**2. Results**

### *2.1. Protein Pattern of Collagens*

The protein pattern between ASC and PSC was compared by using the SDS-PAGE method. ASC and PSC had similar molecular patterns, having α1, α2, β and γ chains, which confirm that both collagens belong to the type I category (Figure 1).

By comparing the standard molecular weight marker and standard analysis, the molecular weights of α1, α2, and β were about 135, 120 and 250 kDa, respectively. As expected, increasing collagen concentration from 0.5 mg/mL to 1 mg/mL had increased the bandwidth. Specifically, the pepsin treatment increased α and β band thickness compared to ASC, which claims the efficiency of pepsin in collagen extraction. As evidence, the final yield of PSC was higher (17.78% ± 0.64%) than ASC (15.46% ± 0.42%) (data are shown as mean ± standard deviation, *n* = 3, *p* < 0.05). However, the intact structure of collagen was more stable in ASC than in PSC, since the pepsin treatment had triggered the strong hydrolysis of collagen; as a result, many smaller peptide fractions were generated in PSC compared to ASC. From the gel image, it was clear that the higher molecular weight of collagen β and γ could be disintegrated into smaller fractions in PSC, which was not obvious in ASC.

**Figure 1.** SDS-PAGE patterns of ASC and PSC from the skin of blacktip reef shark on 7.5% gel. Lane 1: protein markers; lane 2: BS-ASC (0.5 mg/mL); lane 3: BS-ASC (1 mg/mL); lane 4: BS-PSC (0.5 mg/mL); lane 5: BS-PSC (1 mg/mL).

### *2.2. Amino Acid Composition of Collagens*

The total amino acid residues present in collagens were determined to understand the pattern of amino acid composition in ASC and PSC. In general, the pattern of the amino acid profile was similar between ASC and PSC. For instance, both collagens had a higher content of glycine as a major amino acid, only the percentage varies in between collagens, having higher content in PSC (293 residue/1000 residues) than ASC (283 residue/ 1000 residues) (Table 1). The second major amino acid was hydroxyproline (195 and 202 amino acid residue/1000 residues for ASC and PSC, respectively), followed by alanine, proline and glutamic acid, respectively.

**Table 1.** Amino acid composition of acid-soluble and pepsin soluble collagens from blacktip reef shark skin (residues/1000 residues).



**Table 1.** *Cont.*

All values are shown as mean ± standard deviation (*n* = 3, a and b in the same row indicate significant differences, *p* < 0.05).

There was no sulfur-containing amino acid observed in either collagen. From the results, it was clear that the hydrophobic amino acids such as glycine, proline, alanine, valine, leucine, isoleucine, and phenylalanine were more dominant than hydrophilic amino acids such as serine and threonine in both collagens. Compared to ASC, the content of amino acid was in general higher in PSC, which also supports the higher yield and liberation of peptide fragments in PSC (Figure 1). The content of imino acids such as proline and hydroxyproline in PSC (311.68 residues/1000 residues) was much higher than in ASC (288.14 residues/1000 residues).

### *2.3. Maximum Absorption of Collagens*

This experiment was performed to investigate the two characteristic features of collagens: (1) to identify the maximum absorption (nm) of collagen and (2) to verify the contamination of non-collagenous protein presence in extracted collagens. The results showed that the collagens had maximum absorbance at 234 nm, respectively (Figure 2A). The absorption intensity was more in PSC at 230 nm than in ASC. There was no absorbance at 280 nm that usually corresponds to a sulfur-containing amino acid, cysteine, which is normally absent in collagen. From the above finding, it was further confirmed that the extracted collagen was pure.

**Figure 2.** (**A**) UV–Vis spectrum of ASC and PSC made from the skin of the blacktip reef shark. (**B**) Fourier transform infrared spectra of BS-ASC and BS-PSC. Secondary structure analysis of ASC (**C**) and PSC ( **D**) through the deconvolution of amide I band (between 1600 and 1700 cm<sup>−</sup>1).

### *2.4. Secondary Structure Analysis*

The structural changes of collagens were investigated by using FTIR spectra. FTIR transmission spectra of ASC and PSC were shown in Figure 2B and Table 2. Both collagens had general transmission patterns in major amide bands such as amide A, amide B, amide I, amide II and amide III, respectively. The maximum transmission wave numbers of amide A and amide B (which represent N-H stretch and CH2 asymmetric stretch, respectively), were at 3298 and 2926 cm<sup>−</sup>1, and 3298 and 2930 cm<sup>−</sup><sup>1</sup> for ASC and PSC, respectively. ASC and PSC had maximum transmission for amide I at 1639 cm<sup>−</sup>1, amide II at 1542 and 1546 cm<sup>−</sup><sup>1</sup> and amide III at 1237 cm<sup>−</sup>1, respectively. There were not many differences observed in amide I, II and III bands between ASC and PSC. The IR absorption ratios of two collagens were 1.11 (ASC), and 1.03 (PSC), respectively.

