Adsorption of frog foam nest proteins at the air-water interface

Abstract

The surfactant properties of aqueous protein mixtures (ranaspumins) from the foam nests of the tropical frog Physalaemus pustulosus have been investigated by surface tension, two-photon excitation fluorescence microscopy, specular neutron reflection, and related biophysical techniques. Ranaspumins lower the surface tension of water more rapidly and more effectively than standard globular proteins under similar conditions. Two-photon excitation fluorescence microscopy of nest foams treated with fluorescent marker (anilinonaphthalene sulfonic acid) shows partitioning of hydrophobic proteins into the air-water interface and allows imaging of the foam structure. The surface excess of the adsorbed protein layers, determined from measurements of neutron reflection from the surface of water utilizing H(2)O/D(2)O mixtures, shows a persistent increase of surface excess and layer thickness with bulk concentration. At the highest concentration studied (0.5 mg ml(-1)), the adsorbed layer is characterized by three distinct regions: a protruding top layer of approximately 20 angstroms, a middle layer of approximately 30 angstroms, and a more diffuse submerged layer projecting some 25 angstroms into bulk solution. This suggests a model involving self-assembly of protein aggregates at the air-water interface in which initial foam formation is facilitated by specific surfactant proteins in the mixture, further stabilized by subsequent aggregation and cross-linking into a multilayer surface complex.

FIGURE 1

(a) Fluorescence emission spectrum of diluted foam fluid (0.1 mg ml⁻¹ protein) in water (λ_exc = 290 nm) with emission maximum (λ_em) ∼336 nm, characteristic of tryptophan side chains in a nonpolar environment. Water baseline shown for comparison. (b) Far-UV circular dichroism (arbitrary units, raw data, and smoothed fit) spectrum of foam fluid (0.5 mg ml⁻¹ protein, 0.1-mm pathlength), typical of a protein mixture containing predominantly β-secondary structure.

FIGURE 2

Comparison of surfactant properties of dilute frog foam proteins. (Upper panel) Side-on images illustrating the wetting of a hydrophobic surface (Nescofilm) by 50-μl drops of foam nest fluid, compared to water, and 1% w/v sodium dodecyl sulfate (SDS) solution. (Lower panel) Surface-tension data for serial dilutions of frog foam fluid in water, compared to lysozyme (in acetate buffer, pH 4.5) and BSA (in phosphate buffer, pH 7). (Note: the small differences in surface tension at very low concentrations reflect the different buffers used for each sample. Lysozyme was measured in acidic buffer to avoid the aggregation known to occur with this protein at neutral pH and above.)

FIGURE 3

Variation of surface tension with time for aqueous solutions of foam proteins with concentrations (mg ml⁻¹): 1 × 10⁻³ (○), 7 × 10⁻³ (•), 5 × 10⁻² (▵), and 0.5 (×). The continuous lines are drawn to guide the eye.

FIGURE 4

Fluorescence emission spectra of ANS (λ_exc = 390 nm) in nest foam and dilute foam fluid, compared to the parent spectrum in water alone. (The structure of ANS is inset.)

FIGURE 5

Fluorescence volume imaging of ANS-treated foam: montage of 30 planar images, in 3-μm steps, starting from point of contact with the microscope slide (bottom right) progressing sequentially to a total depth of ∼90 μm (top right). Each frame is ∼250 μm². λ_exc = 810 nm (200-fs pulses); λ_obs = 440–500 nm, z-series: 3-μm slices ; 2.8-μm pixels. Trapped air bubbles appear dark in these representations.

FIGURE 6

Examples of two-photon excitation microscope images of bubbles in nest foam treated with ANS. The increased fluorescence intensities show congregation of fluorescently labeled protein(s) at the air-water interface of individual bubbles within the foam.

FIGURE 7

The neutron reflectivity profiles from NRW solutions with protein concentration (mg ml⁻¹) of 1 × 10⁻³ (○), 7 × 10⁻³ (•), 5 × 10⁻² (▵), and 0.5 (×). The continuous lines are the best fits with structural parameters listed in Table 1.

FIGURE 8

Plots of protein partial structure factors versus momentum transfer (κ) at bulk protein concentrations (mg ml⁻¹) of 1 × 10⁻³ (○), 7 × 10⁻³ (•), 5 × 10⁻² (▵), and 0.5 (×). The continuous lines are the best fits calculated using a single uniform layer model with structural parameters given in Table 1.

FIGURE 9

Neutron reflectivity profiles measured in D₂O solutions with bulk protein concentrations (mg ml⁻¹) of 1 × 10⁻³ (○), 7 × 10⁻³ (•), 5 × 10⁻² (▵), and 0.5 (×). The continuous lines represent the best fits using multilayer models and structural parameters listed in Table 4.