Ice Nucleation Inhibition

Abstract

The effect of antifreeze protein type III (one type of fish antifreeze protein) on ice crystallization was examined quantitatively based on a "micro-sized ice nucle-ation" technique. It was found for the first time that antifreeze proteins can inhibit the ice nucleation process by adsorbing onto both the surfaces of ice nuclei and dust particles. This leads to an increase of the ice nucle-ation barrier and the desolvation kink kinetics barrier, respectively. Based on the latest nucleation model, the increases in the ice nucleation barrier and the kink kinetics barrier were measured. This enables us to quantitatively examine the antifreeze mechanism of antifreeze proteins for the first time. Antifreeze proteins are found in the blood and tissues of organisms that live in freezing environments (1). In these organisms , ranging from fish to bacteria, the effect of freezing is retarded or the damage incurred upon freezing and thawing is reduced (2-4). Applications of the antifreeze effect of these antifreeze proteins (AFPs), 1 which is the capacity to inhibit ice crystallization, have been sought for maintaining the texture in frozen food, improving storage of blood, tissues, and organs, cryosurgery, and protecting crops from freezing (4). Freezing is a process of ice crystallization from supercooled water. In this process, water should undergo the stage of ice nucleation, followed by the growth of ice (5). Actually, whether or not freezing takes place is determined to a large extent by ice nucleation. In other words, there would be no ice growth if ice nucleation did not occur. The freezing inhibition brought about by antifreeze proteins is actually to impede the nucleation and the growth of ice by reducing the associated kinetics. Previous studies of the AFPs were mainly focused on the modification of the crystal morphology of ice and the inhibition of ice crystal growth in terms of the adsorption of antifreeze protein molecules on specific surfaces of ice (6-8). It is believed (9-11) that antifreeze proteins lower the freezing point of water merely by adsorbing their residues onto the ice crystal surfaces and thereby inhibiting their growth. Although some reports show the modification of the ice morphology caused by AFPs (7), no study has thus far been carried out to show how AFPs inhibit ice crystallization, in particular ice nucleation. We notice that the neglect of the initial and key stage of ice crystallization, i.e. nucleation, is likely due to the fact that a well defined kinetics measurement is a difficult and challenging task. In this paper, we will present the first study on the effect of AFPs on the nucleation of ice in supercooled water using a newly developed technique, the so-called ''double oil layer mi-cro-sized ice crystallization technique'' (12). This technique allows us to minimize the influence of the wall of the container on the nucleation of micro-sized ice, therefore obtaining reliable and reproducible data on ice nucleation. Because the water is confined in a micro-sized droplet, it is a model system used to mimic the freezing of organisms and ice crystallization in the air, where water is normally distributed in micro or sub-micro sized droplets. Apart from this, our new model on nucleation (13) will be applied to analyze quantitatively the effect of AFP III on ice crystallization. This will be achieved by a quantitative measurement of the change of the free energy barrier associated with different dynamic steps in ice nucleation. Based on these results, we wish to obtain a new and comprehensive understanding on the AFP antifreeze mechanism, in particular that of the effect of AFPs on ice nucleation. We hope that this study will provide fresh physical insight into the phenomenon of AFP antifreeze, which will shed light on the identification of new and effective antifreeze proteins/agents. THEORY In most cases, the formation of a new crystalline phase from the ambient phase proceeds via nucleation followed by growth (5). This implies that in the case of ice crystallization, nucle-ation is the initial and one of the most important steps toward creating ice. Without this step, ice will never occur in super-cooled water. In the following discussion, we will examine ice nucleation based on the newly developed model (13). For ice crystallization, a positive thermodynamic driving force is required, which is defined as shown in Equation 1 (14), kT f s kT H m /T m T kT (Eq. 1) where f and s are the chemical potential of solute molecules in the fluid phase and in the solid phase, respectively; H m denotes the enthalpy of melting per molecule; T m denotes the melting temperature; T is supercooling (T T m T, where T is the actual temperature); and k is the Boltzmann constant. The nucleation process can be regarded as a kinetic process for ice nuclei to overcome a kinetics barrier, the so-called nu-cleation barrier under a given thermodynamic driving force /kT (14). By taking into account the effect of foreign particles on nucleation, the nucleation rate of ice, which is defined as the number of nuclei generated per unit of time-volume, is given according to the model shown in Equations 2-4, J 4a kink (R s) 2 N 0 f mfm 1 ⁄2 B exp(f (m)/(T) 2) (Eq. 2)