*2.2. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analyses of T. flavidus Collagen*

The electrophoretic pattern of *T. flavidus* collagen was similar to the authentic standard of rat type I collagen (Figure 1), indicating an intact collagen profile after electrodialysis. It consisted of four major protein components with molecular weights of 122, 130, 250, and 310 kDa. The first two components had mass values close to α1 and α2 subunits of rat type I collagen. The ratio of 122/130 kDa components (~1:2) was on a level similar to rat collagen standard and consistent with a previous report on type I collagen extract of *T. rubripes* skin [23]. Therefore, 122 and 130 kDa proteins were identified as α2 and α1 subunits of type I collagen, respectively. On the other hand, the last two protein components (250 and 310 kDa) were tentatively identified as β subunit (dimer) and γ subunit (trimer) of type I collagen, respectively. These two proteins had counterparts with similar molecular weights in rat type I collagen standard (Figure 1) as well as other fish collagen [24]. They were possibly formed via intermolecular and/or intramolecular cross-linking of collagen subunits [25]. As starvation is believed to stimulate collagen cross-linking of fish, the difference in β and γ subunit contents between *T. flavidus* and *T. rubripes* collagen were probably due to variations in both species and feeding conditions [23]. In addition, quantification of stained protein bands showed that α1, α2, β, and γ subunits contributed to 96.1 ± 1.3% of total *T. flavidus* collagen. This indicated that extraction by salting-out electrodialysis was able to produce pure type I collagen for further application.

**Figure 1.** SDS-PAGE of molecular weight standard (lane M), authentic type I collagen standard from rat tail (lane A), and *T. flavidus* collagen extract (lane B).

#### *2.3. Amino Acid Composition of T. flavidus Collagen*

*T. flavidus* collagen demonstrated the characteristic amino acid composition of type I collagen. Gly was the most abundant residue of the collagen, accounting for a quarter of the total amino acid components (Table 2, 268 ± 0.08 residues/1000 amino acid residues). This observation is consistent with the common understanding that Gly content is the highest among all amino acid residues as type I collagen is featured with repeating Gly–Pro–X and/or Gly–X–Hyp sequence (X can be any amino acid residues other than Gly, Pro, and Hyp) [26].

Imino acids (Pro and Hyp) had the second highest amino acid content among all residues of *T. flavidus* collagen (246 ± 0.04 residues/1000 amino acid residues), consistent with a previous study on *T. rubripes* skin collagen (170 residues/1000 amino acid residues) [2,23]. They have been reported to decrease the entropic cost of collagen folding by preorganizing individual poly-Pro II chain [27]. They can also stabilize collagen triple helices via interchain hydrogen bond through hydroxyl groups [28]. The content of imino acids is therefore an important factor modulating collagen thermal stability [27,29]. The imino acid content of *T. flavidus* collagen was higher than many fishes, including big eye snapper, grass carp, and tiger pufferfish (167–195 residues/1000 amino acid residues), but similar to those from their porcine counterparts (220 residues/1000 amino acid residues) [16,24,30]. This suggests that *T. flavidus* collagen might have nutritional values similar to those of common mammalian collagen and could therefore potentially be used as an alternative source of gelatin.


**Table 2.** Amino acid compositions of *T. flavidus* collagen (residues/1000 amino acid residues).

#### *2.4. Spectrophotometric Characterization*

The spectrophotometric characterization was in agreement with SDS-PAGE and amino acid composition analyses, further confirming that electrodialysis maintained the physicochemical integrity of type I collagen from *T. flavidus* skin. In Figure 2A, it can be seen that *T. flavidus* collagen and authentic type I collagen standard both have bell shape UV spectra, with the maximum absorption wavelength around 234 nm. This strong absorption can be attributed to peptide bond absorptions by n → π\* transitions within C=O, COOH, and CONH2 groups of collagen peptide chains [25]. Similar to type I collagen of rat tail, *T. flavidus* collagen also absorbed weakly around 250 and 280 nm. The inability to absorb at higher UV regions is related to the deficiency of Tyr and Phe in *T. flavidus* collagen (<30 residues/1000 residues, Table 2) because Tyr and Phe are the major chromophores responsible for absorption at 251 and 276 nm [31]. This phenomenon suggests a possible deprivation of protease-labile

telopeptides from type I collagen [31] during isolation and purification with salting-out electrodialysis extraction. In addition, circular dichroism (CD) analysis was in line with UV results, showing identical spectra between *T. flavidus* collagen and rat type I collagen standard (Figure 2B). Both samples had spectra with a positive amplitude at 221 nm and a negative amplitude at 197 nm. It is also in agreement with recent research on scale collagen of pacific saury [32].

Consistent with UV analysis, *T. flavidus* collagen demonstrated a typical Fourier transform infrared (FTIR) spectrum of type I collagen. Five characteristic peaks were identified in both *T. flavidus* collagen and rat type I collagen: amide A, B, I, II, and III (Figure 2C). The wavenumber of amide A (3311 cm<sup>−</sup>1) was lower than that of free N–H stretching vibration (3400–3440 cm−1) [33]. This red-shift is in agreement with the extensive distributions of N–H(Gly···O=C(X) hydrogen bonds among *T. flavidus* collagen helices during the formation of triple-helical structures [34]. Amide B reflected asymmetrical CH2 stretching within collagen peptides [35], and it had a wavenumber (2926 cm−1) similar to theoretical values. As an indicator of C=O stretching vibration [33], amide I of *T. flavidus* collagen (1645 cm<sup>−</sup>1) was modestly red-shifted from the calculated value (~1660 cm−1) toward lower wavenumber [33]. This was again a by-product of intercollagen cross-linking by N–H(Gly)···O=C(X) hydrogen bonds [34]. N–H bending coupled with C–N stretching vibration actively contributed to amide II formation, inducing FTIR absorption at 1550–1600 cm−<sup>1</sup> [35]. The amide II wavenumber of *T. flavidus* collagen (1551 cm<sup>−</sup>1) was at the lower region of this range, further confirming the influence of interhelical hydrogen bonds. In addition, C–H stretching and N–H bending vibrations were detected in *T. flavidus* collagen [36] with evidence on amide III (1242 cm<sup>−</sup>1) absorption.

**Figure 2.** Spectrophotometric characterization of *T. flavidus* collagen extract and type I collagen of rat tail. (**A**) UV spectra; (**B**) circular dichroism (CD) spectra, and (**C**), FTIR spectra.
