Isolation of intact chloroplasts with high CO2 fixation capacity from sugarbeet leaves containing calcium oxalate

Intact chloroplasts were isolated from sugarbeet leaves by the mechanical disruption technique normally used for spinach. The chloroplast pellet contained a ring of white irregularly shaped crystals which were identified as calcium oxalate. The chloroplasts were greater than 90% intact yet good rates of CO2 fixation were only obtained when inorganic pyrophosphate or 3-phosphoglycerate were added to the assay medium. Chloroplasts free of calcium oxalate were prepared by purification on a three step Percoll gradient. These purified chloroplasts were highly intact and showed high rates of CO2 fixation without adding inorganic pyrophosphate or 3-phosphoglycerate. With optimal assay conditions (0.2 mM orthophosphate and pH 8.0) rates of 110-130 μ mole per milligram chlorophyll per hour were routinely obtained. It is concluded that intact chloroplasts capable of high rates of CO2 fixation can be prepared from sugarbeet leaves using a simple three step Percoll gradient.