*2.4. SDS-PAGE*

The resulting native and acetylated PPC were analysed by SDS-PAGE (Figure 1). Electrophoretic analyses revealed that the PPC contained a predominant protein of about 35 kDa·MW. As we can see on the electropherogram, proteins of about 18–22 kDa·MW accompanied this main protein. According to previous research studies, cucurbitin, the major storage protein in pumpkin seed, is a hexamer with a molecular size of about 325 kDa, consisting of two major bands (MW d 22 kDa) corresponding to acidic and basic polypeptides [38–41]. The vast majority of cucurbitins are salt-soluble globulins with a sedimentation index of about 11–12S [42]. They are accompanied by albumin with a sedimentation index of 2S and a mass of about 12.5 kDa, composed of two subunits: a smaller one, about 4.8 kDa and above, and another with a mass of 7.9 kDa [43,44]. Moreover, cucurbitins have proven antimicrobial, antitumour and anti-inflammatory effects [45]. Analysis revealed that in the acetylated pumpkin protein the content of the heaviest protein (35 kDa) decreased in line with increasing concentrations of modifying reagent. The addition of 0.4–2.0 mL acetic anhydride per g of protein resulted in removal of a protein of about 18 kDa·MW and, at the same time, resulted in an increase in the participation of a protein of about 20 kDa·MW in the composition of acetylated pumpkin protein preparations. Additionally, the proteins of about 35 W and 18 kDa·MW were more susceptible than the protein of about 22 kDa·MW to the presence of the modifying reagent. Furthermore, the intensity of the protein patterns decreased in line with increasing concentrations of modifying reagent, which confirms the reduction in protein content in acetylated pumpkin protein preparations. These results are not in line with those of Zhao et al. [16], who found that acetylation of oat protein isolate with different anhydride-to-protein ratios left the molecular weight of the proteins almost unchanged, as compared to native protein isolate.

**Figure 1.** SDS-PAGE of native and acetylated PPC. PPC—pumpkin protein concentrate; 0.4, 1.0, 2.0—pumpkin protein preparations after acetylation conducted with different concentrations of acetic anhydride (mL/g); MW—molecular weight marker prepared at laboratory (18.4 kDa (βlactoglobulin), 42, 45 kDa (ovalbumin), 66 kDa (bovine serum albumin).
