Unexpected Rise of Glass Transition Temperature of Ice Crystallized from Antifreeze Protein Solution

Antifreeze protein (AFP) is known to bind to a single ice crystal composed of hexagonally arranged waters, hexagonal ice. To investigate the effect of the AFP binding to a general ice block that is an assembly of numerous hexagonal ice crystals, thermodynamic properties, dynamics, and the crystal structure of the ice block were examined in the presence of type I AFP (AFP-I). Previously, it was found that hexagonal ice has a glass transition based on the proton ordering in the ice lattice at low temperature. Measurements of heat capacity under adiabatic conditions, dielectric permittivity, and powder X-ray diffraction revealed that the glass transition occurs around 140 K in the ice containing 0.01–1% (w/w) of the AFP-I, which is greater than the value for the pure hexagonal ice (ca. 110 K). These data imply that AFP affects the glass transition kinetics, i.e., the slowness of the proton migration in the ice block. Hence, adsorption of AFP molecules to each hexagonal ice is thought to change the physicochemical properties of the bulk ice

Time-dependent change in the survival rate (%) of rat insulinoma cell-line, RIN-5F, under hypothermic exposure (+4°C).

<p>A. The survival rate evaluated by counting the number of living cells (Fig. 1B). B. The survival rate evaluated by measuring the amount of insulin secreted from living cells (Fig. 1C). A freshly made Euro-Collins solution (EC) was used as a basic cell-preservation fluid to dissolve AFPI–III, trehalose, and BSA.</p

Analyses of purity and structural integrity of the AFP samples.

<p>A. Electrophoretogram of AFPI–III and AFGP samples provided by Nichirei Foods Inc. on 16.5% tricine-SDS-PAGE. The AFGP sample only contains molecules less than 10 kDa owing to a filtration step. B. The shape of an ice crystal and TH activity obtained for each AFP sample dissolved into the Euro-Collins solution. All protein concentrations were 10 mg/ml.</p

Flow-charts showing the preparation of RIN-5F cells and their survival rate evaluation.

<p>A. Preparation of RIN-5F cells before starting 1–5-day preservation experiments at +4°C. B. Procedures to estimate the number of living cells using the hemocytometer. C. Procedures to measure the amount of insulin secreted from the living cells. Medium-A consists of RPMI-1640 medium containing 10% of fetal bovine serum (FBS). Medium-B consists of the medium containing 1% of FBS. Trypsin-A consists of 0.25% bovine trypsin plus 1 mM of EDTA dissolved in phosphate buffered saline (PBS).</p

Confocal photomicroscope images of human hepatoma cells (HepG2) and rat insulinoma cells (RIN-5F) in Euro Collins-solutions containing 0.4 mg/ml of BSA (A), AFPIII (B and C), and AFPI (D) labeled with fluorescence.

<p>The images A and B show the slice data of 1 µm-width of HepG2, and A’ and B’ an intact cell images synthesized by stacking of the slices. These 4 images were reproduced from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073643#pone.0073643-Tsuda1" target="_blank">[22]</a> with permission. The images C and D are the slice data for RIN-5F cells. All the images were captured after 1 h-preservation at 37°C. Accumulation of AFPIII on the cell-surface is more evident compared with BSA.</p