Assay and Electrophoresis of Superoxide Dismutase from Red Spruce ( Picea rubens Sarg.), Loblolly Pine ( Pinus taeda L.), and Scotch Pine ( Pinus sylvestris L.)

Abstract

This paper reports a method for extracting the antioxidant enzyme superoxide dismutase (SOD) from the needles of red spruce (Picea rubens Sarg.), loblolly pine (Pinus taeda L.), and scotch pine (Pinus sylvestris L.) with high efficiency and free from interfering compounds. The extraction employs phosphate buffer with polyvinylpolypyrrolidone and Triton X-100 followed by dialysis overnight. The isozymes of SOD in each species were separated electrophoretically and tested for their sensitivity to KCN and H(2)O(2). An isozyme resistant to these inhibitors was found in the spruce but not the pine needles. The isozymes from the spruce needles were examined for individual responses to aging and H(2)O(2) inhibition. Four of the five CuZn isozymes in spruce were found to have increased significantly but equally by October of their first year and two of those four isozymes were found to be more sensitive to H(2)O(2). The response of the SOD isozymes in loblolly pine seedlings to O(3) was also examined and the isozymes were found to be induced equally. Because the SOD activity in the young pine needles was too low to electrophorese, the SOD activity from the pines in the O(3) experiment had to be partially purified using CHCl(3) and ethanol, then concentrated.