Enhanced Antifreeze Effect of Antifreeze Protein on Ice Nucleation by Electrolyte Ning Du and Xiang Y. Liu* Biophysics and Micro/nanostructures Laboratory, Department of Physics, National UniVersity of Singapore, 2 Science DriVe 3, Singapore 117542
CRYSTAL GROWTH & DESIGN 2008 VOL. 8, NO. 9 3290–3294
ReceiVed January 31, 2008; ReVised Manuscript ReceiVed April 17, 2008
ABSTRACT: It is found that ice nucleation can be effectively suppressed by antifreeze protein at certain concentrations of electrolyte, because of the neutralization of the surface charge of the antifreeze protein molecules by the counterions. It follows from our ice crystallization experiment that the enhancement of antifreeze efficiency is based on the increase of the kink kinetics barrier of surface integration of water molecules and the disruption of the interfacial match between water and foreign particles by antifreeze protein molecules in the presence of electrolyte. This effect is attributed to the optimal packing of the antifreeze protein molecules on the surface of the ice nuclei as well as of the foreign particles when electrolyte ions are added. This work could provide a model for the study of the specific role of an electrolyte on the antifreeze efficiency of antifreeze proteins. The new understanding suggests another way to increase the antifreeze efficiency of the antifreeze proteins and provides us with a new insight into the antifreeze mechanism of antifreeze proteins on ice crystallization. Introduction The discovery of antifreeze proteins (AFPs) and glycoproteins (AFGPs) three decades ago1 yielded the explanation for the survival of teleost fish at temperatures below the colligative freezing point.2 Analysis of the blood plasma of these fish showed that salts and some ions in the body fluids are only responsible for -0.6 to -0.8 °C of the observed freezing point depression (-1.7 to -2 °C);3 the remainder of the protective effect was attributed to the presence of AFPs and AFGPs. At the present time, four different types of AFPs have been identified from the plasma of sera of several species of teleost fish. Type I AFPs are reported from the winter flounder,4 which are alanine-rich, amphipathic R-helices. Type II AFPs are globular proteins with mixed secondary structure from the sea raven, smelt and Atlantic herring.5 Type III AFPs are from the ocean pout,6 which are made of short β-strands and one helix turn that give it a unique flat-faced globular fold. The type IV AFP from the longhorn sculpin is thought to be a helix-bundle protein.7 It is found that fish blood contains different kinds of salts, primarily made of sodium, potassium, calcium, and chloride. Fish can absorb calcium directly from the water or from food, and the plasma calcium concentration in the teleost fish is about 2-7 mM.8 According to our recent work,9,10 AFPs can inhibit the ice nucleation process (an initial and essential step followed by the growth process during ice crystallization) by adsorbing onto the surface of ice and foreign particles. This indicates that the antifreeze activity of antifreeze proteins is related to their adsorption behavior at interfaces, which is mainly decided by their surface activity. The protein molecules that have high surface activity tend to adsorb strongly onto the interfaces, such as air-liquid or solid-liquid interfaces, and will reduce the surface energy of the system. The main parameters that cause the surface activity to vary are molecular size, hydrophobicity, protein flexibility, and surface charge. Among these factors, the surface charge of a protein is one of the parameters most readily * Corresponding author. Tel.: 65-65162812. Fax: 65-67776126. E-mail:
[email protected].
