC‐pure nfeAFP fractions are bipyramidal in shape with different ratios of c and a axes, suggesting that all the isoforms are able to bind ice. We expressed five recombinant isoforms of nfeAFP and analyzed the thermal hysteresis (TH) activity of each as a function of protein concentration. We also examined the change in activity on mixing the isoforms. TH was estimated to be 0.60 °C for the QAE1 isoform, 0.11 °C for QAE2, and almost zero for the SP isoforms when the concentrations of these isoforms was standardized to 1.0 mm. Significantly, the TH activity of the SP isoforms showed concentration dependence in the presence of 0.2 mm QAE1, indicating that the less active SP isoform becomes ‘active’ when a small amount of QAE1 is added. In contrast, it does not become active on the addition of another SP isoform. These results suggest that the SP and QAE isoforms of type III AFP have different levels of TH activity, and they accomplish the antifreeze function in a co‐operative manner.

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Type III antifreeze protein is naturally expressed as a mixture of sulfopropyl‐Sephadex (SP) and quaternary aminoethyl‐Sephadex (QAE)‐binding isoforms, whose sequence identity is approximately 55%. We studied the ice‐binding properties of a SP isoform (nfeAFP6) and the differences from those of a QAE isoform (nfeAFP8); both of these isoforms have been identified from the Japanese fish Zoarces elongatus Kner. The two isoforms possessed ice‐shaping ability, such as the creation of an ice bipyramid, but nfeAFP6 was unable to halt crystal growth and exhibited no thermal hysteresis activity. For example, the ice growth rate for nfeAFP6 was 1000‐fold higher than that for nfeAFP8 when measured for 0.1 mm protein solution at 0.25 °C below the melting point. Nevertheless, nfeAFP6 exhibited full thermal hysteresis activity in the presence of only 1% nfeAFP8 (i.e. [nfeAFP8]/[nfeAFP6] = 0.01), the effectiveness of which was indistinguishable from that of nfeAFP8 alone. We also observed a burst of ice crystal growth from the tip of the ice bipyramid for both isoforms on lowering the temperature. These results suggest that the ice growth inhibitory activity of an antifreeze protein isoform lacking the active component is restored by the addition of a minute amount of the active isoform.