**Table 2.** FTIR spectra peak position and assignments for blacktip reef shark acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC).


In addition, the secondary structural pattern of collagen was determined by using PeakFit Version 4.12 software and the Gaussian peak fitting algorithm. The data showed that ASC and PSC contained 27.72 and 26.32% of α helix, 22.24 and 23.35% of β-sheet, 21.34 and 22.08% of β-turn, 14.11 and 14.13% of triple helix and 14.59 and 14.12% of the random coil, respectively (Figure 2C).

### *2.5. Solubility against pH and Salt*

The functional behavior of collagen is generally investigated by determining pH and salt solubilities. For this intention, collagen was solubilized with different salt concentrations (ranging from 0–6%) and pH (ranging from 1 to 11), respectively. In general, increasing salt concentration from 0–6% decreased the solubility of collagen from 100% to 70% (Figure 3A). Both collagens reach the exponential phase (optimum) at 3% NaCl; after that, a sudden decrease in the solubility of collagens was observed. On the other hand, a typical sigmoid curve was observed in a collagen solubility pattern against pH (Figure 3B). Similar to salt, both collagens had no significant changes in solubility against pH, and the maximum solubility was obtained at pH 4.

**Figure 3.** Relative solubility (%) at different pH values (**A**) and NaCl concentrations (**B**) of ASC and PSC isolated from blacktip reef shark skin extracted using different methods.

### *2.6. Peptide Mapping*

Peptide mapping was used to understand the hydrolysis pattern of collagen by proteolytic enzymes. Based on the earlier protocol, the collagen was hydrolyzed by trypsin with two different pHs at 2.5 and 7.8 for 3 h and 3 min. Due to the higher activity of trypsin at neutral or slightly basic pH (6–7.5), the reaction was carried-out at pH 7.8 for a shorter time. As shown in Figure 4A, the peptide map was not so obvious in all the collagens hydrolyzed by trypsin at pH 2.5 and 7.8, except ASC at pH 2.5. The hydrolytic pattern of ASC was significantly different from PSC, which had no peptide bands, unlike ASC (Figure 4A). The unseen peptide bands of collagens were visible as the gel concentration increased from 7.5 to 12% (Figure 4B). The data showed that proteolytic hydrolysis of ASC released the low MW peptide fragments ranging from 200 to 5 kDa MW at 2.5 pH, whereas the peptide fragments from 55 to 23 kDa MW were observed at pH 7.8 for ASC (Figure 4B). In general, all the collagens had a major peptide fragment at 23 kDa, both collagens had similar peptide fragments ranging from 55 to 23 kDa at pH 7.8, and no higher molecular component was seen in any of the groups.

**Figure 4.** Peptide maps of ASC and PSC from the skin of blacktip reef shark digested by trypsin using 7.5% ( **A**) and 12% (**B**) gels. Lanes 1 and 6: protein markers; lanes 2 and 4: ASC; lanes 3 and 5: PSC. Lanes 2 and 3: peptide fragments of collagens with trypsin digestion at pH 2.5; lanes 4 and 5: peptide fragments of collagens with trypsin digestion at pH 7.8.

### *2.7. Microstructural Analysis*

The microstructural features of collagens were determined by using SEM at different magnifications (100, 50 and 10 μm). It was seen that both collagens had a fibrillary and more condensed network-like structure (Figure 5). The distribution of fiber was more homogeneous and alveolate porous. There were more interconnected filaments with varying thicknesses in both collagens. As seen, there were not many changes in the microstructural characteristics of either collagen. The fibril bundle structure of collagen was further confirmed by the AFM experiment. Similar to SEM, the fibril bundle structure was denser and more condensed in nature in both ASC and PSC (Figure 6). However, the AFM image of PSC showed longer filaments with loosely connected intra-structure than ASC, where the filaments were shorter and more connected to each other.

**Figure 5.** Scanning electron microscopic structure of ASC (**A**) and PSC (**B**) from blacktip reef shark skin. SEM image with different magnifications: (**a**) (100 μm), (**b**) (50 μm), (**c**) (10 μm).

**Figure 6.** High-resolution AFM image of blacktip reef shark skin collagen fiber bundle. The ASC (**A**) and PSC (**B**) fibril display the natural structure.