affected by external conditions, such as the pH of the solution, and the type and concentration of the electrolyte ions occurring in the solutions. AFP III is a negatively charged molecule of which the PI is about 6.11 Experimentally, adding an electrolyte may reduce the “net charge” of the protein molecules and result in the minimization of the electrostatic repulsion between the charged adsorbed molecules. In this paper, considering the physiological relevance of electrolytes in fish and their screening effect on the surface charge of protein molecules, we examined the effect of Ca(NO3)2 on the antifreeze mechanism of AFP III based on the novel model.9 We choose Ca(NO3)2 here because the Ca2+ ion is the most abundant divalent ion in the fish body. Being a divalent ion, the Ca2+ ion is more effective in neutralizing the surface charge of protein molecules than monovalent ions. The model applied in this work allowed us to quantitatively analyze the adsorption of AFP III molecules on the surface of the ice nuclei and foreign particles in the presence of Ca(NO3)2. In addition, we examined the effect of Ca(NO3)2 on the interaction between AFP III molecules and the conformation change of AFP III in solution by using multiple techniques, including surface tension, fluorescence spectroscopy and zeta potential measurement. Experimental Procedures Ice Nucleation Kinetics Measurement. The experiments on ice nucleation were carried out by employing the so-called double oil layer microsized microscopic crystallization technique.12 This technology allows us to minimize the influence of the container on ice nucleation, and to examine the ice nucleation kinetics under well controlled conditions. The experiments of microsized ice crystallization were performed in a microsized water droplet, which was suspended between two layers of immiscible oil (Silicon Oil AR 1000 from Fluka for lower oil and 200/500cS Fluid from Dow Corning for upper oil) in a circular quartz cell. A glass coverslip was then placed on top of the cell to prevent evaporation. Because of the density difference, the water droplet is suspended between the two layers of immiscible oils. To minimize the effect of dust particles, before the water and oils were injected into the cell, they were filtered twice with 20 nm filters to remove big particles. The water used in the experiments was in a highly pure deionized form (18.2 MΩ). The ice crystallization was controlled by a Linkam THMS600 Heating and Freezing stage, which is capable of controlling the temperature where the cell was mounted within 0.1 °C
10.1021/cg800118z CCC: $40.75 2008 American Chemical Society Published on Web 07/23/2008
Effect of Antifreeze Protein on Ice Nucleation
Crystal Growth & Design, Vol. 8, No. 9, 2008 3291
the ice nucleation kinetics, we examine the correlation between the nucleation induction time τ, i.e., the time required for the first nucleus to appear in the drop of water with a given volume V, and the supercooling ∆T. According to the generic nucleation model, the kinetics of nucleation can be taken into account by examining the correlation between the induction time of ice nucleation and supercooling as follows17
ln(τV) ) κf(m, R ′ )/(T∆T2) - ln{f ′′ (m, R ′ )[f(m, R ′ )]1⁄2BN0} (1)
Figure 1. Correlation between ln(τV) and 1/(T∆Τ2) for (a) DI water filtrated by a 20 nm filter, (b) 3 mg/mL AFP III solution, (c) 3 mg/mL AFP III solution with 10 mM Ca(NO3)2, and (d) 3 mg/mL AFP III solution with 100 mM after adding Ca(NO3)2. Point A is defined as the “effective nucleation temperature”. with the range -192 to + 600 °C. Nucleation was observed by using a polarized transmission microscope (Olympus, BX60-F) to which a 3 CCD color video camera (Panasonic, KY-F55BE) with an S-VHS video recorder (Panasonic AG-MD830) was attached. Any ice crystal occurring in the drop could immediately be detected by a polarized microscope. Surface Tension Measurement. Because of their amphiphilic nature, AFP III molecules will accumulate and self-assemble on the surface of water in aqueous solution. This accumulation of AFP III on the water surface leads to a lowering of the surface tension γ.13 Note that when the surface is fully occupied (saturated) by AFP III molecules, γ will then reach its minimum. In analogy with surfactants, the further addition of AFP will cause the aggregation of AFP. The concentration corresponding to the lowest γ is defined as the critical aggregation concentration (CAC) of AFP. Similar analysis is used in the study of the correlation between protein aggregation and surface tension in refs. 14-16. According to our previous work, the CAC for AFP III is 2.5 mg/mL.10 To study the influence of an electrolyte on the adsorption behavior of AFP III molecules at interfaces in a more transparent way, we add Ca(NO3)2 to the solution of which the surface has already been saturated by AFP III molecules (concentration g2.5 mg/ml). In our experiment, we fixed the AFP III concentration at 3 mg/mL. The surface tension measurements were performed for 16 AFP III solutions at different Ca(NO3)2 concentrations using a single fiber tensiometer (model K14, Kruss). In these measurements, the Dynamic Wilhelmy method, which is a universally applied method especially suited to check the surface tension over long time intervals, is employed. This method uses a vertical plate of known perimeter attached to a balance to detect the force change due to wetting when the plate touches the solution surface. The surface tension can then be calculated from the observed forces. For AFP III solutions, all our measurements take 3 h to reach their equilibrium on the surface as well as in the bulk solution. Zeta Potential Measurement. To check the influence of Ca(NO3)2 on the effective surface charge of the AFP III molecules, and hence the repulsion forces between them, we measured the zeta potentials of the AFP III solutions with Ca(NO3)2 by the zetasizer (Malvern, UK). Measurement of Intrinsic Fluorescence. Fluorescence spectra were recorded on a Cary Eclipse Fluorescence spectrophotometer. The excitation was set at 279nm and the emission was recorded from 300 to 500 nm at a 1 nm intervals. Slit widths were 5 nm for both excitation and emission. The spectra for AFP III (3 mg/ml) were obtained and recordings were repeated subsequent to the addition of Ca(NO3)2 stock solution.
