Purification and characterization of anticoagulation factors from the venom of Agkistrodon acutus

Two anticoagulants from five-pace snake (Agkistrodon acutus) venom,, anticoagulation factor I (ACF I) and anticoagulation factor II (ACF II), have been purified by a multiple-step chromatography procedure of anion-exchange chromatography, gel permeation chromatography and cation-exchange chromatography. Each of them is shown to be homogeneous as judged by PAGE, SDS-PAGE and mass spectrometry. In vitro, both proteins show equivalent anticoagulant activity, and are devoid of proteolytic, esterolytic, L-amino acid oxidase, phospholipase A, thrombin-like, fibrinolytic, hemorrhagic and lethal activities. They have similar amino acid compositions with similar absorption coeffecients (A(280)(1%)) (30.5 for ACF I and 30.0 for ACF II). Both are disulfide-linked consisting of two 14.7 kD chains for ACF I and two 14.6 kD chains for ACFII. ACF I has a molecular mass of 29,604 +/- 8 atomic mass units (amu) compared to 29,468 +/- 6 amu for ACF II, determined by mass spectrometry. The isoelectric points of ACF I and ACF II are 5.7 and 7.0, respectively. We conclude that the two isoforms possess equivalent biological activities with similar amino acid compositions and molecular masses, but different isoelectric points. (C) 2000 Elsevier Science Ltd. All rights reserved.