*2.3. Infrared Spectroscopic Analysis*

In the IR spectra of the isolated collagenous materials, all characteristic absorption bands of amides I, II, and III, as well as amides A and B (Table 3), indicative of the secondary structure of the different materials [33], were observed. The amide A absorption band, associated with the hydrogen-bonded N-H stretching vibration [34], was observed at lower frequencies (3279–3294 cm−1), as opposed to the free N-H stretching vibration that appears in the range of 3400–3440 cm−1. This peak is shifted at lower frequencies than the ones reported for the collagen of the marine sponge *C. reniformis* [25] and the calf skin type I collagen, indicating that the N-H group is involved in extensive hydrogen bonding, which stabilizes the helical structure of collagen [34–36]. On the other hand, the amide B band, related to the asymmetrical stretch of CH2 and NH3 <sup>+</sup> [36,37] remained relatively constant (~2924 cm−1), pointing to the absence of major differences in the lysine content in all of the examined samples [37].

**Table 3.** IR spectra peak position and assignments for insoluble collagen before (InSC) and after (SF-InSC) spicules removal, intracellular collagen (ICC), and spongin-like collagen (SlC) isolated from *A. cannabina* and *S. carnosus*. For comparison reasons, the respective peaks for bovine collagen (BOC) [34] are also included.


The amide I band, mainly associated with the C=O stretching vibration coupled with the N-H bending vibration along the polypeptide backbone or with hydrogen bonding coupled with COO−, C-N stretching, and CCN deformation, is the most intense band in proteins and, therefore, the most sensitive and useful marker for the analysis of the secondary structure of proteins with IR spectroscopy [38]. Normally resonating in the range of 1600–1700 cm−<sup>1</sup> [39,40], bands around 1630 cm−<sup>1</sup> indicate imide residues, and bands around 1660 and 1675 cm−<sup>1</sup> are assigned to intermolecular crosslinks and b-turns, respectively [38]. In our samples, the amide I peaks are shifted to lower frequencies, indicative of higher hydrogen bonding potential [37], less intermolecular cross-linking, and decreased molecular order [39]. The lowest frequencies were observed in both InSCs, before and after treatment with HF, whereas the frequencies increased in the cases of the SlC and ICC samples, concomitantly to the molecular order increase. Additionally, the amide II band, associated with the N-H bending vibration coupled with the C-N stretching vibration, was also shifted to lower frequencies (1527–1547 cm−1), indicative of the involvement of the N-H group in hydrogen bonding [35]. Finally, the amide III band, attributed to the C-N stretching vibration in combination with the N-H deformation, is considered as the collagen fingerprint because it is accredited to the characteristic collagen repeating tripeptide (Gly-X-Y) [38]. Furthermore, in the IR spectra of the isolated collagenous materials, additional bands at about 1030 cm−<sup>1</sup> appeared, mostly attributed to C-O vibrations due to the presence of carbohydrates [25,41]. In the case of the SF-InSCs, in the recorded IR spectra (Figure 2) a less intense peak at ~1030 cm−<sup>1</sup> appeared, possibly corresponding also to the Si-O-Si asymmetric bond stretching vibration, known to absorb in the range of 1030–1100 cm<sup>−</sup>1.

The absorption intensity ratio between the amide III band (1238 cm−1) and the band at approximately 1450 cm−<sup>1</sup> was 0.88 and 0.89 for the ICCs of *A. cannabina* and *S. carnosus*, respectively, indicating that the triple helix has been adequately preserved during the isolation procedure [34,36,37,40]. Generally, a ratio of approximately 1 indicates that the triple helical structure of collagen is intact [42]. In the case of the InSCs, this ratio is low for both sponges (~0.7), indicating that the triple-helical

structure might be slightly affected during the extraction procedure. It was shown earlier that this ratio might be lower when the collagen triple helix is affected by cleavage of telopeptides through pepsin digestion [36]. Moreover, upon treatment for the removal of spicules, the absorption intensity ratio between amide III band and the band approximately at 1450 cm−<sup>1</sup> increased to 0.96 and 0.94 for *A. cannabina* and *S. carnosus*, respectively, demonstrating the removal of other impurities.

**Figure 2.** IR spectra of insoluble collagen before (InSC; upper) and after (SF-InSC; lower) spicule removal isolated from *A. cannabina* (**A**) and *S. carnosus* (**B**).
