Influence of pH on protein extraction from Sus scrofa pancreas

Abstract

The porcine pancreas contains various enzymes, structural, regulatory, secretory, receptor and other biologically active substances that ensure both the functioning of the organ and its biological role in the organism. The aim of this work was to study the influence of pH changes in 0.9% sodium chloride solution used as an extractant on the efficiency of bioactive protein isolation from the porcine pancreas. The extraction was carried out with the 0.9% NaCl, 0.9% NaCl pH=4 and 0.9% NaCl pH= 8.5 with a stirring speed of 400 rpm for 150 min at 4 ºC; the ratio of pancreas: extractant was 1:5, the supernatant was separated by centrifugation. The protein concentration was measured by a biuret reaction on a semi-automatic biochemical analyzer Biochem SA. The proteomic composition of extracts and native pancreas was evaluated by 10% SDS-PAGE according to Laemmli method in the “VE10” chamber. Digital images of electrophoregrams were obtained using a Bio-5000 Plus scanner, edited in a graphic editor and analyzed using ImageJ software. When determining the intensity of protein fractions, it was noted that the use of 0.9% NaCl contributed to a greater yield of proteins with molecular weights of 200 kDa, 150 kDa, 69 kDa, 52 kDa and 33 kDa into the extractant; a pH shift to the acidic area stimulated the yield of fractions with molecular weights of 130 kDa, 50 kDa, 49 kDa, 45 kDa, 40 kDa, 30 kDa and 27kDa, and a pH shift to the alkaline area — only 47 kDa and 42 kDa. Most pancreas proteolytic enzymes have a molecular weight in a range of 34–23kDa, excepting the immature form of carboxypeptidases with MW 45–47kDa. The greatest intensity of protein bands was observed in the region with MW less than 33kDa on the obtained electrophoregrams. The presence of intense protein fractions in the region of molecular weights of less than 50–52kDa and 40kDa was also noted, which may correspond to enzymes such as pancreatic lipase and phospholipase A2, and the presence of protein fractions with MW above 130 kDa corresponding to various types and isoforms of collagen and laminin. In addition, such processes as protein aggregation and proteolysis can also influence the molecular weight distribution of protein fractions.