Partial characterization of digestive proteases in sheepshead, Archosargus probatocephalus (Spariformes: Sparidae)

ABSTRACT Digestive proteases were partially characterized in sheepshead juveniles, using biochemical and electrophoretic techniques. Results showed higher activity level of the stomach proteases (2.39 ± 0.02 U mg protein-1) compared to the intestinal proteases (1.6 ± 0.1 U mg protein-1). The activity of trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A was also recorded. The optimum temperature of the stomach proteases was recorded at 45 °C, while for intestinal proteases was recorded at 55 °C. Stomach proteases showed less stability to temperature changes than intestinal proteases. An optimum pH of 2 was recorded for stomach proteases with high stability under acidic conditions, while an optimum pH of 9 was recorded for intestinal proteases showing high stability under alkaline conditions. Stomach proteases were inhibited around 78% with Pepstatin A, indicating the presence of pepsin as the main protease. The stomach proteases zymogam revealed one active band with Rf of 0.49, this enzyme was completely inhibited by Pepstatin A. The intestinal proteases zymogram revealed four active proteases (51.3, 34.9, 27.8 and 21.2 kDa) that were inhibited by TLCK, which mainly represent a trypsin-like serine proteases. It can be conclude that digestion in sheepshead can be considered as a carnivorous species with an omnivorous tendency.摘要：本研究采用生物化学与电泳技术，对羊头鱼（sheepshead）幼鱼的消化蛋白酶进行了部分性质表征。结果显示，胃部蛋白酶的活性水平（2.39±0.02 U·mg蛋白⁻¹）显著高于肠蛋白酶（1.6±0.1 U·mg蛋白⁻¹）。本研究同时测定了胰蛋白酶（trypsin）、胰凝乳蛋白酶（chymotrypsin）、亮氨酸氨肽酶（leucine aminopeptidase）及羧肽酶A（carboxypeptidase A）的活性。胃部蛋白酶的最适温度为45℃，肠蛋白酶的最适温度则为55℃。胃部蛋白酶对温度变化的稳定性弱于肠蛋白酶。胃部蛋白酶的最适pH为2，在酸性条件下稳定性较高；肠蛋白酶的最适pH为9，在碱性条件下稳定性优异。胃部蛋白酶可被胃蛋白酶抑制剂A（Pepstatin A）抑制约78%，表明胃蛋白酶（pepsin）是其主要的蛋白酶组分。胃部蛋白酶酶谱显示一条比移值（Rf）为0.49的活性条带，该酶可被胃蛋白酶抑制剂A完全抑制。肠蛋白酶酶谱显示四种活性蛋白酶（分子量分别为51.3、34.9、27.8及21.2 kDa），均可被TLCK抑制，其主要成分为类胰蛋白酶丝氨酸蛋白酶。综上可推断，羊头鱼为兼具杂食倾向的肉食性鱼类。