An Investigation into the Structure of Protein Toxins of Australian Snake Venoms

• Main Author: Harrison, Julian Alexander
• Format: Text
• Language: unknown
• Published: School of Chemistry and Molecular Bioscience 2020
• Subjects: snake venoms; mass spectrometry; enzyme kinetics; Antarc*; antarcticus
• Online Access: https://ro.uow.edu.au/theses1/1113; https://ro.uow.edu.au/cgi/viewcontent.cgi?article=2112&context=theses1

Australian elapid venoms are composed of multiple protein toxins. There is a paucity of data for the structure of these toxins. Mass spectrometry provides avenues for investigating the proteins found within Australian snake venoms. In this thesis, native mass spectrometry was used to analyse the whole venoms of Acanthophis antarcticus (common death adder), Notechis scutatus (mainland tiger snake), Oxyuranus microlepidotus (inland taipan) and Oxyuranus scutellatus scutellatus (coastal taipan). Whole venoms were fractionated using size exclusion chromatography prior to mass spectrometry analysis. The protein fractions in the venoms could be separated into four mass categories (red > 100 kDa; yellow 100 to 40 kDa; green, 40 to 6 kDa; blue, < 6 kDa). Variations in the number and masses of venom proteoforms were reported between this thesis and previous investigations. This was attributed to differences in venom protein expression between individual snakes, and from different geographical locations. The next objective of this thesis was to screen the Australian elapid venoms for trimeric phospholipase A2 (PLA2). For this analysis, snake species from the genera Acanthophis, Notechis, Oxyuranus and Tropidechis were investigated. Size exclusion chromatography for each of the venoms yielded peaks with elution volumes expected for trimeric phospholipase A2. These size exclusion fractions were then analysed for phospholipase A2 activity using a colorimetric assay. All size exclusion fractions suspected to contain trimeric phospholipase A2 were found to have PLA2 activity. SDS-PAGE and sequence analysis of these fractions indicated that they contained PLA2, as well as other venom proteins (such as factor Xa-like proteins). Subsequent native mass spectrometry of these size exclusion fractions confirmed that all of the venoms contained heterotrimers, which had genera-specific characteristics. Investigations into the glycosylation of these toxins indicated that the trimeric PLA2 from the Notechis genera had two glycosylated subunits, and one of the trimer isoforms from the venom of Tropidechis carinatus (rough-scaled snake) was not glycosylated. All other trimers had a single glycosylated subunit. Mass differences between trimer isoforms were primarily due to differences in glycosylation. Ion mobility mass spectrometry analysis of the trimeric phospholipase A2 indicated that they all have similar three-dimensional architectures based on collisional cross sections. A continuous mass spectrometry assay was developed to measure the enzymatic activity of phospholipase A2 against phosphatidylcholine liposomes. The experimental setup allowed for the measurement of the activity of paradoxin (PDx), a trimeric phospholipase A2 from the venom of the inland taipan, at a toxicologically relevant concentration. Further experimentation revealed that the kinetic parameters obtained for paradoxin were similar to those obtained for a viper toxin using a pH stat method. These values for PDx were Vmax, 14.9 nMs-1, Km 97.1 μM and kcat 2.5 s-1. The activity of paradoxin was also measured in the presence of different divalent metals. In agreement with the literature, these experiments showed that paradoxin required calcium to be active, and that the greatest activity for paradoxin was observed when both calcium and magnesium were present. The instrument conditions for the analysis of intact oligomeric complexes using the Agilent 6560 ion mobility Q-TOF, a drift tube ion mobility mass spectrometer, were determined. These settings were used to measure the collisional cross sections of some standard native proteins over a mass range ~8-330 kDa, the results of which were mostly comparable to previous investigations. The Agilent 6560 ion mobility Q-TOF was then used to determine the collisional cross sections of paradoxin and its subunits. This was done to determine the effectiveness of paradoxin and other venom proteins as calibrants for collisional cross section estimates using travelling wave ion mobility mass spectrometry. When paradoxin and its subunits were used as calibrants, the collisional cross section calculations obtained for most protein ions over a mass range of ~12-70 kDa were within three percent of the literature values determined previously using drift tube ion mobility mass spectrometry (DTIM-MS). Travelling wave ion mobility mass spectrometry (TWIM-MS) investigations revealed that paradoxin is highly resistant to conformational changes induced by organic solvents or collisional energy. These structural properties made it ideal for use as a calibrant for measuring the collisional cross section of other proteins using TWIM-MS.