| Summary: | The venoms of snakes and insects are complex mixtures of toxins and enzymes that produce profound physiological and behavioural consequences when injected into prey creatures. Understanding how these consequences are achieved from very small amounts of venom in short time intervals sheds light on the principles of venom action and the defence mechanisms that may have to be overcome. In addition to the possibility of better treatments for envenomed humans, wider insights into drug discovery are also promised. This thesis describes an investigation into the chemical effects of Honey Bee (Apis mellifera) venom. The toxic effects of some bee venom components (such as melittin and phospholipase A2) are well known, but the venom’s capacity to respond to potential defensive reactions (as represented by the biomolecules commonly released by mammals in response to wounding and the injection of toxins) has not been so well studied. Using the technique of Real-time NMR, specially adapted to this research, purified whole bee venom was challenged with a range of bioactive molecules that a mammalian victim could potentially release in response to the presence of the venom (i.e. adrenalin, cortisol, angiotensin, bradykinin, substance P and opioid peptides). Reactions were carried out in the NMR spectrometer under physiological conditions (pH 7.4, 37°C) and full spectrum “snapshots” were taken at frequent intervals. Subsequent analysis revealed whether or not any transformation had taken place, and, if so, what intermediates and products were generated. It was found that only substance P and the opioid peptides were acted upon by the bee venom, implying the venom contained one or more appropriate peptidase activities. Further experimentation with the opioids Met- and Leu-enkephalin, and their N-terminal fragment Tyr-Gly-Gly, established that the peptidase activity was primarily that of a dipeptidyl dipeptidase, supported by a dipeptidase. Subsequent kinetic studies implied that these substrates were not ideal and so other opioid peptides (Endomorphin I, Endomorphin II and Casomorphin 1-7) were also tested. It was found that Endomorphin I was the “best” substrate. The substrate specificity of the venom dipeptidyl peptidase shows consistencies with the known mammalian dipeptidyl peptidases III and IV. The presence of a DPP IV – like enzyme (i.e. an evolutionary homologue of the human version) in bee venom has been predicted from gene sequence analysis and the generation of melittin from its precursor. This bee protein is also known as Api m 5 and is a notable allergen. Consequently, the bee venom was challenged with a model substrate and a model inhibitor for human DPP IV. The model substrate was readily converted and the model inhibitor reduced the rate of conversion of the opioid peptides. Two synthetic variants of the inhibitor were tested, but they were not as effective. Although bee venom Api m 5 shares structural and preferred substrate/inhibitor similarities with human DPP IV, some contrasts remain (for example the former’s ability to act on enkephalin peptides). It is proposed that the role of the bee venom DPP-IV like enzyme (i.e. Api m 5) is primarily to destroy opioid peptides released by mammalian sting victims in response to the physical and toxicological impact of the venom. Opioid peptides bind to skin nociceptors to limit pain perception following injury, so fragmentation of these peptides could lead to prolonged pain from the sting site(s) and therefore behaviour that serves the interests of the bees (i.e. avoidance, retreat or stopping in the case of a mammal foraging a hive). Preliminary experiments were conducted to follow the action of the hyaluronidase in bee venom which breaks up the extra cellular matrix carbohydrate to speed up the tissue spread of the venom components. The NMR method again proved to be useful.
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