Characteristics and determination scheme of Worthington mushroom polyphenol oxidase

Polyphenol oxidase （ Tyrosinase ）（TY） It is a bifunctional copper containing oxidase , It has CATECHOLASE and cresolase activities （Malmström  and  Rydén 1968）.

Jolly et al .(1974)  Call it oxygen and  4  Electron transfer phenol oxidase . It is responsible for the browning reaction on the whole phylogenetic scale . Although tyrosinase from Neurospora crassa has been purified （Fling et al . 1963）, But most of the work is done with mushroom enzyme , Although the output and consistency are very poor ;Smith and Krueger (1962)  Proved its diversity .Bouchilloux wait forsomeone .(1963)  Four enzymes were obtained .

Worthington  Characteristics of mushroom polyphenol oxidase ：

Molecular weight ：128,000 (Duckworth and Coleman 1970).

form ： The enzyme is a tetramer , Each molecule contains  4  Gram copper atom  (Jolley et al . 1974), And two binding sites of aromatic compounds including phenolic substrates . There is also a significantly different oxygen binding site , Copper site （Duckworth and Coleman 1970）. Copper may be in cuprous state ; The inactivation of this enzyme is related to Cu 2+ Related to the increase of .（Kertész wait forsomeone ,1972  year ）. The amino acid composition has been determined .Jolley Extensive structural studies have been reported by et al .（1969）; And Davos and Coleman （1970）. See also  Jolley wait forsomeone .（1972  year 、1973  Years and  1974  year ）.

The best  pH  value ：6.0-7.0.

Extinction coefficient ： Extinction coefficient = 24.9（ Immediately after purification ）（Duckworth  and  Coleman 1970）.

Inhibitors ： Compounds complexed with copper . The enzyme is also competitively inhibited by benzoic acid p-catechol and cyanide p-oxygen （Duckworth  and  Coleman 1970）.

AI Meijie Worthington  Polyphenol oxidase activity ：

Polyphenol oxidase is an oxygen transferase . Besides using  O 2 Catalytic dehydrogenation of catechol to produce o-quinone and o-hydroxylation of phenol to produce catechol ,Strothkamp  and  Mason (1974)  Peroxidase activity was also reported .Kertész Kinetic studies have been reported by et al .（1971）. See also  Malmström  and  Rydén  The comments .

Specificity ： A large number of para substituted catechins are oxidized （Duckworth  and  Coleman 1970）.

stability ： The lyophilized preparation is  -20°C  It can be stored stably under  6-12  Months .

AI Meijie Worthington  Polyphenol oxidase assay ：

Method ： Polyphenol oxidase oxidizes tyrosine to dihydroxyphenylalanine , Dihydroxyphenylalanine is oxidized to o-quinone . The latter is accompanied by  280 nm  Increase in absorbance at . The rate of increase is proportional to the enzyme concentration , And after the initial lag  5-10  Linear relationship within minutes . Under specified conditions , stay  25°C、pH 6.5  Next , A unit in  280 nm  Cause every minute  0.001  Change in absorbance .

Reagents ：

0.5 M  Potassium phosphate buffer ,pH 6.5

0.001 M L- Tyrosine

enzyme ：

With  1 mg/ml  The concentration of is dissolved in reagent grade water . Further dilute in reagent grade water to  200-400  Company / ML concentration .

Program ：

Adjust the spectrophotometer to  280 nm  and  25°C. Move the pipette into each cuvette , As shown below ：

0.5 M  Phosphate buffer ,pH 6.5 1.0  ml

0.001 M L- Tyrosine  1.0  ml

Reagent grade water  0.9  ml

Bubble oxygen into the cuvette through the capillary  4-5  minute , Oxygenate the reaction mixture . Transfer the cuvette to the spectrophotometer and record  A 280 4-5  Minutes to reach temperature equilibrium and determine the blank rate （ If there is ）. Join in  0.1 ml  Properly diluted enzyme , Record  A 280 10-12  minute . Determine from the linear part of the curve ΔA 280 . There is expected to be  2-3  Minute nonlinearity “ lagging ”.