Fractionation of snake venom metalloproteinases by metal ion affinity: A purified cobra metalloproteinase, Nk, from Naja kaouthia binds Ni2+-agarose

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 Ni2+-agarose, and was eluted by ∼10 mM imidazole, confirming the validity of the approach. Nk cleaved the platelet von Willebrand factor (VWF) receptor, glycoprotein (GP)Ibα, with similar activity to the previously reported cobra metalloproteinase, mocarhagin, as shown by EDTA-inhibitable Nk-dependent proteolysis of a purified GPIbα extracellular fragment (glycocalicin), and inhibition of 125I-VWF binding to GPIbα on washed human or canine platelets. Second, crude venom from the viper, Trimeresurus albolabris, was fractionated on Ni2+-agarose. Samples of flow-through, wash, and imidazole-eluted (0–30 mM 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 Ni2+-agarose to fractionate snake venom metalloproteinases.