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Nest foams of Leptodactylus pentadactylus are very stable and do not shear easily. Testing showed that the foam does not dissolve in any of the used solvents. Vortexing of the foam with 100 µl ddH₂O did not dissolve the material. Moreover, the foam could not be dissolved by vortexing (up to 4 min) with any other tested solvent: neither with 30% (v/v), 50% (v/v), 70% (v/v) nor with 96% EtOH homogenous solutions could be achieved. Also, increasing the ratio of organic solvent by addition of MeOH and DMSO did not lead to solution of the foam. The foam mass could not be dissolved in 100% MeOH nor in 100%

DMSO, respectively. Summing up, it could neither be dissolved in ddH2O nor in the organic solvents MeOH or DMSO by vortexing.

With the sonicator the best suspension was seen after sonication for 70 min with ddH2O in 1.5 ml tubes. With DMSO and the pure sample without solvent, a viscous suspension was also achieved, however, still with foam fragments that were not homogenised, while on the other hand, with EtOH or MeOH, fragments of the foam (possibly proteins) were precipitated after 70 min. Moreover, heat development that was observed during sonication intensified the precipitating effect of the solvents MeOH and EtOH. Heating of the material led to temperatures of approx. 40°C on the outer tube side, however, not showing the inner temperature. Because of the long duration of the process (70 min), efficient cooling of the substances was not possible, while heat development during homogenisation of the foam with the Bead Beater could efficiently be prevented by cooling the tubes on ice before and immediately after homogenisation.

Moreover, the use of the Bead Beater resulted in the generation of clear suspensions containing no residual fragments, while grinding of the frozen material with mortar and pestle did not result in homogenous suspensions. Despite long and carefully grinding of the frozen material, foam fragments were still contained in the suspension, making the following filtration with the 0.2 µm filter impossible.

The use of the three different homogenisation methods – sonicator, mortar and pestle and the Bead Beater – resulted in the same protein pattern of the extracts as can be seen in the SDS-PAGE in Figure 11. Thus, after considering the disadavantages and quality of homogenisation, the Bead Beater was used as method of choice for all further approaches.

Additionally, the quality of homogenisation with the Bead Beater did not depend on the time of homogenisation (2 x 20 sec, 1 x 20 sec, 2 x 10 sec or 1 x 10 sec). The protein pattern was the same, and the protein concentration did not show any significant difference between the different durations of homogenisation. However, as with the homogenisation for 2 x 10 sec the fewest heat development, with cooling in between, was achieved, it became the method of choice.

FIGURE 11: Protein content of the foam mass after N₂-method, Bead Beater or sonication.

Crude extracts after grinding with mortar and pestle (N₂), homogenisation with the Bead Beater (BB) or with the sonicator (S) (ddH₂O as solvent). S and S_F: Comparison of the protein composition of the crude extract before (left) and after filtration (right) with a 0.2 µm filter. No loss of protein bands can be observed, only loss of intensity of the protein bands in the sample before and after filtration. Coomassie Blue-stained SDS-PAGE (12% T). Molecular masses are indicated in kDa. Peqgold Protein Marker V (10–250 kDa) (M).

Figure 12 shows that between the singular steps of homogenisation, no difference in the protein composition can be detected: the untreated extract, whether after use of the Bead Beater, sonication or frozen grinding, showed no difference to that after centrifugation in the ultracentrifuge for 30 min (16000 x g), to that with two times 30 min centrifugation or to that with additional filtration. Thus, no distinctive proteins (i.e. a distinctive band, in a distinctive kDa-range) were lost in any of the steps. Only a loss of intensity was seen between the individual steps, e.g. after centrifugation and filtration (Figure 11).

M N2 BB SM M SF

250 130 95 72 55

36

28 kDa

3.2 PROTEIN CHARACTERISATION

Analysis of the protein concentration and results of SDS-PAGE showed that Leptodactylus pentadactylus foam nests are rich in proteins with sizes ranging from 180 to approx. 20 kDa. The pattern can be clearly distinguished from that of Polypedates leucomystax not showing proteins in the 20 kDa range, for example, where L. pentadactylus is giving strong bands in SDS-PAGE (Figure 12). Protein concentrations of the Bead Beater crude extract (centrifuged once for 30 min at 16,000 x g) of Leptodactylus pentadactylus foam were between 1.5 and 2.2 mg/ml and between 1 and 1.5 mg/ml for the additionally filtered extract, when using ~0.5 g foam material and 1100 µl liquid without further dilution prior to filtration, thus, giving a protein concentration of ~3.5–5.5 mg protein/g foam, depending on the sample. As the eggs were removed before homogenisation of the extract, the proteins of the foam did not come from disrupted eggs. Moreover, as with each approach the protein concentrations varied, the components seemed to be not regularly distributed in the foam nest.

Moreover, the proteins found in the foam nests showed stability for several weeks, without degradation or loss of protein bands in SDS-PAGE (tested after one, and three weeks) during storage at -20°C, and thawing for several times in between (Coomassie stained gels not shown).

FIGURE 12: Protein composition of the foam nests of L. pentadactylus (L.p.) and Polypedates leucomystax (P.l.). L. pentadactylus and P. leucomystax show different proteins in their foam nests. No difference in the protein composition between the homogenised (H), additionally centrifuged (30 min, 16000 x g) (C) and the centrifuged and filtered extract (CF) can be seen. 10 µg protein per lane. Coomassie Blue-stained SDS-PAGE (12% T). Molecular masses are indicated in kDa. Peqgold Protein Marker V (10–250 kDa) (M).