PURIFICATION OF AN APPLE POLYPHENOLOXIDASE ISOFORM RESISTANT TO SDS PROTEINASE-K DIGESTION

An active proteolysed isoform of apple pulp polyphenoloxidase (PPO) was purified by a very quick three-step method based on its resistance to further sodium dodecyl sulphate-proteinase K digestion. After extraction from a thylakoid membrane pellet and pre-purification by temperature-induced phase partitioning, PPO was subjected to sodium dodecyl sulphate-proteinase K digestion, and then 388-fold to homogeneity purified by DEAE-cellulose column chromatography with a yield greater than 40%. This purified, enzymatically active PPO isoform was used to raise polyclonal antibodies. High titred specific serum was obtained and immunoblots were performed to detect active and latent forms of the enzyme.