After each turtle had been injected with ketamine and was no longer responsive to a pain

stimulus, it was decapitated. Portions of the right lobe of the liver and the left pectoral muscle

were removed with forceps and snap-frozen in liquid nitrogen no more than three minutes after

decapitation. Tissues were maintained at -80 C until they were homogenized as described

below.

 Subsamples of liver and pectoral muscle were homogenized in 1.0 ml of Sigma T-6789

buffer (0.05 M Tris, 0.138 M NaCl, 2.7 mM KC1, pH 8.0 with 1% bovine serum albumin)

yielding a 10% weight:volume tissue solution for each sample. Liver and muscle homogenates

were further diluted to 10% and 33%, respectively, to insure that enzyme activities would be

within the range of the standard curves used. Total protein concentration of each tissue solution

was evaluated using a Bradford assay with standard curves constructed using dilutions of bovine

serum albumin (BSA, 2 mg/ml undiluted). Concentrations of standards (in duplicate) and tissue

solutions (in triplicate) were determined by absorbance at 595 nm with a 695 nm reference

wavelength using a microplate reader.

Glutathione Peroxidase Activity Assay

 Glutathione peroxidase activity was evaluated using a total GPX assay modified from

Nakamura et al. (1974). Diluted muscle and liver homogenate solutions (n = 46) were incubated

for three minutes at 25 C in a reaction cocktail containing 0.297 U/ml glutathione reductase,

1.25 mM glutathione, and 0.188 mM NADPH in a 100 mM potassium phosphate buffer with 10

mM EDTA (pH 7.4). T-butyl hydroperoxide (12 mM) was then added to the reaction mixture

and the absorbance of the resulting solution at 340 nm was recorded every minute for four

minutes using a microplate reader. A blank consisting of 100 mM potassium phosphate buffer

with 10 mM EDTA (pH 7.4) was also assayed to evaluate glutathione-independent reaction rates.