UNFOLDING AND REFOLDING OF ACTIVE APPLE POLYPHENOL OXIDASE

For the first time, unfolding (6 M guanidine) and refolding of partial ly proteolysed purified polyphenol oxidase (PPOr) was achieved, with 8 8% of activity recovered. Optimal refolding conditions consisted in st epwise dialysis of guanidine treated extracts, the dialysis buffers co ntaining I M (NH4)(2)SO4 and 100 mu M CuSO4. However, CuSO4 had limite d effect on the recovering of PPOr activity, whereas (NH4)(2)SO4 was e ssential. Concerning the PPO tertiary structure, denaturing conditions (combinations of boiling and reducing agent) used on SDS-PAGE have sh own (i) a compact tertiary structure and (ii) the presence of disulfid e bonds in PPOr, accounting for the shift between 27 and 41 kDa, and 4 1 and 42 kDa, respectively. Resistance to proteolytic cleavage was use d to study the conformational changes induced by the denaturing treatm ents. Folded PPOr was resistant to further proteolysis whereas unfolde d 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.