An efficient protocol for the extraction of pigment-free active polyphenol oxidase and soluble proteins from plant cells.

The elimination of brownish pigments from plant protein extracts has been a challenge in plant biochemistry studies. Although numerous approaches have been developed to reduce pigments for enzyme assays, none has been able to completely remove pigments from plant protein extracts for biochemical studies. A simple and effective protocol was developed to completely remove pigments from plant protein extracts. Proteins were extracted from red anthocyanin-rich transgenic and greenish wild-type tobacco cells cultured on agar-solidified Murashige and Skoog medium. Protein extracts from these cells were brownish or dark due to the pigments. Four approaches were comparatively tested to show that the diethylaminoethyl (DEAE)-Sephadex anion exchange gel column was effective in completely removing pigments to obtain transparent pigment-free protein extracts. A Millipore Amicon^® Ultra 10K cut-off filter unit was used to effectively desalt proteins. Moreover, the removal of pigments significantly improved the measurement accuracy of total soluble proteins. Furthermore, enzymatic assays using catechol as a substrate coupled with high-performance liquid chromatography analysis demonstrated that the pigment-free proteins not only showed polyphenol oxidase (PPO) activity but also enhanced the catalytic activity of PPO. Taken together, this protocol is effective for extracting pigment-free plant proteins for plant biochemistry studies. A simple and effective protocol was successfully developed to not only completely and effectively remove anthocyanin and polyphenolics-derived quinone pigments from plant protein extracts but also to decrease the effects of pigments on the measurement accuracy of total soluble proteins. This robust protocol will enhance plant biochemical studies using pigment-free native proteins, which in turn increase their reliability and sensitivity.