A A C C   2 0 0 0   A n n u a l   M e e t i n g

Polyphenol oxidase (EC 1.14.18.1) is a copper containing enzyme that is widely distributed in plant species and in animals. This enzyme catalyzes in the presence of molecular oxygen: Hydroxylation of monophenols to o-diphenols (monophenolase activity) and oxidation of o-diphenols to o-quinones (diphenolase activity) which then polymerize non-enzymatically into red, brown or black pigment (melanin). It is this latter property that makes polyphenol oxidase (PPO) an important enzyme in food industry as it causes browning in some of the wheat-based food products. In wheat, this enzyme can be found in either latent or active form, the former dominating in the bran. The latent PPO is often unintentionally activated during isolation. The activated enzyme may initiate a host of unwanted reactions culminating in the formation of o-quinones. These highly reactive quinones may condense with a variety of amino acid residues in proteins and/or carbohydrates. This normally renders it impracticable to purify any protein from such sources. We have taken special precautions to avert this situation by minimizing the chances of quinone formation. PPO inhibitors and antioxidants, protease inhibitors and non-ionic detergent are utilized as a cocktail in the isolation buffer that enables isolation of PPO in zymogen form. Purification is subsequently achieved by employing a combination of ammonium sulfate precipitation, hydrophobic interaction chromatography, ion exchange chromatography and affinity chromatography. The isolated isozymes have been biochemically characterized and the results will be presented in this poster.