Purification and Characterization of a Glutathione S-Transferase from Pome Fruits

in HortScience
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  • 1 Univ. of Guelph, Plant Agriculture, Guelph, Ontario, N1G 2W1, Canada
  • | 2 Univ. of Guelph, Plant Agriculture, Guelph, Ontario, N1G 2W1, Canada
  • | 3 Ontario Ministry of Agriculture and Food, Ontario Ministry of Agriculture and Food, Simcoe, Ontario, N3Y 4N5, Canada

Glutathione S-transferase (GST) is a ubiquitous and constitutive enzyme that is involved in numerous cellular activities including the amelioration of oxidative stresses caused by the presence of xenobiotics and reactive oxygen species. In the present study, a glutathione S-transferase was extracted, purified, and partially characterized from two types of pome fruits. Pear (Pyrus × communis L., cultivar D'Anjou) and apple (Malus × domestica Borkh., cultivar Delicious) fruit were tested. The glutathione S-transferase was extracted using traditional methods, and purified using a combination of ammonium sulfate precipitation, dialysis, and GST-specific affinity chromatography. The GST enzyme was subsequently eluted from the column and concentrated prior to characterization studies. A purified fraction from the column was loaded onto an SDS-PAGE gel, and resulted in a single band with an apparent molecular weight of ≈26 kDa. This band was excised and used for MALDI-TOF/MS peptide mass fingerprint studies, and also served to confirm the apparent mass of the protein (25969 Da). The ExPASy software was used for the peptide mass fingerprint study, where the digest of the GST using trypsin was compared to a theoretical digest of an Arabidopsis GST, and resulted in two peptides of significant mass homology. The purified GST was also tested for enzyme activity using the standard assay substrate of 1-chloro-2,4-dinitrobenzene and reduced glutathione. Total GST protein extracted from `D'Anjou' pear was 0.532 mg·mL-1, while `Delicious' apple contained 0.127 mg·mL-1. The activity of GST enzymes may play a role in minimizing oxidative stress injury in stored pear and apple tissues.

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