**3. Materials and Methods**

#### *3.1. Materials and Chemicals*

Fresh orange (*Citrus sinensis* Osbeck) used in this study was procured from a local market (Chongqing, China). All chemicals used were of analytical grade.

#### *3.2. Extraction and Partial Purification of Protein*

Orange tissue (250 g) was homogenized in 700 mL of Tris-HCl buffer (0.5 mol/L) containing 10% polyvinylpyrrolidone (PVPP). The homogenate liquid was stored for 8 h at 4 ◦C and then subjected to centrifugation at 2057× *g* for 10 min using a centrifuge machine (Eppendorf centrifuge 5804 R, Eppendorf, Hamburg, Germany). The resultant supernatant was fractionated with 25% solid ammonium sulfate to remove impurities. The process was repeated using 90% ammonium sulfate saturation to precipitate proteins with PPO activity. The precipitate was re-dissolved in Tris-HCl (0.5 mol/L) and dialyzed against the same buffer for 34 h. The buffer was changed every hour during the whole process of dialysis. Ultrafilter was used to concentrate the crude extract of protein. Then, DEAE Sepharose Fast Flow and Sepracryl S-200 columns were used to purify the crude protein [5]. The fractions containing the highest activity of PPO were selected, concentrated and stored for further analysis.

#### *3.3. Electrophoresis Assay*

Native PAGE was carried out on preparative 12% polyacrylamide gels using the method described by Davis [30] with slight modifications. After running, the gels were stained with 0.1 mol/L Catechol (50 mL) and Coomassie Brilliant Blue R-250. The gels were analyzed for activity and estimation of molecular weight. The molecular weight and subunit of purified PPO enzyme were determined by SDS–PAGE [31].
