The metalloenzyme involved in this study was manganese superoxide dismutase (MnSOD). MnSOD is found in both prokaryotes and eukaryotes utilises manganese as the central metal ion. It is involved in the oxidation and reduction of superoxide, generating oxygen and hydrogen peroxide. The aims of this study were to purify and characterise MnSOD, to draw temperature stability and structural comparisons between Mus musculus and Homo sapiens MnSODs, and to determine the optimal conditions for crystallisation.
Using E. coli cells transformed with M. musculus MnSOD cDNA, recombinant MnSOD was produced and purified by MCAC. The purity of each pure protein fraction was calculated using Doc-It®Ls software (UVP LLC) and the activity of each pure protein fraction was determined via Native PAGE. Protein concentration measurements were obtained using both the BCA™ assay and absorbance measurements at 280 nm.
Protein characterisation was achieved in various ways, these being: measuring the metal content of MnSOD as well as determining its molecular weight, specific activity, transition temperature and elucidating its proposed structure. The characteristic absorbance of manganese at 480 nm showed that the purified protein was metallated while SDS PAGE revealed the protein’s monomeric molecular weight to be 23.3 kDa. The specific activity of MnSOD was determined by two methods utilising the cytochrome c assay: through a Vs value approximately equal to ½ Vb and through the Vb/Vs against 1/ dilution method (Vanneste and Ysebaert-Vanneste, 1980). Through the former, the specific activity was found to be 8794 U/mg while through the latter it was found to be 6974 U/mg.
Both the transition temperature and the structure of the protein were compared to that of H. sapiens MnSOD. Using Native PAGE, the transition temperature of M. musculus MnSOD was found to be 86.76 °C ± 0.32 °C while that of H. sapiens MnSOD was found to be 88.29 °C ± 0.19 °C. A t-test showed that the two values were significantly different. Finally, the proposed structure of M. musculus MnSOD was drawn using both Vector NTI® Advance software (Invitrogen) and PyMOL software (Schrödinger) after the amino acid sequence of the two proteins was aligned using Vector NTI® Advance software (Invitrogen).