UV-Visible spectrophotometry
UV-visible spectroscopy data of crude sample 1 and 2 showed absorbance between 280 and 300 nm as shown in Fig. 1 (Anbuchezian et al. 2018)
Thin Layer Chromatography: Thin layer chromatography for the two crude sample was performed using the mobile phase Butanol: Ethanol: Water (5:3:2). The plate was spotted with sample and immersed in the solvent tank. After the mobile phase reached the solvent front, it was sprayed with 0.2% ninhydrin and heated at 110º C for 5 min to identify the pink spots that indicate the existence of amino acids and peptides. R f value was calculated for each bands as shown in Fig. 2a,b (Anbuchezian et al. 2018; Ogbole et al. 2020)
Fourier Transform Infrared Spectroscopy: The FTIR spectrum of the crude samples were characterized by Fourier transform infrared spectroscopy shown in Fig. 3. Strong signals were seen to occur at 3290.56 cm−1 and 1635.64 cm−1 for the crude sample-1 and peaks were identified at 3309.85 cm−1 and 1635.64 cm−1 for crude sample 2. Amide I bands range from 1620-1700 cm−1 in which proteins attributed to β-sheet structure it represents N-H bending with a contribution from C–N stretching vibrations. (Anbuchezian et al. 2018). Amide II bands range from 3300-3200 cm−1 that represents N-H stretching.
Ultra performance liquid chromatography: The crude sample 1 and 2 was analyzed on C18, 1.7 µm, 2.1x100 mm column eluted with crude samples at a flow rate of 0.5 ml/min (Xie et al.2009). The separation was monitored at 215 nm. Fig. 4 a,b demonstrates the UPLC chromatogram of the crude peptide from hemolymph.
SDS-PAGE
SDS-PAGE and Coomassie brilliant blue staining analysis of the crude samples (Fig. 5) depicted a variety of proteins with molecular weights ranging from 3.5 to 205 kDa (Hou et al. 2010).
Nuclear Magnetic Resonance
1H NMR spectra are suitable for proofing the presence or absence of individual amino acids from the crude sample. The wavelength range 7.3 ppm confirms the presence of peptide in the NMR spectra of Scylla serrata hemolymph which was absent in the crude 2 and also contained amino acids (3.25 to 3.8ppm) (e.g., Isoleucine, leucine, valine, threonine, alanine, proline, glutamine, lysine, Glycine. (Sumalatha et al. 2016). Figure 6 shows the NMR pattern of Crude Sample 1(1H NMR (400 MHz, D2O) δ 7.32 (dd, J = 23.2, 13.8 Hz, 1H), 7.13 (s, 1H), 6.84 (s, 1H), 6.21 (d, J = 64.9 Hz, 1H), 5.95 (s, 1H), 5.85–5.56 (m, 1H), 5.53–5.27 (m, 2H), 5.15 (dt, J = 29.0, 22.6 Hz, 8H), 5.01–3.43 (m, 1239H), 3.32 (d, J = 20.8 Hz, 1H), 3.19 (s, 1H), 2.96 (s, 1H), 2.66 (s, 1H), 2.39–1.97 (m, 1H), 1.85 (s, 1H), 1.65 (s, 1H), 1.40 (s, 1H), 1.25 (d, J = 6.1 Hz, 1H), 1.15–0.97 (m, 1H), 1.08–0.52 (m, 1H)) and Crude Sample 2 (1H NMR (400 MHz, D2O) δ 5.85–5.54 (m, 1H), 5.52 (s, 1H), 5.44 (s, 1H), 5.25 (t, J = 17.3 Hz, 1H), 5.20–5.08 (m, 1H), 5.02 (d, J = 7.1 Hz, 1H), 4.93 (d, J = 8.9 Hz, 1H), 4.83 (d, J = 8.6 Hz, 1H), 4.97–4.44 (m, 1H), 4.48 (dd, J = 22.0, 12.3 Hz, 1H), 4.35–4.21 (m, 1H), 4.20 (d, J = 10.7 Hz, 1H), 4.13–4.00 (m, 1H), 3.99 (s, 1H), 3.94 (s, 1H), 3.85 (s, 1H), 3.79 (s, 1H), 3.70 (s, 1H), 3.68–3.58 (m, 1H), 3.53 (s, 1H), 3.32 (s, 1H), 3.23 (s, 1H), 3.14 (s, 1H), 2.69 (s, 1H), 2.56 (s, 1H), 2.39 (s, 1H), 2.19 (s, 1H)). Since the crude sample 2 showed absence of peptide groups, further analysis and purification are proceeded only with crude sample 1.
Column chromatography
Crude sample 1 showed strong signals of peptide presence in NMR on comparison with that of crude sample 2, hence further analysis was carried out for crude sample 1. Column chromatography was carried out using sephadex G-25 to elute smaller molecular peptides. The column was equilibrated with PBS and also used as mobile phase. (Hu et al. 2020). The fractions was collected at the flow rate of 1ml/min. Totally 12 fractions was collected and the concentration of protein was quantified by Bradford assay. 6 of the 12 fractions had good protein concentration as shown in Table 1 and UV-spectra of the protein are depicted in Fig. 7. Further these fractions were tested for antioxidant activity.
Table 1
represents the concentration of fractions
S.No
|
Fractions
|
Concentration (µg/ml)
|
1.
|
3
|
0.6356
|
2.
|
5
|
0.115
|
3.
|
6
|
-0.02192
|
4.
|
7
|
-0.02973
|
5.
|
8
|
-0.005
|
6.
|
9
|
-0.0569
|
Antioxidant Activity (Dpph)
Many hypotheses state that oxidative stress is one of the hallmarks associated with AD pathology however the clinical trials attempting to investigate the action of anti-oxidants on AD progression have been rendered unsatisfactory to date. Crude and partially purified protein samples from the crab Scylla serrata haemolymph were evaluated for its capacity to scavenge free radicals using DPPH assay. To check radical scavenging activity, protein samples were treated with DPPH reagent in methanol. The results were analysed in UV-spectrophotometer at 517 nm and the intensity of colour change was measured (Sumalatha et al. 2016).
The radical scavenging activity of crude hemolymph showed 54.03% at 100 µg/ml concentration. Among the 6 fractions, fraction 3 (42.4%) showed higher radical scavenging activity. Further, the radical scavenging activity for the fractions were significantly similar to that of crude sample 1. However, further purification of the fractions is required to identify the peptide and its properties.