Antioxidant enzyme activities were spectrophotometrically quantified in blood samples (SOD = 50 µL, CAT = 20 µL, GST = 50 µL) and in animal tissues from the experimental groups. Around 100 mg of frozen samples from the digestive gland, the gill, and the lung of control, hibernation, and arousal-hib animals were homogenized (UltraTurrax®,, IKA Werke, Staufen, Germany) in buffered saline solution and centrifuged (10,500× g at 4 °C for 5 min). Supernatants were collected, aliquoted, and frozen for the determination of enzymes and proteins.
SOD activity was determined according to Misra and Fridovich [33 (link)]. The inhibition of the auto-oxidation of epinephrine was measured at 480 nm (30 °C), and a unit (U) was defined as the amount of SOD that inhibits 50 % of adrenochrome formation. The activity of SOD was expressed as Units per milligram of protein (U/mg). CAT activity was measured by the method described by Aebi [34 ] through the decomposition of 10 mM H2O2 by the sample in 50 mM phosphate buffer (pH 7.0). The decrease in the absorbance of H2O2 for 60 s at 240 nm represents the enzyme activity, and it was expressed as Units of CAT per milligram of protein (U/mg). GST activity was calculated by the Habig et al. [35 (link)] method, where the increase in the absorbance for 180 s at 340 nm from a mixture of buffer solution (20 mM Tris base, 1 mM EDTA, 1 mM dithiothreitol, 0.5 M sucrose, 0.15 M KCl, and 0.1 mM phenylmethylsulfonyl fluoride; pH 7.6), 50 mM 1-chloro-2,4-dinitrobenzene, 100 mM reduced glutathione, and the sample. Results of GST activity were expressed as milliUnits per milligram of protein (mU/mg).
SOD activity was determined according to Misra and Fridovich [33 (link)]. The inhibition of the auto-oxidation of epinephrine was measured at 480 nm (30 °C), and a unit (U) was defined as the amount of SOD that inhibits 50 % of adrenochrome formation. The activity of SOD was expressed as Units per milligram of protein (U/mg). CAT activity was measured by the method described by Aebi [34 ] through the decomposition of 10 mM H2O2 by the sample in 50 mM phosphate buffer (pH 7.0). The decrease in the absorbance of H2O2 for 60 s at 240 nm represents the enzyme activity, and it was expressed as Units of CAT per milligram of protein (U/mg). GST activity was calculated by the Habig et al. [35 (link)] method, where the increase in the absorbance for 180 s at 340 nm from a mixture of buffer solution (20 mM Tris base, 1 mM EDTA, 1 mM dithiothreitol, 0.5 M sucrose, 0.15 M KCl, and 0.1 mM phenylmethylsulfonyl fluoride; pH 7.6), 50 mM 1-chloro-2,4-dinitrobenzene, 100 mM reduced glutathione, and the sample. Results of GST activity were expressed as milliUnits per milligram of protein (mU/mg).
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