SIMPLE DETERMINATION OF THE CO2/O-2 SPECIFICITY OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE BY THE SPECIFIC RADIOACTIVITY OF [C-14] GLYCERATE 3-PHOSPHATE

A new method is presented for measurement of the CO2/O2 specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The [C-14]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. (CO2)-C-14 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 [C-14]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.