PURIFICATION AND ANALYSIS OF WHEAT GRAIN POLYPHENOL OXIDASE PROTEIN

Authors: Anderson, James; Morris, Craig

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/1/2002
Publication Date: 4/29/2002
Citation: Anderson, J.V., Morris, C.F. Purification and analysis of wheat grain polyphenol oxidase protein. Cereal Chem. 80(2): 135-143.

Interpretive Summary: The darkening of wheat noodles in the Asian Pacific Rim is of tremendous importance, and confers various levels of acceptability on particular varieties or lots of grain. The enzyme polyphenol oxidase (PPO) has long been associated as the factor leading to the discoloration of raw noodles (mainly white salted and yellow alkaline). Based on this association, many wheat breeding programs have developed various assays for monitoring the activity of PPO in wheat germplasm. Interestingly, until now, no published reports existed on the purification of wheat protein linked with PPO activity observed in assays used for selecting grain with superior noodle quality traits. In this study, we purified a wheat protein linked to the PPO activity observed in assays currently used by breeders for screening wheat germplasm. Based on early observations that the majority of PPO activity was located in the bran layer of mature seeds, we used the milled bran fraction to purify protein associated with PPO activity. Our purified wheat protein migrated at approximately 67 kDa under denaturing conditions and approximately 45 kDa under non-denaturing and non-reducing conditions. N-terminal protein sequence from our purified wheat protein showed the best sequence identity to other plant PPO sequences from grape and pineapple. Polyclonal antibody developed from our purified PPO protein reacted with wheat proteins exhibiting molecular weights of approximately 67 kDa and 83 kDa. We propose that the 67 kDa protein is a mature, processed form of wheat PPO lacking a transit peptide for import into plastids and the 83 kDa wheat protein is an immature, unprocessed form still containing a 14-16 kDa transit peptide. In addition, our data suggests that immature seeds contain multiple isoforms of PPO but appear to be inhibited (inactive) unless activated by SDS or aging.

Technical Abstract: Wheat breeding programs have used various whole-seed assays for estimating polyphenol oxidase (PPO)-like activity and thereby identify germplasm that has a greater chance of producing consumer products with superior color characteristics. Yet, the biochemistry underlying these assays is poorly understood and the purification and characterization of a wheat caryopsis PPO has not been reported. Using continuous-elution electrophoresis as a final purification step, a protein was isolated from wheat bran that migrated at 67 kD on SDS-PAGE under denaturing and reducing conditions. The purified protein exhibited PPO activity in the presence of SDS, and eluted at 45 kD on SDS-PAGE under non- denaturing and non-reducing conditions. In the absence of SDS and reducing agents, a major peak of wheat PPO activity migrated in the 50 to 55 kD range during gel filtration chromatography. N-terminal sequence analysis of peptide fragments obtained from tryptic- digests confirmed the purified wheat bran protein as PPO. This wheat PPO protein showed the greatest sequence identity to grape and pineapple PPOs. Compared to the deduced amino acid sequence of a partial wheat PPO sequence which was recently isolated, the current PPO shows no more sequence identity than other plant taxa widely divergent from wheat. Based on immuno-blot analysis, purified PPO from wheat bran of mature caryopses appears to be a processed, mature form lacking an estimated 14 to 16 kD transit peptide required for plastid localization.