Quantification and comparison of chloramphenicol acetyltransferase activity in transformed plant protoplasts using...

Rice and petunia leaf and cell suspension protoplasts were transformed by electroporation in the presence of pDW2. This plasmid contains a chloramphenicol acetyltransferase (CAT) coding region under the control of a promoter constructed from sequences of the cauliflower mosaic virus genome. We have compared two different approaches to measuring CAT activity in this system, namely high-performance liquid chromatography (HPLC) and a radioisotope-based method. Our results show that both techniques have a similar detection limit of 10 mU CAT and (with an activity greater than 10 mU CAT) the standard error for measuring known amounts of CAT activity was less than +/- 12% for both assays. The HPLC technique, however, has a greater linear response range of 10-600 mU CAT than the radioisotope method, which has a range of 10-400 mU CAT. The HPLC assay also requires a shorter assay time. As a result of this work we believe that HPLC is a viable alternative to the radioisotope-based assay described.