title: Unfolding and refolding of active apple polyphenol oxidase.
abstract: For the first time, unfolding (6 M guanidine) and refolding of partially proteolysed purified polyphenol oxidase (PPOr) was achieved, with 88% of activity recovered. Optimal refolding conditions consisted in stepwise dialysis of guanidine treated extracts, the dialysis buffers containing 1 M (NH4)2SO4 and 100 microM CuSO4. However, CuSO4 had limited effect on the recovering of PPOr activity, whereas (NH4)2SO4 was essential. Concerning the PPO tertiary structure, denaturing conditions (combinations of boiling and reducing agent) used on SDS-PAGE have shown (i) a compact tertiary structure and (ii) the presence of disulfide bonds in PPOr, accounting for the shift between 27 and 41 kDa, and 41 and 42 kDa, respectively. Resistance to proteolytic cleavage was used to study the conformational changes induced by the denaturing treatments. Folded PPOr was resistant to further proteolysis whereas unfolded PPO was totally digested, indicating the role of tertiary structure of PPOr in the resistance to proteases.
authors.affiliation: Laboratoire de Biotechnologie et Physiologie végétales Appliquées, Université Montpellier II, France.
authors.forename: S
authors.initials: S
authors.lastname: Mari
authors.forename: L
authors.initials: L
authors.lastname: Marquès
authors.forename: F
authors.initials: F
authors.lastname: Breton
authors.forename: Y
authors.initials: Y
authors.lastname: Karamanos
authors.forename: J J
authors.initials: JJ
authors.lastname: Macheix
mesh_terms: D004156:Catechol Oxidase / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D010940:Plant Proteins / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D019685:Rosales / Q000201:enzymology*; D012967:Sodium Dodecyl Sulfate
chemical_list: D004220:Disulfides; D010940:Plant Proteins; D012967:Sodium Dodecyl Sulfate; D004156:Catechol Oxidase