Ice Nucleation Inhibition

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 nucleation” 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 nucleation 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. 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 nucleation” 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 nucleation 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 (1Davies P.L. Sykes B.D. Curr. Opin. Struct. Biol. 1997; 7: 828-834Crossref PubMed Scopus (188) Google Scholar). In these organisms, ranging from fish to bacteria, the effect of freezing is retarded or the damage incurred upon freezing and thawing is reduced (2Davies P.L. Hew C.L. FASEB J. 1990; 4: 2460-2468Crossref PubMed Scopus (375) Google Scholar, 3Knight C.A. Nature. 2000; 406: 249-251Crossref PubMed Scopus (129) Google Scholar, 4Knight C.A. Devries A.L. Oolman L.D. Nature. 1984; 308: 295-296Crossref PubMed Scopus (290) Google Scholar). Applications of the antifreeze effect of these antifreeze proteins (AFPs), 1The abbreviation used is: AFPs, antifreeze proteins. 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 (4Knight C.A. Devries A.L. Oolman L.D. Nature. 1984; 308: 295-296Crossref PubMed Scopus (290) Google Scholar). 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 (5Mutaftschiev B. Hurle D.T.J. Handbook of Cryst. Growth la Fundamentals: Thermodynamics and Kinetics. North-Holland, Amsterdam1993: 187-248Google Scholar). 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 (6Raymond J.A. Devries A.L. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 2589-2593Crossref PubMed Scopus (676) Google Scholar, 7Antson A.A. Smith D.J. Roper D.I. Lewis S. J. Mol. Biol. 2001; 305: 875-889Crossref PubMed Scopus (106) Google Scholar, 8Knight C.A. Crystal Growth & Design. 2001; 1: 429-43814Crossref Scopus (25) Google Scholar). It is believed (9Chao H. Sonnichsen F.D. Deluca C.I. Sykes B.D. Davies P.L. Protein Sci. 1994; 3: 1760-1769Crossref PubMed Scopus (109) Google Scholar, 10Yeh Y. Feeney R.E. Chem. Rev. 1996; 96: 601-617Crossref PubMed Scopus (468) Google Scholar, 11Raymond J.A. Wilson P. Devries A.L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 881-885Crossref PubMed Scopus (153) Google Scholar) 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 (7Antson A.A. Smith D.J. Roper D.I. Lewis S. J. Mol. Biol. 2001; 305: 875-889Crossref PubMed Scopus (106) Google Scholar), 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 micro-sized ice crystallization technique” (12Du N. Liu X.Y. Appl. Phys. Lett. 2002; 81: 445-447Crossref Scopus (21) Google Scholar). 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 (13Liu X.Y. Sato K. Furukawa Y. Nakajima K. Advances in Crystal Growth Research. Elsevier Science Publishers B.V., Amsterdam2001: 42-61Crossref Google Scholar) 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. In most cases, the formation of a new crystalline phase from the ambient phase proceeds via nucleation followed by growth (5Mutaftschiev B. Hurle D.T.J. Handbook of Cryst. Growth la Fundamentals: Thermodynamics and Kinetics. North-Holland, Amsterdam1993: 187-248Google Scholar). This implies that in the case of ice crystallization, nucleation is the initial and one of the most important steps toward creating ice. Without this step, ice will never occur in supercooled water. In the following discussion, we will examine ice nucleation based on the newly developed model (13Liu X.Y. Sato K. Furukawa Y. Nakajima K. Advances in Crystal Growth Research. Elsevier Science Publishers B.V., Amsterdam2001: 42-61Crossref Google Scholar). For ice crystallization, a positive thermodynamic driving force is required, which is defined as shown in Equation 1 (14Zettlemoyer A.C. Nucleation. Marcel Dekker, Inc., New York1969Google Scholar), ΔμkT=μf-μskT=(ΔHm/Tm)ΔTKT(Eq. 1) where μf and μs are the chemical potential of solute molecules in the fluid phase and in the solid phase, respectively; ΔH m denotes the enthalpy of melting per molecule; T m denotes the melting temperature; ΔT is supercooling (ΔT = 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 nucleation barrier under a given thermodynamic driving force Δμ/kT (14Zettlemoyer A.C. Nucleation. Marcel Dekker, Inc., New York1969Google Scholar). 