*3.3. Recombinant FhSOD1 and FhSOD3 Are Highly Active*

To investigate the role of *F. hepatica* SODs in parasite metabolism and host defence, we exploited E. coli to recombinantly express both FhSOD1 and FhSOD3 antioxidant proteins. SDS-PAGE and Western blot analysis using monoclonal anti-histidine antibodies revealed soluble rFhSOD1 and rFhSOD3 purified at ~16 and ~17 kDa, respectively, which are predicted to have a conserved secondary structure consisting of a β-sheet made up of eight antiparallel β-strands typical of SODs (Figures S3 and S4). rFhSOD1 and rFhSOD3 had similar activity against superoxide (~400 U/mg each, as defined by the standard curve), where one unit of enzyme activity is defined as the amount of protein required to exhibit 50% dismutation of superoxide under physiological conditions (150 mM salt, pH 7.4, 37 ◦C), albeit they were ~10 times less active than the positive control (Figure S3). Size exclusion (gel filtration) chromatography revealed distinct peaks corresponding to molecular weights of ~36 kDa (rFhSOD1), ~31 kDa (rFhSOD3), and ~76 kDa (rFhSOD3), indicative of homodimers and a mix of homodimers and homotetramers for rFhSOD1 and rFhSOD3, respectively (Figure 3). Analysis of the individual fractions collected from each peak demonstrate that both the dimeric (rFhSOD1 and rFhSOD3) and tetrameric (rFhSOD3) forms of the recombinant proteins are enzymatically active (Figure 3).

**Figure 3.** Structural organisation and enzymatic activity of rFhSOD1 and rFhSOD3. Size exclusion chromatography of (**A**) FhSOD1 and (**B**) FhSOD3. The corresponding enzyme activity of each elution fraction is expressed as the percentage inhibition of formazan dye formation. (**C**) The predicted molecular sizes of each recombinant protein calculated against the molecular weight markers. Markers are indicated by dotted lines—M1; conalbumin, M2; carbonic anhydrase, M3; aprotinin.
