Abstract
BackgroundRubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase) is a Calvin Cycle enzyme involved in CO2 assimilation. It is thought to be a major cause of photosynthetic inefficiency, suffering from both a slow catalytic rate and lack of specificity due to a competing reaction with oxygen. Revealing and understanding the engineering rules that dictate Rubisco’s activity could have a significant impact on photosynthetic efficiency and crop yield.ResultsThis paper describes the purification and characterisation of a number of hydrophobically distinct populations of Rubisco from both Spinacia oleracea and Brassica oleracea extracts. The populations were obtained using a novel and rapid purification protocol that employs hydrophobic interaction chromatography (HIC) as a form I Rubisco enrichment procedure, resulting in distinct Rubisco populations of expected enzymatic activities, high purities and integrity.ConclusionsWe demonstrate here that HIC can be employed to isolate form I Rubisco with purities and activities comparable to those obtained via ion exchange chromatography (IEC). Interestingly, and in contrast to other published purification methods, HIC resulted in the isolation of a number of hydrophobically distinct Rubisco populations. Our findings reveal a so far unaccounted diversity in the hydrophobic properties within form 1 Rubisco. By employing HIC to isolate and characterise Spinacia oleracea and Brassica oleracea, we show that the presence of these distinct Rubisco populations is not species specific, and we report for the first time the kinetic properties of Rubisco from Brassica oleracea extracts. These observations may aid future studies concerning Rubisco’s structural and functional properties.
Highlights
Rubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase) is a Calvin Cycle enzyme involved in CO2 assimilation
In order to test the suitability of hydrophobic interaction chromatography (HIC), lysed samples from both S. oleracea and B. oleracea were subjected to (NH4)2SO4 precipitation, with the majority of Rubisco protein precipitating between 35 and 60% saturation of (NH4)2SO4 (Figure 1)
HIC reveals the presence of hydrophobically distinct Rubisco fractions As the HIC column binds proteins at high salt concentration, the (NH4)2SO4 fraction containing Rubisco was directly subjected to our HIC protocol
Summary
Rubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase) is a Calvin Cycle enzyme involved in CO2 assimilation. Rubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase) is the CO2-fixing enzyme in the Calvin cycle, the primary pathway of carbon assimilation in photosynthetic organisms. It catalyses the reaction between Ribulose-1,5-bisphosphate (RuBP) and CO2 to produce two molecules of 3-phosphoglycerate [1,2]. Form I Rubisco is the most common, found in higher plants, cyanobacteria and eukaryotic algae [2], and its structure has been solved in many species, but first reports were for tobacco [10] and spinach [11]. Hybrid enzymes which contain large subunits and small subunits from different species have been reported to show differences in their stability and specificity [15,16,17]
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