*2.3. Effect of Pepsin Digestion*

Samples were treated with pepsin in order to evaluate whether some proteins or the collagen telopeptides were responsible for the gel consistency of the obtained materials and to confirm the collagenous materials of the bands observed in the SDS-PAGE.

We found that there were no qualitative macroscopic differences between untreated extracts and the ones treated with pepsin; on the other hand, the supernatant obtained after a rapid centrifugation, was significantly different. Supernatants of undigested samples formed a nearly 90◦ meniscus while the digestion caused a change in the meniscus angle that was >90◦ (Figure 3).

**Figure 3.** Effects of pepsin digestion on choanosome (Ch) collagen extract. (**a**) Images of the materials soon after the digestion/control treatment; and (**b**) meniscus details of the supernatants obtained after the removal of the insoluble component. Tubes legend: A = Ec digested; B = Ec control; C = Ch digested; D = Ch control.

SDS-PAGE analysis was carried out on all the samples, including the supernatants obtained after the removal of the insoluble parts. In terms of composition, the material obtained from Ch was less complex than that extracted from Ec (Figure 4, compared undigested Ec and Ch).

**Figure 4.** 7.5% (**a**,**b**) and 15% (**c**,**d**) sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) stained with R-250 Coomassie (**a**,**c**) and alcian blue (**b**,**d**) of digested and undigested samples. Lanes 1/5 = digested Ec; 2/6 = undigested Ec; 3/7 = digested Ch; 4/8 = undigested Ch. Lanes 1–4 = pellet; 5–8 = supernatant. Pellets obtained after centrifugation were resuspended in 800 μL of loading buffer and freeze-dried supernatants in 80 μL; 40 μL of the resulting dispersions were added in each well. Arrowhead = presumptive collagen; broken line rectangle = possible glycan.

Moreover, the SDS-PAGE profile of collagens treated with pepsin revealed a significant difference with the untreated ones. In particular, several bands in the higher (>100 kDa) molecular weight appeared in the Ec (compare lane 12 s—tained with Coomassie of Figure 4) suggesting that pepsin treatment was able to solubilize different proteins. Both Ec and Ch-solubilized collagens had a lower molecular weight when treated with pepsin suggesting that the enzyme was able to remove collagens telopeptides. Our data suggests that a slight difference between Ec and Ch collagens does exist, while both seems to be homotrimeric. Pepsin treatment seems to increase the solubility and the Coomassie sensitivity of other kind of collagens present in the ectosome. Considering the evident effect of pepsin treatment on the supernatant (Figure 4) it is likely to be that some pepsin-sensitive molecules not stained with the employed dyes were present in the samples.

The combined analysis of Coomassie/alcian blue staining (Figure 4) revealed the presence of a glycan that, since it was not found in the supernatant, should be stably associated with the material itself.

The materials obtained from either Ch or Ec were completely dissolved after few hours when treated with papain at 50 ◦C. After the precipitation of GAGs and the following freeze-drying step, quite a large amount of material was obtained. About 3 mg per mL of starting materials (Ec 14.4 mg/mL and Ch 10.6 mg/mL) were recovered; however, when run trough Tris/borate/EDTA polyacrylamide gel electrophoresis (TBE-PAGE), only a small band was detected (Figure 5).

Neither of the bands were susceptible to an extensive incubation with Chondroitinase ABC (data not shown), and thus the nature of this molecules could not be assessed.

**Figure 5.** 10% Tris/borate/EDTA polyacrylamide gel electrophoresis (TBE-PAGE) of glycosaminoglycans (GAG) extracted from choanosome (Ch; lane 2) and ectosome (Ec; lane 3), after staining with alcian blue. A similar quantity (170 μg) of shark chondroitin sulfate was loaded into lane 1 as reference.
