Purification and preliminary characterization of a plasma kallikrein inhibitor isolated from sea hares Aplysia dactylomela Rang, 1828

Abstract

An inhibitor active against pancreatic trypsin was found in the crude extract from the sea hares Aplysia dactylomela Rang, 1828. A stronger inhibitory activity against human plasma kallikrein was detectable after treating this extract at 60 T, for 30 min. the plasma kallikrein inhibitor (AdKI) purification was achieved by acetone fractionation (80%) v/v, ion-exchange chromatography on Mono Q column and gel filtration chromatography on Superdex 75 column (FPLC system). By the latter a molecular mass of 2,900 Da was estimated. the purified inhibitor strongly inhibits human plasma kallikrein with a K-i value of 2.2 X 10(-10) M, while human plasmin and pancreatic trypsin were inhibited with K-i values of 1.8 X 10(-9) and 4.7 x 10(-9) M, respectively. Chymotrypsin, pancreatic elastase, pancreatic kallikrein and thrombin are not inhibited. the effect of AdKI on plasma kallikrein was confirmed by the prolongation of activated partial thromboplastin time, using a clotting time assay. the inhibitor did not affect prothrombin time or thrombin time. AdKi is a more specific inhibitor than other serine proteinase inhibitors from marine invertebrates. (C) 2003 Elsevier B.V. All rights reserved.