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=4παβkink|(Rs)2N0|f''(m)[f(m)]1/2Bexp(-κf(m)/(ΔT)2)(Eq. 2) κ=16πγcf3Ω2/3kTSm2(Eq. 3) B=14πα2ΩγcfkT1/2(Eq. 4) where γcf denotes the specific interfacial free energy between the crystals and the mother phase; Ω is the volume of the growth units; S m is the entropy of melting per molecule; a is the dimension of a growth unit; Rs and N 0 are the radius and the density of foreign bodies, respectively; βkink denotes the kink kinetic coefficient; n 1 indicates the density of growth units in the system; and m is a function of the interfacial free energy difference between the different phases shown in Equation 5, m=(γsf-γsc)/γcf(Eq. 5) where γ is the interfacial free energy; subscripts f, c, and s denote the fluid phase, the cluster of the crystalline phase, and the foreign body, respectively; and Equations 6 and 7 show f(m)=1/4(2-3m+m3)(Eq. 6) f''(m)=1/2(1-m)(Eq. 7) Notice that f(m) (Equation 8) is a factor describing the lowering of the nucleation barrier ΔG * due to the occurrence of foreign bodies (or substrate). f(m)=ΔG*/ΔGhomo*(Eq. 8) In the equations, ΔG * is the actual nucleation barrier, and ΔG *homo is the homogeneous nucleation barrier; m (-1 ≤ m ≤ 1) can be approximately regarded as cosθ (θ is the contact angle between the nucleating phase and the substrate (15Liu X.Y. J. Phys. Chem. 2001; 105: 9949-9955Google Scholar, 16Liu X.Y. Appl. Phys. Lett. 2001; 79: 39-41Crossref Scopus (13) Google Scholar)). Both f(m) and f ″(m) change from 0 to 1, depending on the correlation and the structure match between the nucleating phase (ice) and the substrate (foreign bodies) (17Liu X.Y. Langmuir. 2000; 16: 7337-7345Crossref Scopus (45) Google Scholar, 18Liu X.Y. J. Phys. Chem. 1999; 111: 1628-1635Crossref Scopus (122) Google Scholar). When the interaction between the nucleating phase and the substrate is optimal, one has m → 1 and f(m) → 0(cf. Equation 6). On the other hand, if the interaction between the nucleating phase and the substrate is very poor, one has m → -1 and f(m) → 1(cf. Equations 6 and 7), meaning that the substrate exerts almost no influence on nucleation. Under such conditions, the nucleation of ice will become very difficult. As demonstrated by our experiments (see “Results and Discussion” and Table I), we cannot completely eliminate the influence of foreign bodies, such as dust particles, that will in most cases promote ice nucleation. Therefore, in order to inhibit ice nucleation any antifreeze agent should be able to disrupt the interaction between ice nuclei and foreign bodies.Table IThe freezing temperature (for a droplet of constant volume) is dependent on the number and size of dust particlesFilter pore size (nm)20010020Freezing temperature (°C)-53-58-65 Open table in a new tab Apart from surpassing the nucleation barrier, the nucleation of ice is also affected by the incorporation of H2O molecules onto the surface of ice nuclei at the kink sites (cf. Fig. 1a). The rate of kink kinetics is described by βkink. βkink is associated with ΔG kink‡, the energy barrier to be overcome in order to remove other molecules adsorbed at the kink sites (cf. Fig. 1c), and is given by Equation 9, βkinkαexp(-ΔGkink/kT)(Eq. 9) Obviously, the adsorption of additives on the surface of ice, in particular at the kink sites (cf. Fig. 1b), will enhance ΔGkink‡ by Δ(ΔGkink‡)=ΔGkink‡'-ΔGkink‡(cf.Fig.1c,ΔGkink‡' denotes the kink kinetics barrier attributed to the adsorption of impurities/additives on the surface). Consequently, the integration of H2O units into ice crystals will be significantly slowed down or even terminated due to a very low βkink (or high ΔGkink‡) (cf. Equation 9). Notice that one of the most common ways to examine the nucleation kinetics is to measure the induction time t nucl of nucleation at different supercoolings instead of a direct measurement of the nucleation rate (cf. Equation 2) (13Liu X.Y. Sato K. Furukawa Y. Nakajima K. Advances in Crystal Growth Research. Elsevier Science Publishers B.V., Amsterdam2001: 42-61Crossref Google Scholar). However, due to the crystallization sequence, what one then measures is the induction time t i for crystallization, which is defined as the mean time lapse for the appearance of the first crystal in the liquid. Actually, t i includes the nucleation induction time t nucl and the time t g necessarily required for ice crystals to grow from the critical size r c to an observable size. Because the free energy barrier of three-dimensional nucleation is much higher than that of growth, the growth of crystals then becomes in most cases much easier than nucleation. This is exactly the case for ice crystallization; once ice nuclei are formed, the rapid growth rate leads to instant freezing of