The performance of the carbon-fixing enzyme, ribulose 1, 5-bisphosphate carboxylase/oxygenase (EC 18.104.22.168, Rubisco), controls biomass accumulation in green plants, algae and most autotrophic bacteria. In particular, the carboxylase activity of Rubisco incorporates carbon from CO2 to ribulose 1, 5-bisphosphate (RuBP) producing two molecules of 3-phosphoglycerate. Here a detailed protocol is given for the assay of the carboxylase activity of Rubisco from Chlamydomonas reinhardtii, a model organism for chloroplast studies and a fitting host for biotechnologically oriented genetic manipulation of the enzyme. Rubisco has to be pre-incubated with Mg2+ ions and bicarbonate to induce the catalytically competent active center (Laing and Christeller, 1976). Once Rubisco is activated, the assay of its carboxylase activity described here is based on the fixation of 14C-carbon dioxide/bicarbonate into acid-resistant radioactivity (Lorimer et al., 1977). Although a spectrophotometric assay is also available (Lilley and Walker, 1974), the method based on fixation of a radioactive substrate is irreplaceable when processing a large number of samples, and it is still the technique most often used for the determination of Rubisco activity.
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