A trypsin inhibitor from Maclura pomifera (Raf.) Schneid. seeds

LAZZA C.M., VAGHI G., TORRES M.J. Y LÓPEZ L.M.I

Mar del Plata

Congreso; XLIII Reunión Anual de la SAIB; 2007

Sociedad Argentina de Investigaciones Bioquímicas y Biología Molecular

 Plant proteinase inhibitors (PIs) plays a central role in the diversification of both the insect herbivores as well as the host plants. These proteins are fundamental in the control and/or the protection against proteolytic action of the digestive enzymes of seed predators. One way to obtain better inhibitors of insect proteinases is, to search among plant species unrelated to host plant for PI families that do not exist in the host plants. Crude extract was prepared by grinding of twenty gram of  M. pomifera seeds, previously washed and dried, with 100 ml of saline Tris buffer (50 mM Tris, pH 7.2; 0.15 M NaCl) in a mixer. The homogenate was filtered through a two-folded piece of gauze to remove plant debris, and then centrifuged for 20 min at 10000 x g. The crude extract was partially purified by precipitation with 5 volumes of acetone and  centrifugation at 16000 ´ g for 30 min. The final acetone precipitate was redissolved with 2.5 volumes of  saline Tris buffer and applied to a gel filtration column (Sephadex G-50 Fine). The fraction with antitrypsin activity was submitted to an anion exchanger column (Q-Sepharose FF) pre-equilibrated with 50 mM Tris buffer, pH 7.2 and eluted using a liner gradient of NaCl.  The purified inhibitor was homogeneous by  SDS-PAGE and IEF, with  pI 5.2 and  Mr 14 kDa, common features of Kunitz inhibitors. 1CONICET fellow, 2 CONICET Researcher Career