the whole water droplet in less than 0.5 s. This implies that we have t g << t nucl, and then t i ≈ t nucl. According to the definition of the nucleation rate (Equation 10), one has J=1/(tnuclV)=1/(tiV)(Eq. 10) where V is the volume of the water droplet in the experiment. Combining Equations 2, 9, and 10 yields Equation 11, 1n(tnuclV)=kf(m)/(ΔT)2+ΔGkink‡/kT-1n{f''(m)[f(m)]1/2B'}(Eq. 11) where B′ = 4πa(Rs)2 N 0 CB, which remains constant under a given condition (C is constant). It follows from Equation 11 that for ice nucleation, the plot ln(τV) ∼ 1/(ΔT)2 will give rise to a straight line for a given f(m) (and f ″(m)) (cf. Fig. 3a) (f(m) can be utilized to derive the key parameters associated with the kinetics of ice nucleation. See the discussions in the following sections.). This equation will be applied in the following discussion to analyze the antifreeze effect of AFPs. In our experiments, AFP III (A/F Protein Canada) was used to examine the antifreeze effect. The protein is a compact, angular structure in which the overall fold composes numerous short β-strands and one turn of α-helix. To measure the induction time of ice nucleation, a newly developed experimental technique, the so-called double oil layer micro-sized crystallization technique (12Du N. Liu X.Y. Appl. Phys. Lett. 2002; 81: 445-447Crossref Scopus (21) Google Scholar), was employed. This technique can minimize the influence of the container and dust particles on ice nucleation and also allows us to examine the effect of antifreeze proteins on ice nucleation kinetics quantitatively. The experiments of micro-sized ice crystallization were performed in a micro-sized water droplet, which was suspended in two layers of immiscible oil in a circular quartz cell (12Du N. Liu X.Y. Appl. Phys. Lett. 2002; 81: 445-447Crossref Scopus (21) Google Scholar). First, the lower layer of oil (Silicon Oil AR 1000 from Fluka), which has a larger density than water, was injected into the quartz cell up to one-half of its volume. Second, a drop of pure water or AFP III solution was carefully injected onto the surface of the oil using a microsyringe. Third, an oil (200/500 cS fluid from Dow Corning) with a density smaller than water was injected to fill the cell, which covers the water droplet and the lower oil layer. A glass coverslip was then placed on the top of the cell to avoid evaporation. Due to the density differences, the water droplet is suspended between the two layers of immiscible oils. In order to minimize the effect of dust particles, before the water and oils were injected into the cell, they were filtered twice using 20-nm filters to remove big particles. The water used in the experiments was in a highly pure deionized form (18.2 megohms). The ice crystallization was controlled by a Linkam THMS 600 heating and freezing stage, which is capable of controlling the temperature within 0.1 °C from the range of -192 to +600 °C where the cell was mounted. Nucleation was observed using a polarized transmitted microscope (Olympus, BX60-F) to which a 3CCD color video camera (Panasonic, KY-F55BE) with an S-VHS video recorder (Panasonic AG-MD830) was attached (Fig. 2). Any ice crystal occurring in the drop could immediately be detected by a polarized microscope. Our experiments show that under normal crystallization conditions, it is almost impossible to eliminate the influence of dust particles. This is evidenced by the fact that the freezing temperature (for a constant droplet volume) decreases progressively as the pore size of the filters is decreased progressively from 200 to 100 to 20 nm (cf. Table I). Actually, in most cases, the term “homogenous ice crystallization,” to which most authors refer (19Liu X.Y. J. Phys. Chem. 2000; 112: 11550-11558Google Scholar), is a ice nucleation process by dust particles. This implies that the effect of the dust particles on ice crystallization is and should be into account in our as an ice nucleation foreign bodies lower the nucleation barrier by a factor f (cf. Equation In the case of nucleation the adsorption of additives on foreign particles will the interaction the match between the substrate (foreign and the nucleating This will then in m → 1 and f → for a given nucleation is constant under a given condition (see Equations and such a change can then be from the lowering of the and the increase of the of ln(τV) ∼ 1/(ΔT)2 Equation The from 0 to 1 in Fig. this if the adsorption of additives leads to a and an interfacial structure between the substrate