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Purification of an apple polyphenoloxidase isoform resistant to SDS-proteinase K digestion

1994; Elsevier BV; Volume: 36; Issue: 5 Linguagem: Inglês

10.1016/s0031-9422(00)89623-5

1873-3700

L. Pons Marqués, Annie Fleuriet, Jean-Claude Cleyet-Marel, Jean‐Jacques Macheix,

Postharvest Quality and Shelf Life Management

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.