Antioxidant and metabolic enzymes

Mussels were individually weighted and then pulverized in liquid nitrogen. The pulverized sample was transferred to a glass tissue homogenizer (Tenbroeck type) containing 0.1 M Tris (pH 7.6), 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and 1:1,000 (v/v) protease inhibitor cocktail (P8340; Sigma Aldrich, St. Louis, MO, USA). After homogenization, samples were centrifuged at 10, 000 × g for 15 min at 4 °C and the supernatants were collected for the assessment of enzymatic activities. The activities of catalase, glutathione transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPX), glucose 6-phosphate dehydrogenase (G6PDH), pyruvate kinase (PK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) were measured using spectrophotometric kinetic assays as described in Moreira et al. (2021b). The activities of opine dehydrogenase (ODH), strombine dehydrogenase (SDH), and alanopine dehydrogenase (ADH) were also measured using spectrophotometric kinetic assays, as described in Schiedek (Schiedek, 1997).

The activity of all enzymes was expressed as U per milligram of protein in the supernatant, which was measured using Coomassie brilliant blue G-250 and a standard curve built with bovine serum albumin as standard (Bradford, 1976).