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Metabolism and Enzymology

Simple Determination of the CO₂/O₂ Specificity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by the Specific Radioactivity of [¹⁴C]Glycerate 3-Phosphate ¹

Department of Biochemistry, University of Arizona, Tucson, Arizona 85721, Department of Plant Science, University of Arizona, Tucson, Arizona 85721

A new method is presented for measurement of the CO₂/O₂ specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The [¹⁴C]3-phosphoglycerate (PGA) from the Rubisco carboxylase reaction and its dilution by the Rubisco oxygenase reaction was monitored by directly measuring the specific radioactivity of PGA. ¹⁴CO₂ fixation with Rubisco occurred under two reaction conditions: carboxylase with oxygenase with 40 micromolar CO₂ in O₂-saturated water and carboxylase only with 160 micromolar CO₂ under N₂. Detection of the specific radioactivity used the amount of PGA as obtained from the peak area, which was determined by pulsed amperometry following separation by high-performance anion exchange chromatography and the radioactive counts of the [¹⁴C]PGA in the same peak. The specificity factor of Rubisco from spinach (Spinacia oleracea L.) (93 ± 4), from the green alga Chlamydomonas reinhardtii (66 ± 1), and from the photosynthetic bacterium Rhodospirillum rubrum (13) were comparable with the published values measured by different methods.