2.1.1. Collagens Electrophoretic Patterns

The electrophoretic patterns of collagens from smooth-hound skin (ShS) performed under denaturing condition are presented in Figure 1 and showed that there are no differences between the ASC and pepsin solubilised collagen (PSC).

As native collagen molecule is constituted of three polypeptide chains (α-chains) organised in a triple-helix, the denaturising sodium dodecyl sulfate (SDS) break the H-bonds yielding peptides [32]. In both types of ShS-collagen, the α (α1, α2) and their cross-linked dimer β-chains are the major components with low content of γ-chain. Thus, the electrophoresis mobility and subunit composition may suggest that ASC and PSC isolated from ShS should most likely be classified as type I collagen. The SDS-PAGE, revealed two bands of chains α1 and α2 with a molecular weight of about 101 kDa and 83 kDa respectively; however with different intensities (α1 intensity higher than α2 by approximately ratio 2:1). Such results suggest that α1 is formed by 2 subunits as collagen type I characterised by the existence of 2 identical subunits of α1 and one of α2 [33].

Additionally, high molecular weight (MW) components, β-chains were clearly detected in both ShS-ASC and PSC with a mean molecular weight of 226 kDa. Such results are in conformity with several findings reported for other elasmobranches skins of brownbanded bamboo shark [34], skate [21] and shark [35].

**Figure 1.** Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of (1): acid soluble collagen (ASC) and (2): pepsin soluble collagen (PSC) from hound-smooth skin M: high molecular weight marker (KDa).

#### 2.1.2. Peptide Mapping

The ShS-ASC and PSC were markedly digested by Lysyl endopeptidase which cleaves peptide bonds at the carboxyl side of lysyl residues [36]. Generally, band intensity of major components α, β and γ of ShS-ASC and PSC decreased after digestion and degraded into smaller peptides with molecular weight ranging from 100 to 13 kDa (Figure 2).

**Figure 2.** Peptide mapping of acid soluble collagen ASC and pepsin soluble collagen PSC digested by *Lysyl endopeptidase* with different hydrolysis time 1: ASC, 2: ASC-5 min, 3: ASC-25 min, 4: PSC, 5: PSC-5 min, 6: PSC-25 min from hound smooth skin, M: high molecular weight marker (KDa).

When comparing the effect of hydrolysis duration, an enhanced enzymatic hydrolysis was found with an incubation time of 25 min for ShS-ASC and PSC. This was evidenced by the appearance of higher number of peptides bands with low molecular weight (Figure 2, lines 3 and 6); and a decreased band's intensity for PSC collagen. This might be caused by the pepsin action on the telopeptide region inducing its cleavage and thus facilitating the changes in configuration, which may favour the hydrolysis by lysyl endopeptidase.

#### 2.1.3. Viscosity Measurement

The temperature of denaturation (Td) of ShS-ASC and PSC, referred as the temperature at which the variation in viscosity is half completed, was calculated from a plot of temperature-induced variation in viscosity (Figure 3).

**Figure 3.** Change in fractional viscosity with temperature of acid soluble collagen (ShS-ASC) and pepsin soluble collagen (ShS-PSC) from the skin of hound smooth.

The viscosities of both collagens were higher at temperature ranging from 15–20 ◦C then decreased with increased heating up to 25 ◦C.

The Td's of ASC and PSC were 26.68 ◦C and 26.66 ◦C respectively. The similarity in the denaturation temperature may be related to the resemblance of the major peaks wavelength of ASC and PSC [37]. Such values were comparable to Td reported for collagen from other marine species such as the Japanese sea bass (26.5 ◦C) [23], edible jellyfish exumbrella (26.0 ◦C) [14], chub mackerel (25.6 ◦C), bullhead shark (25.0 ◦C) [23] but lower than porcine skin collagen (37.0 ◦C) [11]. However, the Td's of both ASC and ASC smooth hound skin were higher than those of Spanish marckel skin (15.12 and 14.66 ◦C, respectively) [38].
