In Situ Observation of Antifreeze Glycoprotein Kinetics at the Ice Interface Reveals a Two-Step Reversible Adsorption Mechanism

Antifreeze glycoproteins (AFGPs) are a necessary tool for the survival of fish that live in subfreezing environments [ Yeh, Y.; Feeney, R. E. Antifreeze proteins—Structures and mechanisms of function. Chem. Rev. 1996, 96 (2), 601−617]. Although scientists agree that these proteins arrest ice crystal growth by a surface adsorption mechanism, the exact nature of the interaction remains an open question. Here, we study the adsorption kinetics of AFGPs during solution ice crystal growth using confocal fluorescence microscopy within and just below the freezing−melting temperature hysteresis region. The AFGP kinetics at the ice surface reveal a two-step inhibition process: (i) incomplete adsorption or a weak interaction that modifies the surface for (ii) a stronger interaction to achieve the complete adsorption necessary to halt growth. The growth is modified from a rough interface to a faceted one, and growth is halted at supercoolings less than 0.05 °C. However, growth resumes, and the proteins desorb, return to the solution phase, and are not incorporated into the ice crystal as previously proposed. Our findings are contrary to an AFGP mechanism described by the Gibbs−Thomson model. We argue that an alternative explanation must include a solvated protein interacting with a solvated ice surface. While thermodynamics considerably alter the interfacial region, antifreeze action is a purely kinetic phenomenon.