Results and Discussion The antifreeze effect of antifreeze protein can be examined in terms of nucleation experiment as described in the experiment part. The nucleation process can be regarded as a kinetic process for ice nuclei to overcome the so-called nucleation barrier ∆G* under a given supercooling∆T (∆T ) Tm - T, T and Tm are the actual and the melting temperatures, respectively).17 To explore
rc ) 2Ωγcf/∆T∆Sm
(2)
2 κ ) 16πγ3cfΩ2/3k∆Sm
(3)
R ′ ) Rs/rc
(4)
and
where rc is the radius of a critical nucleus and ∆Sm denotes the ice melting entropy per molecule; k is the Boltzmann constant; B′ is a constant under a given condition, Ω is the volume of a growth unit, Rs and N0 are the radius and the number density of the foreign particles, respectively; γcf denotes the interfacial free energy between water and ice. In eq 1, the interfacial correlation function17
R ′ -m 1 1 1 - mR′ 2 1 3 f(m, R ′ ) ) + + R′ 2 - 3 + 2 2 w 2 w R ′ -m 2 R ′ -m 3 + mR′2 -1 w 2 w
(
)
(
[ ( )] (
w ) [1 + R′2 - 2R ′ m]1⁄2
)
)
(5) (6)
is important, as it describes the lowering of the nucleation barrier ∆G* because of the occurrence of foreign particles,
f(m, R ′ ) ) ∆G * /∆G/homo
(7)
* where ∆Ghomo denotes the homogeneous nucleation barrier. f(m,R′) varies from 0 to 1, depending on R′ and m. m (-1e m e 1) describes the structural match and interaction between the nucleating phase (ice) and the foreign particles, and can be approximated by cos θ (θ is the contact angle between the nucleating phase and the substrate). (f′′(m,R′) is a function similar to f(m,R′)). For an optimal interaction and structural match between the nucleating phase and the substrate m f 1 and f(m,R′) f 0, meaning that the nucleation barrier is completely eliminated due to the occurrence of foreign particles. When f(m,R′) ) 1 (extremely poor structure match/interaction between ice and foreign particles), the nucleation barrier is the * highest (∆Ghomo ) under the given conditions even when the foreign particles are still present, in which case the foreign particles do not play any role in lowering the nucleation barrier. As shown in Figure 1, ln(τV) is plotted vs 1/(T∆T2) for the following systems: DI water filtrated by a 20 nm filter (a); 3 mg/mL AFP III solution (b); 3 mg/mL AFP III solution with 10 mM Ca(NO3)2 (c); and 3 mg/mL AFP III solution with 100 mM Ca(NO3)2 (d). As shown in curve a at low ∆T (high 1/(T∆T)2), one has a straight line segment (line 2) with the largest slope within the measurable range of ∆T, whereas at high ∆T (low 1/(T∆T2)), one has a straight line segment (line 1) with a much smaller slope. The two straight line segments meet at point A. Beyond this point the nucleation barrier rises * abruptly to the highest level (∆G* ≈ ∆Ghomo , f(m,R′) ) 1) so that ice nucleation becomes extremely difficult after point A in the region of the reversed homogeneous-like nucleation.9 Therefore, here we define the temperature at point A as the
3292 Crystal Growth & Design, Vol. 8, No. 9, 2008
Du and Liu
Table 1. Measured Effective Nucleation Temperature for Different Systems
DI water (20 nm filter) AFP III (3 mg/mL) AFP III (3 mg/mL) dissolved in 10 mM Ca(NO3)2 AFP III (3 mg/mL) dissolved in 100 mM Ca(NO3)2
equilibrium melting temperature Tm (°C)
effective nucleation temperature T (°C)
∆T ) Tm T (°C)
0 0 -0.5 ( 0.1
-38.7 ( 0.1 -39.9 ( 0.3 -41.0 ( 0.2
38.7 ( 0.1 39.9 ( 0.3 40.5 ( 0.2
-0.8 ( 0.1
-41.2 ( 0.2
40.4 ( 0.2
