An improved spectrophotometric determination of the activity of ribulose 1,5-bisphosphate carboxylase

Spectrophotometric assay of ribulose 1,5-bisphosphate carboxlase, Rubisco, is a very useful method for the routine determination. However, when the freshly synthesized ribulose 1,5-bisphosphate, RuBP, is used as substrate there is a lag time between the carboxylation and NADH oxidation in the assay. In this study, we found firstly that the lag time was closely related to the concentrations of phosphoglycerate kinase, PGK, glyceraldehyde 3-phosphate dehydrogenase, GAP-DH, and phosphocreatine kinase, PCK, in the assay mixture. After increasing the concentrations of the coupling enzymes of PGK and GAP-DH, the lag time was shortened, but could not be eliminated completely. By increasing the concentration of PCK, lag time decreased significantly until there was no lag time. So, to overcome the lag time, the key is to increase the use of PCK but not the coupling enzymes. In addition, the accumulation of ADP in the assay mixture proved to be the most important factor in the assay mixture that produced the lag time in the spectrophotometric assay, and an optimum condition for activation and catalysis of Rubisco were also established. With the assay conditions established, high initial and total Rubisco activities were obtained.