## 3 Results and Discussions
### Evidence for Intermolecular Disulphide Bond Formation  
#### 3.2.1 The Impact of Extraction on Hempseed Protein Subunits  
As discussed in the introduction, previous research focused on maximising protein yield has encountered an HPI that exhibits poor functionality [4]. As aforementioned, it is highly likely that the intermolecular disulphide bond formation may occur as a side reaction during HPI extraction and that this could explain the poor solubility of the resultant protein. However, direct evidence of intermolecular SS bonds and information about the rate at which they are formed during the extraction process are lacking. Hence, the effect of extraction on hempseed proteins was determined for the hempseed system using native and reducing SDS-PAGE of samples subjected to AE-IEP extraction at pH = 10.0 and 37 \({}^{\circ}\)C for different times (0 min, 5 min, and 120 min) (Figure 4A). Non-reducing SDS-PAGE provides information on the native protein-protein interactions, while reducing PAGE cleaves the SS bonds to provide complementary information. Therefore, monitoring SDS-PAGE profiles of the protein solutions at both conditions during the alkaline extraction can reveal the protein conjugation due to intermolecular disulphide bond formation.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Extraction pH** & \begin{tabular}{c} **Extraction Yield** \\ **(\%)** \\ \end{tabular} & \
\begin{tabular}{c} **Protein Recovery** \\ **Yield (\%)** \\ \end{tabular} &
\begin{tabular}{c} **Free -SH Group in HPI** \\ **(\(\mathbf{\mathbf{\mathbf{\mathbf{\mathbf{\mathbf{\mathbf{\mathbf{\mathbf{\mathbf{\mathbf{\mathbf{\mathbf{\mathbf{\mathbf{\mathbf{\Prior to alkaline extraction (t = 0 min), the SDS-PAGE profiles revealed the typical banding of edestin monomer located at 50.0 kDa (Lane 2), from which acid and base subunits at 34.0 and 18.0-20.0 kDa were dissociated under the reducing conditions (Lane 3). The results are in good agreement with other studies [15; 35]. Across the entire extraction process, reducing SDS-PAGE (Lane 3, 5, and 7) showed a similar band profile. In contrast, native SDS-PAGE profiles (Lane 2, 4, and 6) showed a progressive decline of the band intensity of the edestin monomer as the protein extraction progressed further. Instead, high MW components are visible at higher MW areas (high-intensity smearing, 50.0-250.0 kDa in this study) and at the top of the gel (>250.0 kDa). This, in combination with the absence of smearing at the high MW area under reducing conditions, provides clear evidence that edestin oligomers are indeed caused by intermolecular SS bonding during extraction. In addition, there is smearing between 10 kDa and 50 kDa, indicating that  
Figure 4: (**A**) SDS-PAGE profiles of hempseed protein after different extraction times (0, 5, and 120 min) with (reducing, R) or without (non-reducing, NR) the addition of \(\beta\)-ME. The protein was extracted under pH = 10.0 at 37 °C. Different bands were identified and marked on the profile image, including a large extent of smearing (>50.0 kDa), edestin (50.0 kDa), edestin AS (34.0 kDa), edestin BS (18.0 and 20.0 kDa), and albumin (10.0–15.0 kDa). (**B**) The relative band intensities of hempseed protein subunits (50 and >250 kDa) at 0, 5, and 120 min extraction times, which were analysed by Gel-doc software. Means denoted by a different letter indicate significant differences (\(p<0.05\)).  
the low MW albumin might also form disulphide bonds. These findings are also evident in the quantified band intensities (Figure 4B). In summary, high MW protein aggregates were confirmed to be formed during extraction due to disulphide bonding, which can explain the decline in the functionality of HPI.