Mobina Maleki - Faculty of basic sciences, University of Mazandaran, Babolsar, Iran
Bagher Seyedalipour - Faculty of basic sciences, University of Mazandaran, Babolsar, Iran
Saman Hosseinkhani - Faculty of biological sciences, University of Tarbiat Modares, Tehran, Iran
Ehsan Nazifi - Faculty of basic sciences, University of Mazandaran, Babolsar, Iran

Superoxide dismutase ۱ (SOD۱) is an antioxidant enzyme. Mutation in the SOD۱ enzyme cause ALS. So far, more than ۱۷۰ mutations in the SOD۱ enzyme with the inherited pattern of ALS have been reported, and the accumulation of this protein is associated with pathological features of the disease. In this study, by replacing aspartic acid with glycine (G۴۱D), the activity and structure of the mutated enzyme and its comparison with the wild-type enzyme was investigated. In this study, plasmid pET۲۸a (+), which contains superoxide dismutase gene, was used. Then, the primers were designed using Oligo۷ software and the site directed mutagenesis (Quick-change PCR) method was used to create the mutation. The product was digested using Dpn۱ enzyme and then transferred to E.coli DH۵α strain by chemical method (heat shock). Induction of protein expression was influenced by IPTG and lactose and investigated SDS-PAGE electrophoresis gel. After purification of the protein by nickel-Sepharose column chromatography and dialysis, the activity of the enzyme was measured using pyrogallol at ۴۲۰ nm by spectrophotometry method. Structural comparison of mutant and wild-type enzymes was performed by intrinsic and external fluorescence spectra at a concentration of ۰.۰۲ mg/ml. The highest specific activity of wild-type and G۴۱D mutant were ۷۰۳۱ U/mg ۵۷۴۴ U/mg, respectively. Intrinsic fluorescence studies showed that the intensity of fluorescence in the mutant enzyme increased compared to the wild-type enzyme, indicating the location of the amino acid tryptophan in a more non-polar environment. External fluorescence studies showed that the intensity of mutant fluorescence was reduced compared to the wild-type enzyme, indicating a more compact structure and a reduction in hydrophobic pockets at the surface. As a result, it was found that this mutation caused local changes in the structure of the enzyme, which caused its compression.

G۴۱D mutant enzyme, Site directed mutagenesis, Specific activity, Fluorescence