The digestive glands of clams were rapidly dissected whilst frozen in liquid
nitrogen and then stored at 80 C. Three sub-samples were prepared by pooling
digestive glands from three to five clams, for each site and sampling campaign.
Hepatopancreatic tissues were homogenized with a Potter Elvehjem tissue grinder
in ice cold buffer, comprising 10mMof TriseHCl (pH 7.6) containing 150mMKCl and
0.5 M sucrose. The homogenate was centrifuged for 15 min at 1480 rpm and at 4 C,
and then the supernatant solution was centrifuged for 45 min at 9800 rpm at 4 C.
The resulting mitochondrial pellet was resuspended in the homogenizing buffer
(3 mL). The supernatant solution obtained after centrifugation at 28 000 rpm for
90 min was considered as the cytosolic fraction (Orbea et al., 2002). Aliquots were
stored at 80 C until analysis. Total protein contents of all fractions were determined
spectrophotometrically at 595 nm according to the method of Lowry et al.
(1951) using bovine serum albumin (BSA, fraction V) as a standard. The progress
of Catalase (CAT) and Superoxide dismutase (SOD) enzymatic reactions was
measured using a UV VIS spectrophotometer (Beckman DU 6400, USA), maintained
at room temperature. Reactions were measured for 120 s and a linear portion of the
decline curve was used to calculate reaction rates.