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Articles

Storage and Maintaining Activity of Ribulose Bisphosphate Carboxylase/Oxygenase ¹

Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824

Purified ribulose-1,5-bisphosphate carboxylase/oxygenase in 50% saturated (NH₄)₂SO₄ 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 CO₂ and Mg²⁺ 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 O₂, 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.

³ Present address: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139.

¹ Supported in part by grants from the National Science Foundation (PCM 78-15891) and from the United States Department of Agriculture/SEA Competitive Grants Program, 5901-0410-8-0173-1. Published as Michigan Agricultural Experiment Station Journal Article number 9453.