A spectrophotometric assay for rubisco activity: Application to the kelp Laminaria saccharina and implications for radiometric assays

We optimized a spectrophotometric method for measuring ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) activity in crude extracts of the kelp Laminaria saccharina Lamour. Activity exhibited a decline during the assays, such that rates determined over a 10-min period were only 70% of activity during the first minute. Activity was significantly enhanced by addition of HCO3- to the extraction buffer and was stable for at least 4 h. Highest activities were determined using small extract loads (1-2.5 mg fresh weight . mL(-1) of assay mixture); extract loads of 10 mg . mL(-1) caused a 25% decrease in activity. Rubisco activities determined for individual kelp plants using the spectrophotometric method were significantly correlated with total protein and gross photosynthetic capacity (P-max) and were 1.5-2 times higher than P-max. Low Rubisco activities relative to photosynthetic rates reported in many previous studies of macroalgae and microalgae may have resulted at least partly from underestimation of enzyme activity by radiometric assays using relatively large volumes of unactivated crude extract and assay periods longer than 1 min.