Six determination methods of Worthington peroxidase activity

Peroxidase  (HRP)  It's a kind of hemoglobin , It can catalyze the oxidation of various substrates by hydrogen peroxide , For example, ascorbate 、 Ferrocyanide 、 Cytochrome  c  And many colorless forms of dyes .HRP  Its molecular weight is  40 kDa, Most suitable  pH  The value is  7.0. stability ：HPOFF  stay  2-8°C  Stable under  9-12  Months .HPOD  stay  2-8°C  Stable under  2  to  3  year .

Unit definition ： One  Worthington  The unit is  25°C、pH 7.0  Use Aminoantipyrine and phenol to decompose every minute  1µmole H 2 O 2 .

Technical specification ：RZ (Reinheitzahl)  Is the absorbance ratio ,A 403 /A 275, Has been used as an indication of purity . However ,Shannon et al., JBC, 241 , 266 (1966)  This ratio of isozymes is reported in  2.50  To  4.19  Change between . This point , Together with buffer and  pH  The impact of value , It seems that the accuracy of this ratio as a purity standard is questioned .

Many different methods are used for the determination of peroxidase activity . The following is a list by emcee Worthington Some approximate transformations determined .

• 1  individual  Worthington  Company  = 4.6  individual  Worthington  O-benzidine units previously used

• 1  individual  Worthington  Company  = 0.62  individual  ABTS  Company （ Micromolar dye oxidized per minute ,pH 6.0,25°C,1.7 mM  dyestuff ）

• 1  individual  Worthington  Company  = 2  individual  ABST  Company （ µmole  Dyes that oxidize every minute ,pH 5.0,25°C,8.7 mM  dyestuff ）

• 1 Worthington  Company  = 0.5  Guaiacol unit （ Oxidized guaiacol per minute  µmole,pH 7.0,25°C）

• 1 Worthington  Company  = 0.5  Pyrogallol to  purpogallin Company （mg  Every product  20  second ,pH 6.0,20°C）

• 1 Worthington  Company  = 5  Pyrogallol to  purpogallin  Company （ stay  pH 6.0,30°C  Micro moles of product per minute ）

AI Meijie Worthington Determination of peroxidase ：

Method ： Traditionally , Peroxidase activity is expressed in the unit of oxidation rate of pyrogallol , This is a  Willstalter  and  Stoll  stay  1917  A method introduced in , Recent research shows that this method has some shortcomings .（Maehly  and  Chance 1954.） A variety of hydrogen donors have been used in peroxidase assay systems , Including compounds that may cause cancer , For example, o-anisidine . use  4- An improved method for the determination of Aminoantipyrine as a hydrogen donor has been adopted （Trinder 1966）. The reaction rate is measured by the decomposition of hydrogen peroxide  510 nm  Increase in absorbance at . Under specified conditions , stay  25°C  and  pH 7.0  Under the condition of , One unit will cause decomposition every minute  1  Micromolar hydrogen peroxide .

Reagents ：

0.2 M  Potassium phosphate buffer  pH 7.0

0.0017 M  Hydrogen peroxide . Use reagent grade water to  1 ml 30%  Hydrogen peroxide （Merck Superoxol  Or equivalent ） Dilute to  100 ml. use  0.2 M  Potassium phosphate buffer  pH 7.0  take  1 ml  The solution is further diluted to  50 ml. Prepare fresh food every day .

0.0025 M 4- Aminoantipyrine and  0.17 M  phenol ： By way of  810 mg  Phenol is dissolved in  40 ml  Prepare in reagent grade water . Join in  25 mg 4- Aminoantipyrine was diluted with reagent grade water to a final volume of  50 ml.

enzyme ：

With  1 mg/ml  Dissolve in reagent grade water . Before use , Further dilute to obtain  0.02-0.04 ΔA/min  Rate .

Program ：

Adjust the spectrophotometer to  510 nm  and  25°C.

Move the pipette into each cuvette , As shown below ：

phenol / Aminoantipyrine solution  1.4  ml

0.0017 M Hydrogen peroxide  1.5  ml

In the spectrophotometer at  25°C  Incubate  3-4  Minutes to reach temperature equilibrium and determine the blank rate （ If there is ）. Join in  0.1 ml  Dilute the enzyme and record  A 510 An increase in 4-5  minute . Calculate from the linear part of the curve ΔA 510 / minute .

Calculation ：

 

notes ： Although the reaction rate obtained by the phenol antipyrine method is lower than that of the previous method  4.5-4.7  times , But peroxidase preparations are the same .

RZ (Reinheitzahl)  Is the absorbance ratio ,A 403 /A 275 (RZ)  Has been used as an indication of purity . However , Shannon et al .(1966)  The report says , This ratio of isozymes ranges from  2.50  Change to  4.19. This point , Together with buffer and  pH  The impact of value , It seems that the accuracy of this ratio as a purity standard is questioned .