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

Two anticoagulants from five-pace snake (Agkistrodon acutus) venom,, antico agulation factor I (ACF I) and anticoagulation factor II (ACF II), have bee n purified by a multiple-step chromatography procedure of anion-exchange ch romatography, gel permeation chromatography and cation-exchange chromatogra phy. Each of them is shown to be homogeneous as judged by PAGE, SDS-PAGE an d 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 activ ities. They have similar amino acid compositions with similar absorption co effecients (A(280)(1%)) (30.5 for ACF I and 30.0 for ACF II). Both are disu lfide-linked consisting of two 14.7 kD chains for ACF I and two 14.6 kD cha ins 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 spectrom etry. The isoelectric points of ACF I and ACF II are 5.7 and 7.0, respectiv ely. We conclude that the two isoforms possess equivalent biological activi ties with similar amino acid compositions and molecular masses, but differe nt isoelectric points. (C) 2000 Elsevier Science Ltd. All rights reserved.