Stock, Naomi L

Chemical cues are used commonly by prey to identify predation risk in aquatic environments. Previous work has indicated that negatively-charged ions of m/z 501 are possibly a kairomone that induces anti-predator responses in tadpoles. This thesis found that this ion species: (i) is produced by injured tadpoles; (ii) exhibits increased spectral intensity with higher tadpole biomass; and (iii) is not produced by starved predators. These results refute the hypothesis that the ion is a kairomone, and rather support its role as an alarm cue released from tadpoles. High resolution mass spectrometry (HR-MS) revealed a unique elemental composition for [M-H]-, m/z 501.2886, of C26H45O7S-. Collision induced dissociation (CID) of ion m/z 501 formed product ions of m/z 97 and m/z 80, HSO4- and SO3-, respectively, indicating the presence of sulfate. Green frog (Lithobates clamitans) tadpoles exposed to m/z 501, and an industrial analogue, sodium dodecyl sulphate (NaC12H25O4S), exhibited similar anti-predator responses, thereby suggesting the potential role of organic sulfate as a tadpole behavioural alterant.

Author Keywords: Alarm cue, Amphibian, Chemical Ecology, Mass spectrometry, Predator-prey interactions

The amitochondrial protozoan, Giardia intestinalis, encodes four members of the cytochrome b5 (CYTB5) family of heme proteins of unknown function. While homology models can predict the likely fold of these proteins, supporting experimental evidence is lacking. The small size of the cytochromes (~16 kDa) makes them attractive targets for structural analysis by Electron Paramagnetic Resonance spectroscopy (EPR) and Nuclear Magnetic Resonance spectroscopy (NMR). EPR measurements are particularly useful in defining the geometry of the coordination environment of the heme iron; such measurements indicated that the planar imidazole rings of the invariant histidine axial ligands are nearly perpendicular to each other, rather than in the coplanar orientation observed within mammalian CYTB5s. This may be due to geometrical constraints imposed by a one-residue shorter spacing between the ligand pair in the Giardia cytochromes b5 (gCYTB5s). Following optimization of sample and instrument conditions for NMR experiments, a comparison of the 1D 1H-NMR spectra of gCYTB5 isotype I to those of three of its heme-pocket mutants (Tyr51→Phe, Tyr61→F, and Cys84→Ala) were used to tentatively assign the heme methyl and vinyl protons. Mutant Tyr61→F had the greatest effect on the wild-type spectrum due to maximum through-space contacts with the heme macrocycle and its proximity to the His63 axial ligand. These experiments are a prelude to further NMR experiments that can lead to solving the complete structures of these proteins.

Author Keywords: cytochrome b5, heme b, mutant protein, paramagnetic iron, resonant spectroscopy, sequence homology