Interfacial adsorption of antifreeze proteins: a neutron reflection study

Abstract

Interfacial adsorption from two antifreeze proteins (AFP) from ocean pout (Macrozoarces americanus, type III AFP, AFP III, or maAFP) and spruce budworm (Choristoneura fumiferana, isoform 501, or cfAFP) were studied by neutron reflection. Hydrophilic silicon oxide was used as model substrate to facilitate the solid/liquid interfacial measurement so that the structural features from AFP adsorption can be examined. All adsorbed layers from AFP III could be modeled into uniform layer distribution assuming that the protein molecules were adsorbed with their ice-binding surface in direct contact with the SiO(2) substrate. The layer thickness of 32 A was consistent with the height of the molecule in its crystalline form. With the concentration decreasing from 2 mg/ml to 0.01 mg/ml, the volume fraction of the protein packed in the monolayer decreased steadily from 0.4 to 0.1, consistent with the concentration-dependent inhibition of ice growth observed over the range. In comparison, insect cfAFP showed stronger adsorption over the same concentration range. Below 0.1 mg/ml, uniform layers were formed. But above 1 mg/ml, the adsorbed layers were characterized by a dense middle layer and two outer diffuse layers, with a total thickness around 100 A. The structural transition indicated the responsive changes of conformational orientation to increasing surface packing density. As the higher interfacial adsorption of cfAFP was strongly correlated with the greater thermal hysteresis of spruce budworm, our results indicated the important relation between protein adsorption and antifreeze activity.

FIGURE 1

Molecular structure of AFP III (1HG7 from Antson et al. (3), left) and cf-AFP (1M8N from Leinala et al. (9), right).

FIGURE 2

CD spectrum obtained from 0.2 mg/ml AFP III at 22°C dissolved in deionized water (solid line) is compared with the profile obtained by Ananthanarayanan et al. (25) measured from an AFP fraction separated from ion-exchange chromatography (SP-A) and dissolved in 0.1 M NH₄HCO₃ (pH 8.0) buffer at 0°C. The close similarity in the amplitude and position of the two bands shows that both protein samples retained their native conformations.

FIGURE 3

Plots of log (neutron reflectivity) measured at the silica/D₂O interface with AFP III (1HZ7) in the solution of 0.01 (□), 0.1 (+), 0.5 (Δ), and 2mg/ml (○). The protein solutions were obtained by directly dissolving AFP lyophilized powder in pure D₂O. The resulted solution pH was generally constant at around pH 7.8. The reflectivity profile from the bare silica/D₂O interface is also given for comparison (dashed line). The solid lines are the best fits with parameters given in Table 1.

FIGURE 4

Plots of log (neutron reflectivity) measured at the silica/D₂O interface with cfAFP-501 (1M8N) in the solution of 0.01(□), 0.1 (+), 1.0 (Δ), and 2.0mg/mL (○). The protein solutions were obtained by directly dissolving AFP lyophilized powder in pure D₂O. The resulted solution pH was generally constant at around pH 7.8. The reflectivity profile from the bare silica/D₂O interface is also given for comparison (dashed line). The solid lines are the best fits with the parameters given in Table 2.

FIGURE 5

Plots of 1/Γ (surface excess) versus 1/C (bulk concentration) for fish AFP III (○) and insect cfAFP-501 (□). The lines were drawn to guide the eye.

FIGURE 6

Thermal hysteresis measured against fish AFP III and insect cfAFP concentrations.

FIGURE 7

Schematic representation of the adsorption from fish AFP III (1GH7) (a) and insect cfAFP (1M8N) (b) in a concentration-dependent manner.