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Manganese Superoxide Dismutase Activity Assay in the Yeast Saccharomyces cerevisiae.

Bio-protocol (2021-03-05)
Louise Thines, Pierre Morsomme
ABSTRACT

Superoxide dismutases (SODs) act as a primary defence against reactive oxygen species (ROS) by converting superoxide anion radicals (O2 -) into molecular oxygen (O2) and hydrogen peroxide (H2O2). Members of this enzyme family include CuZnSODs, MnSODs, FeSODs, and NiSODs, depending on the nature of the cofactor that is required for proper activity. Most eukaryotes, including yeast, possess CuZnSOD and MnSOD. This protocol aims at assessing the activity of the yeast Saccharomyces cerevisiae MnSOD Sod2p from cellular extracts using nitroblue tetrazolium staining. This method can be used to estimate the cellular bioavailability of Mn2+ as well as to evaluate the redox state of the cell.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium chloride, ACS reagent, ≥99.0%
Sigma-Aldrich
Hydrochloric acid, 36.5-38.0%, BioReagent, for molecular biology
Sigma-Aldrich
Nitrotetrazolium Blue chloride, ≥90.0% (HPLC)
Sigma-Aldrich
Ethylenediaminetetraacetic acid disodium salt dihydrate, ACS reagent, 99.0-101.0%
Sigma-Aldrich
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, for molecular biology, ≥97.0%
Supelco
Bicinchoninic Acid solution
Sigma-Aldrich
Glycine, BioUltra, for molecular biology, ≥99.0% (NT)
Sigma-Aldrich
Sodium phosphate monobasic, BioReagent, for molecular biology, anhydrous, ≥98%
Sigma-Aldrich
Sodium phosphate dibasic, BioXtra, ≥99.0%
Sigma-Aldrich
Glycerol, for molecular biology, ≥99.0%
Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
N,N,N′,N′-Tetramethylethylenediamine, BioReagent, suitable for electrophoresis, ~99%
Sigma-Aldrich
Bromophenol Blue