and the nucleating phase, one then has m → -1 and f → This to an increase in the nucleation barrier (cf. Equation The effect can be from the increase in the f(m) of ln(τV) ∼ 1/(ΔT)2 and the of the line 0 to line in Fig. Apart from the AFP III also onto the surface of ice as shown in Fig. 1, and c, will enhance According to Equation 11, the in the of the ln(τV) ∼ 1/(ΔT)2 plot at a constant f(m) to the change in as by Fig. In our experiments, AFP III was to deionized water at and When AFP molecules on these (foreign (cf. Fig. the interaction and the structure match between foreign particles and the nucleating phase will be significantly As this adsorption on the substrate and the on nucleation can be from the ln(τV) ∼ 1/(ΔT)2 For the experiments, the of deionized water AFP III and with AFP III are given in Fig. The and from the for these are in Table It follows that ice nucleation is by AFP III induction at the two The two can be by the in the and the with the deionized water within the range of For the of from the and the of ln(τV) X.Y. Sato K. Furukawa Y. Nakajima K. Advances in Crystal Growth Research. Elsevier Science Publishers B.V., Amsterdam2001: 42-61Crossref Google effect of AFP III on the ice nucleation kinetics and the in the ln(τV) ∼ 1/(ΔT)2 deionized effect of AFP III on the interfacial effect and kink kinetic energy barrier for the nucleation of is the of the water = is the of the water, deionized is the of the ΔG water = is the of the water, deionized water Open table in a new tab us at Fig. and Table The adsorption of AFP III molecules on foreign particles out to the match between the nucleating ice and the dust particles. This can be from the of m (or which decreases from to and and the of the nucleation barrier by a factor and for a and respectively. The given in Fig. and Table show that AFP III will also onto the ice As in Fig. the adsorption will the incorporation of water molecules into the ice nuclei as to inhibit ice crystallization. This can be from the increases in the desolvation kink kinetics barrier ΔGkink‡ for solution and for Table that as in order to change the nucleation kinetics of ice, the AFP molecules should be able to onto the surface of dust particles or the surface of ice nuclei as by Fig. 5, a and This implies that in the experimental AFP III should be (13Liu X.Y. Sato K. Furukawa Y. Nakajima K. Advances in Crystal Growth Research. Elsevier Science Publishers B.V., Amsterdam2001: 42-61Crossref Google Scholar). Our latest that to AFP molecules will and on the surface of water. This is due to the fact that AFP III has both the and the When these molecules are in the water, the of the molecules will avoid water, the molecules will on the surface of water as to have a from water (cf. Fig. 6). According to the equation (see of & Inc., New Scholar) (Equation where is the a is the of AFP in the and is the surface of AFP in the the of AFP III on the surface leads to a lowering of the surface This is by the given in Fig. that the surface is by AFP III γ will then its In with the of AFP will the of This has been by our light and size Y. and Therefore, the point of the in Fig. 6 is defined as the critical of the to other based on the surface of we can also a of AFP on the surface of ice and on the surface of dust particles. The adsorbed AFP III molecules will the water a direct on ice crystallization X.Y. J. Chem. 2002; PubMed Scopus Google Scholar). It is that the of the AFP III molecules on the surface of the also the interfacial free energy γcf between the crystalline phase c and mother phase f to Based on Equation this change will lower and nucleation However, our that this effect is not with the other two inhibition caused by AFP In we have the inhibition effect of AFP III on ice nucleation at two different This effect is caused by the interaction between AFP III and ice as well as that between AFP III and foreign particles. The adsorption of AFP III on foreign particles will the match between the nucleating ice and dust particles, the adsorption on the surface of ice will inhibit the integration of water molecules into the ice two can be from the increase of the ice nucleation barrier and the desolvation kinetics This new understanding of the antifreeze mechanism of AFP has never been examined before and is provide us with in new antifreeze proteins. We are very much to for and critical of the We also Inc., for us the We also for discussions and for the of

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