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

An active proteolysed isoform of apple pulp polyphenoloxidase (PPO) wa s purified by a very quick three-step method based on its resistance t o further sodium dodecyl sulphate-proteinase K digestion. After extrac tion from a thylakoid membrane pellet and pre-purification by temperat ure-induced phase partitioning, PPO was subjected to sodium dodecyl su lphate-proteinase K digestion, and then 388-fold to homogeneity purifi ed by DEAE-cellulose column chromatography with a yield greater than 4 0%. This purified, enzymatically active PPO isoform was used to raise polyclonal antibodies. High titred specific serum was obtained and imm unoblots were performed to detect active and latent forms of the enzym e.