酶活测定.docVIP

Bok-Rye Leea,b，2012 Fresh samples (0.5 g) were extracted with 1.5 ml of 100 mM K-PO4 buffer solution (pH 7.0) containing 2 mM phenylmethylsulfonyl fluoride（苯甲基磺酰化氟）, and centrifuged at 14 000 g at 4?C for 20 min. For the determination of PPO activity, 1-ml reaction mixture containing 20 μl?enzyme extract and 10 mM phosphate buffer (pH 7.0) was aerated for 2 min in a small test tube followed by the addition of 20 mM catechol （苯邻二酚）(Sigma, Saint Louis,MO63103, USA) as the substrate. The change in A420 per min was measured (Siriphanich and Kader 1985). Woo-Jin Junga, 2011 Leaf samples (300 mg fresh weight) were extracted in 4 mL of buffer (50 mM Tris pH 8.5, 14.4 mM 2-mercaptoethanol（巯基乙醇）, 1% (w/v) insoluble polyvinylpolypyrrorolidone) and centrifuged at 6000 × g for 10 min at 4 ?C. The total protein concentration in soluble enzyme extracts was determined using the Bradford assay (1976). The activity of polyphenol oxidase (PPO; EC 1.14.18.1) was assessed using the method of Siriphanich and Kader (1985). In assaying for PPO activity, 1 mL of reaction mixture contained 100 μL enzyme extract and 50 mM Tris–HCl buffer (pH 8.0). Each sample was aerated for 2 min in a small test tube followed by the addition of pyrocatechol（邻苯二酚） (Sigma C-9510), as the substrate, at a final concentration of 20 mM. Increase in absorbance at 420 nm was recorded for 1 min at 25℃using a UV–vis spectrophotometer (Ultrospec 4300Pro, LKB, Cambridge, UK). One unit of enzyme activity was defined as the amount of enzyme that causes the increase of A 0.01 value per min. The results were expressed as change in A min?1 mg?1 protein. Proteins were extracted with 100 mM Na-Pi, pH 7, containing 2% ascorbic acid, and 20% PVPP (w/w) and the extracts were kept at 4_C for 30 min with occasional agitation. After cen-trifugation (32,600 g, 20 min, 4_C) the supernatants were recovered and desalted in PD-10 Sephadex G25 mini-columns (Pharmacia) using 50 mM Na-Pi, pH 6, for the elution. The protein concentration