Superheating of ice crystals in antifreeze protein solutions

Abstract

It has been argued that for antifreeze proteins (AFPs) to stop ice crystal growth, they must irreversibly bind to the ice surface. Surface-adsorbed AFPs should also prevent ice from melting, but to date this has been demonstrated only in a qualitative manner. Here we present the first quantitative measurements of superheating of ice in AFP solutions. Superheated ice crystals were stable for hours above their equilibrium melting point, and the maximum superheating obtained was 0.44 degrees C. When melting commenced in this superheated regime, rapid melting of the crystals from a point on the surface was observed. This increase in melting temperature was more appreciable for hyperactive AFPs compared to the AFPs with moderate antifreeze activity. For each of the AFP solutions that exhibited superheating, the enhancement of the melting temperature was far smaller than the depression of the freezing temperature. The present findings clearly show that AFPs adsorb to ice surfaces as part of their mechanism of action, and this absorption leads to protection of ice against melting as well as freezing.

Fig. 1.

Sequence of a FH and MH experiment in a drop of MpAFP solution. (A) A single ice crystal grown in 36 μM MpAFP solution was stable down to -1.87 °C below the melting point. (B) Dendritic growth indicating the sudden growth of the ice at this supercooling. The FH of the sample was determined as the difference between the temperature at which this dendritic growth commenced and the T_m of the sample. (C) Growth continued until most of the sample was frozen. (D–E) When the frozen sample was warmed to close to the melting temperature, all of the ice melted except for the initial crystal. (F) The remaining crystal was slowly warmed further. (G) The ice crystal remained stable at superheatings of up to 0.18 °C above T_m for over 25 min. (H) The crystal remained stable for 5 s at +0.18 °C before rapidly melting (I) in a 0.14 s time interval, with a velocity of 48 μm/s. The difference between the T_m and the temperature at which the crystal melted was taken as the MH.

Fig. 2.

Fluorescence images of ice superheated in a GFP-MpAFP solution. (A–E) A series of images recorded while the crystal was warmed slowly to temperatures above the equilibrium melting point. (F) At a superheating of 0.04 °C, melting of the superheated ice crystal started at a point on the surface and proceeded through the crystal. (G–J) After melting commenced, the GFP-tagged AFPs that had been adsorbed on the ice diffused away.

Fig. 3.

Analysis of melting nucleation and the melting velocities of ice crystals formed in AFP solutions. (A) Cumulative fraction of melted crystals as a function of superheating in a TmAFP solution. (B) Melting velocities of ice crystals stabilized in AFP solutions. Open squares correspond to the crystals in A. Solid squares represent data points from experiments using TmAFP solutions with different concentrations. Note that the melting velocities recorded in multiple crystal samples were comparable to those obtained using single crystal samples. Results of additional experiments with individual crystals in MpAFP (solid circles) and GFP-MpAFP (open circles) solutions are also shown. The circled data point corresponds to the experiment described in Fig. 1. The melting velocity of ice in pure water at 0.01 °C is marked with the symbol +.

Fig. 4.

Comparison of FH and MH activity for GFP-MpAFP. (A) MH activity as a function of concentration. (B) The measured MH as a function of FH at the corresponding concentrations.