Assay Procedure for Protocatechuate 3,4-Dioxygenase

Principle

The disappearance of protocatechuate is measured at 290nm by spectrophotometry.

Unit definition

One unit causes the oxidation of one micromole of protocatechuate per minute under the conditions described below.

Method

Reagents

 

A. Tris-acetate buffer, pH 7.5 :50mM[Dissolve 6.1g of Tris (MW=121.14) in ca.800ml of H2O and, after adjusting pH to 7.5 at 25℃ with 0.2M acetic acid, fill up to 1,000ml with H2O.]
B. Protocatechuate acid solution :0.4mM[Dissolve 6.16mg of protocatechuate in ca.80ml of buffer (A) and, after adjusting pH to 7.5 at 25℃ with 1.0N KOH, fill up to 100ml with buffer (A).] (Should be prepared fresh)

Procedure

  1. Pipette 3.0ml of protocatechuate solution (B) into a cuvette (d=1.0cm) and equilibrate at 37℃ for about 5 minutes.
Concentration in assay mixture
Tris-acetate buffer 50 mM
Protocatechuate 0.39mM
  1. Add 0.05ml of the enzyme solution* and mix by gentle inversion.
  2. Record the decrease in optical density at 290nm against water for 3 to 4 minutes in a spectrophotometer thermostated at 37℃, and calculate the ΔOD per minute from the initial linear portion of the curve (ΔOD test).

At the same time, measure the blank rate (ΔOD blank) by using the same method as the test except that the enzyme diluent (A) is added instead of the enzyme solution.

* Dissolve the enzyme preparation in ice-cold diluent (A) (1.0mg/ml or more) and dilute to 0.2-0.8U/ml with the same buffer, immediately before assay.

Calculation

Activity can be calculated by using the following formula:

 

Weight activity (U/mg)=(U/ml)×1/C

 

Vt :Total volume (3.05ml)
Vs :Sample volume (0.05ml)
3.8 :Millimolar extinction coefficient of protocatechuate (F/micromole)
1.0 :Light path length (cm)
df :Dilution factor
C :Enzyme concentration in dissolution (c mg/ml)

 

Materials

     

This procedure is for informational purposes. For a current copy of Sigma’s quality control procedure contact our Technical Service Department.

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