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Purification and Analysis of Wheat Grain Polyphenol Oxidase (PPO) Protein

¹USDA/ARS, Biosciences Research Laboratory, State University Station, Fargo, ND 58105. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable. ²USDA-ARS Western Wheat Quality Laboratory, Washington State University, Pullman, WA 99164-6394. ³Corresponding author. E-mail: morrisc@wsu.edu. Phone: +1.509.335.4062. Fax: +1.509.335.8573.

Wheat (Triticum aestivum L.) breeding programs have used various whole kernel assays to estimate polyphenol oxidase (PPO) activity, thereby identifying germplasm that has a greater chance of producing consumer products with superior color. However, the enzymes involved in these assays are poorly understood and the purification and characterization of a wheat kernel PPO protein has not been reported previously. A PPO from wheat bran was purified using ammonium sulfate precipitation, ion and size-exclusion chromatography, and continuous elution electrophoresis. The purified protein migrated at 67 kDa on SDS-PAGE under denaturing and reducing conditions, exhibited PPO activity in the presence of SDS, and eluted at 45 kDa on SDS-PAGE under nondenaturing and nonreducing conditions. N-terminal sequence analysis of peptide fragments obtained from tryptic digests confirmed the purified wheat bran protein as a PPO. This wheat PPO protein showed the greatest sequence identity to grape (Vitis vinifera) and pineapple (Ananas comosus) PPO. The purified wheat PPO shares no more sequence identity with the deduced amino acid sequence of a previously isolated partial wheat PPO sequence than it does to PPO from other plant taxa widely divergent from wheat. Based on immunoblot analysis, purified PPO from wheat bran appears to be a processed, mature form lacking an estimated 14–16 kDa transit peptide required for plastid localization.