title: Fractionation of snake venom metalloproteinases by metal ion affinity: a purified cobra metalloproteinase, Nk, from Naja kaouthia binds Ni2+-agarose.
abstract: Snake venom metalloproteinases represent unique probes for analyzing platelet adhesion receptors regulating hemostasis and thrombosis. Snake venom metalloproteinase-disintegrins consist of a propeptide domain, a catalytic domain containing a metal ion-coordination sequence (HEXXHXXGXXH), a disintegrin domain, and a Cys-rich domain. Here, we investigate whether metal ion-affinity chromatography may be used to fractionate venom metalloproteinases based on the metal ion-coordination motif. First, we showed that a purified cobra metalloproteinase, Nk, from Naja kaouthia bound Ni(2+)-agarose, and was eluted by approximately 10mM imidazole, confirming the validity of the approach. Nk cleaved the platelet von Willebrand factor (VWF) receptor, glycoprotein (GP)Ibalpha, with similar activity to the previously reported cobra metalloproteinase, mocarhagin, as shown by EDTA-inhibitable Nk-dependent proteolysis of a purified GPIbalpha extracellular fragment (glycocalicin), and inhibition of (125)I-VWF binding to GPIbalpha on washed human or canine platelets. Second, crude venom from the viper, Trimeresurus albolabris, was fractionated on Ni(2+)-agarose. Samples of flow-through, wash, and imidazole-eluted (0-30mM gradient) fractions were analyzed by (i) SDS-polyacrylamide gel electrophoresis, (ii) immunoblotting with a rabbit anti-mocarhagin antibody, and (iii) assessing metalloproteinase activity using human fibrinogen as substrate. The combined results support the general concept of using Ni(2+)-agarose to fractionate snake venom metalloproteinases.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, 3008 Victoria, Australia.
authors.forename: Lakshmi C
authors.initials: LC
authors.lastname: Wijeyewickrema
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Gardiner
authors.forename: Yang
authors.initials: Y
authors.lastname: Shen
authors.forename: Michael C
authors.initials: MC
authors.lastname: Berndt
authors.forename: Robert K
authors.initials: RK
authors.lastname: Andrews
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D004285:Dogs; D004546:Elapid Venoms / Q000032:analysis*; D017815:Elapidae; D006801:Humans; D008666:Metalloendopeptidases / Q000032:analysis*; D009532:Nickel / Q000378:metabolism; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000378:metabolism