“effective nucleation temperature”. Notice that the three other curves (b-d) share similar characteristics and their “effective nucleation temperatures” can be measured as well. The values of the “effective nucleation temperature” for various systems are listed in Table 1. According to our measurements, Ca(NO3)2 can decrease the equilibrium melting point of water from 0 °C to -0.5 and -0.8 °C at the concentration of 10 mM and 100 mM respectively. In Table 1, ∆T is the decreased nucleation temperature which has been eliminated the effect of melting point depression by Ca(NO3)2 thermodynamically. It is found that ∆T increased by 1.2 °C upon adding AFP III (from 38.7 to 39.9 °C), and the antifreeze efficiency is enhanced in the presence of Ca(NO3)2, as indicated from the further drop of the nucleation temperature (∆T increased from 39.9 to 40.5 °C in Table 1). This suggests that besides decreasing the equilibrium melting point of water, Ca(NO3)2 could change the adsorption properties of AFP III molecules and thus enhance the antifreeze efficiency of AFP III. Details on how the adsorption behavior of AFP III molecules is changed by Ca(NO3)2 at interfaces are discussed below. The surface activity of AFP III, which determines its adsorption properties at interfaces, can be obtained from the surface tension measurement. In our experiment, we examine the adsorption behavior of AFP III molecules at the air-water interface, and establish the correlation between the concentration of the electrolyte and the surface assembly of AFP III molecules. When surface active molecules, such as AFP III, are adsorbed at the air-water interface, the surface tension is reduced. We can express the lower surface tension of the interface caused by the presence of adsorbed molecules as18
Π ) γ0 - γ 0
(8)
where γ is the surface tension with no amphiphile present and γ its value with adsorbed amphiphile. For lyophilic solutes, such as ionic salts, they are repelled away from the air-water interface and so raise the surface tension (line 1 in Figure 2a). By applying eq 8, we can eliminate the effect of Ca(NO3)2 on the surface tension. The surface pressure of AFP III was obtained in the presence of Ca(NO3)2 (line 3 in Figure 2a). As our previous result showed,10 the surface of 3 mg/ml AFP III solution is already fully occupied by AFP III molecules. The increase of surface pressure from 30.4 to 31.8 mN/m (Point A in Figure 2a) upon adding Ca(NO3)2 (1 × 10-3 mM) to AFP III solution suggests that the AFP III molecules adsorbed on the solution surface pack more compactly with each other than would be the case without addition of Ca(NO3)2. This close packing may be explained by the screening effect of electrolyte on the thickness of electric double layer of AFP III molecule (Figure 2b). The charged protein molecules interact through a screened potential whose range is the Debye length (the thickness of electric double layer),19λd ) κ-1. κ is the Debye-Huckel parameter and is proportional to c1/2,20 where
Figure 2. (a) Surface activity of AFP III molecules in the presence of Ca(NO3)2 measured in the surface tension experiment (T ) 23 °C). The accumulation of AFP III on the solution surface leads to the lowering of the surface tension γ, i.e., γ will decrease with the increase of the adsorption area by AFP III molecules. Surface pressure of AFP III in the presence of Ca(NO3)2 (line 3) is calculated with Π ) γ0-γ, where γ0 is the surface tension of Ca(NO3)2 alone (line 1) and γ is the surface tension of AFP III dissolved in a Ca(NO3)2 solution (line 2). The surface pressure for a 3 mg/ml AFP III solution is 30.4 mN/m. (b) Illustration of the adsorption of AFP III molecules at air-water interface under different Ca(NO3)2 concentrations.
