Unfolding and refolding of active apple polyphenol oxidase

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 I M (NH4)(2)SO4 and 100 mu M CuSO4. However, CuSO4 had limited effect on the recovering of PPOr activity, whereas (NH4)(2)SO4 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. (C) 1998 Elsevier Science Ltd. All rights reserved.