Storage and maintaining activity of ribulose bisphosphate carboxylase/oxygenase

Purified ribulose-1,5-bisphosphate carboxylase/oxygenase in 50% saturated (NH(4))(2)SO(4) was stable when frozen as small beads in liquid nitrogen and stored at -80 C. When stored as a slurry at 4 C most of the activity was lost within four weeks. This loss was due not only to enzyme polymerization. Activity in old preparations purified from spinach leaves, but not tobacco or tomato leaves, can be restored to the level of newly purified enzyme after storage at 4 C by treatment with 50 to 100 millimolar dithiothreitol for several hours followed by dialysis against buffer and 1 millimolar dithiothreitol before CO(2) and Mg(2+) activation and assay. Some enzyme oligomers that had been formed were not converted back to native enzyme by treatment with 100 millimolar dithiothreitol.The purified enzyme contained about 2 gram-atoms iron per mole enzyme that could not be removed by chelating agents. When the enzyme was incubated with 100 millimolar dithiothreitol and exposed to O(2), a purple dithiothreitol-iron complex was formed which could be removed by dialysis. The activities of ribulose-1,5-bisphosphate carboxylase and oxygenase were not altered by reducing the iron content to 0.7 mole per mole enzyme by treatment with dithiothreitol followed by exhaustive dialysis against iron free buffer.