c is the electrolyte concentration. According to the relation λd ∞ c-1/2, the thickness of electric double layer of AFP III decreased when the Ca(NO3)2 concentration increased. This means as more Ca2+ was attracted to the surfaces of the AFP III molecules, the repulsion between protein molecules became even weaker as their surface charge was further neutralized. As a consequence, the protein molecules may pack even closer to each other at air-water interface and the surface pressure increased. It was noticed that the surface pressure increased to a stable value at 10 mM Ca(NO3)2 (Point B in Figure 2). This suggests that the electric double layer is reduced to the lowest value and the protein molecules are most compactly packed on the solution surface at this concentration. To verify this screening effect caused by Ca2+, the zeta potential was measured for AFP III at different Ca2+ concentrations. The zeta potential reflects the effective charge on the particles and it is the potential energy of attraction and repulsion between charged particles.21 Figure 3 shows how the zeta potential of the AFP III molecule varies with Ca(NO3)2 concentration. As the Ca(NO3)2 concentration increases, the negative zeta potential decreased to zero. This suggests that when more positive ions (Ca2+ ions) are attracted to the negatively charged protein surface, the AFP III molecules become neutralized. In this way, the electrostatic repulsion between the protein molecules is minimized to its lowest value when the zeta potential of AFP III reaches zero at 10 mM
Effect of Antifreeze Protein on Ice Nucleation
Figure 3. Plot of the zeta potential of the AFP III solution (3 mg/mL) against the concentration of Ca(NO3)2.
Crystal Growth & Design, Vol. 8, No. 9, 2008 3293
Figure 5. Effect of AFP III and Ca(NO3)2 on the ice nucleation kinetics and the corresponding shift in the ln(τV) ∼1/(T∆Τ2) plot. Table 2. Effect of AFP III on the Interfacial Effect Parameter and Kink Kinetic Energy Barrier for the Nucleation of Ice f
m
DI watera (20 nm filter) AFP III (3 mg/mL) AFP III (3 mg/mL) dissolved in 10 mM Ca(NO3)2 AFP III (3 mg/mL) dissolved in 100 mM Ca(NO3)2
0.168 0.250 0.283
0.48 0.35 0.30
v 17.7 v 22.8
0.266
0.32
v 20.9
a
Figure 4. Fluorescence emission spectrum of AFP III solution upon adding Ca(NO3)2 at 22 ( 1 °C.
Ca(NO3)2 concentration. It follows from Figure 2a that the minimal surface tension of the AFP III solution was reached at the same Ca(NO3)2 concentration. This indicates that the electrolyte can minimize the interactions between the protein molecules by neutralizing their effective surface charge and thus allow them to pack closely to one another when they are adsorbed at the interfaces. Therefore, similarly, the adsorption of AFP III molecules at the interfaces of water-foreign particle and water-ice could block the adding of water molecules effectively thus the antifreeze efficiency is enhanced when the interactions between AFP III is minimized upon adding electrolyte. High salt concentration may induce a conformation change of protein molecules. In our experiment, fluorescence spectroscopy measurements were carried out to examine the effects of Ca(NO3)2 on the conformation change of AFP III. The results are shown in Figure 4. For AFP III solutions with low Ca(NO3)2 concentration (
