The purification and spectral properties of polyphenol oxidase I fromNicotiana tabacum

Abstract

Polyphenol oxidase plays a key role in plant defense systems. We report the first-time purification of polyphenol oxidase (PPO 1.14.18.1) from fresh leaves of tobacco (Nicotiana tabacum) using acetone powder, ammonium sulfate precipitation, and column chromatography with DEAE-Sephadex A-50, CM-Sephadex C-50, and Sephadex G-75. PPO I was purified approximately 71-fold (3200 U/mg). The MALDI-TOF-MS spectrum showed that the enzyme was purified to a pure protein with a molecular weight of 35700 Da. The optimum pH of PPO I was 7, the optimum temperature was 40°C, and the Km value was 6.8 mM using catechol as the substrate at pH 6.5 and with 0.05 M H₃PO₄−NaOH buffer. The maximum emission peak of PPO I was 339 nm with 16 nm of blue-shifted compared with 355 nm of free tryptophan. The UV/VIS spectra and the absence of an EPR signal are indicative of type-3 coppers, but not type-1 or type-2 coppers. PPO I and mushroom PPO have the same active center for a pair of coupled antiferromagnetic copper ions.