The project had two aims:
1) Purification (i.e. separation and isolation) of the key venom molecules involved in Box jellyfish envenomation.
2) Identification of the molecular mechanisms involved in fatal heart failure due to box jellyfish envenomation
Summary of findings:
Venoms are complex mixtures of proteins and other bioactive molecules. Each protein has a distinct set of physical and chemical properties. Those properties can be used as tools to purify a specific protein from a complex mixture while retaining the activity of that protein. Consequently each purification procedure has to be adjusted to each protein.
Previous studies had partially purified the crude toxic fractions CTF-α, -β and –γ (see Figure 1) from box jellyfish venom. However, partially purified proteins still have a high level of complexity, i.e. these fractions have far more than just a single component in them (rough estimate: ± 100 components). While the study of partially purified components can result in valuable data, resulting signals can be unreliable and inconclusive. However, to develop adequate treatment it is imperative to identify the specific components responsible for the box jellyfish induced heart failure.
Over the past year, with the help of Dr. Michael Smout, I have developed a purification procedure that substantially reduces the complexity of CTF-α and –β (see Figure 2 and 3). To develop this procedure several chromatographic columns, and the ideal order for the purification process, had to be tested. The procedure that attained the highest level of purity of the venom proteins, is as follows:
a) Size-exclusion chromatography: In this step, proteins are separated by size. This step uses the Superdex 200 Increase 10/300 GL that was funded by the Lions Foundation. This step has also been previously used to partially purify CTF-α, -β and –γ.
b) Ion-exchange chromatography: Here, proteins are separated by the electric charge state of the molecule. This step, achieves a better degree of separation than size-exclusion chromatography, because many proteins are of similar size but only very few have an equal charge state. For this step a Mono Q 5/50 GL was used (provided by the AITHM). The problem with this method is that the buffers used for ion-exchange chromatography are extremely high in salt, and this can have a damaging effect on the venom components. Therefore, it is important to add another chromatographic step immediately after the ion-exchange chromatography.
c) Size-exclusion chromatography: To remove the salt from the sample and increase the level of purity a final size-exclusion step was added to the procedure. The column used was a HiLoad Superdex 75 PG (provided by Prof. Norelle Daly).
This procedure results in a very low complexity sample of which the individual components can be identified via mass spectrometry (see new grant application).
A description of this procedure will be published shortly (within the next 6 months).
Following Aim 1, the purified samples were to be tested on a ProtoArray. Due to the extensive work associated with Aim 1 and the high level of sample purity required for Aim 2, this aim has not been completed yet. However, the ProtoArrays have been purchased and Aim 2 will be carried out within the next few months.
Additional work – Effect of box jellyfish venom on cancer cells
Because Aim 2 required completion of Aim1, a small additional side project was conducted while Aim 1 was carried out. This project used the size-exclusion column funded by the Lions Foundation.
This project tested the effects of whole venom as well as 3 partially purified fractions (CTF-α, -β and –γ) on a cancer cell line, to
1. test whether the fractions responsible for the heart failure in humans have an effect on other cells
2. identify fractions that have an effect on cancer cells but not on heart cells.
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The results from this project have been submitted for publication to the Journal of Venomous Animals including Tropical Diseases. The Lions Foundation was mentioned in the role of funding body for the study.
Read the article.