Partial purification of cytolytic venom proteins from the box jellyfish, Chironex fleckeri

Venom proteins from the nematocysts of Chironex fleckeri were fractionated by size-exclusion and cation-exchange chromatography. Using sheep erythrocyte haemolysis as an indicator of cytolytic activity, two major cytolysins, with native molecular masses of ∼370 and 145 kDa, and one minor cytolysin (∼70 kDa) were isolated. SDS-PAGE and western blot protein profiles revealed that the 370 kDa haemolysin is composed of CfTX-1 and CfTX-2 subunits (∼43 and 45 kDa, respectively); the most abundant proteins found in C. fleckeri nematocyst extracts. The 145 kDa haemolysin predominately contains two other major proteins (∼39 and 41 kDa), which are not antigenic towards commercially available box jellyfish antivenom or rabbit polyclonal antibodies raised against whole C. fleckeri nematocyst extracts or CfTX-1 and -2. The kinetics of CfTX-1 and -2 haemolytic activities are temperature dependent and characterised by a pre-lytic lag phase (∼6–7 min) prior to initiation of haemolysis. Significant amino acid sequence homology between the CfTX proteins and other box jellyfish toxins suggest that CfTX-1 and -2 may also be lethal and dermonecrotic. Therefore, further in vivo and in vitro studies are required to investigate the potential roles of CfTX-1 and -2 in the lethal effects of C. fleckeri venom.