Assay Procedure for p-Hydroxybenzoate Hydroxylase

Principle

The disappearance of NADPH is measured at 340nm by spectrophotometry.

Unit definition

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

Method

Reagents

 

A.Tris-malate buffer, pH 8.2 :50mM[Dissolve 3.03g of Tris (M.W=121.14) in ca.300ml of H2O and, after adjusting the pH to 8.2 at 25℃ with 1.0M maleic acid, fill up to 500ml with H2O.]
B. p-hydroxybenzoate solution :5.0mM[80mg p-hydroxybenzoate (Na salt)/100ml of buffer solution (A)] (Should be prepared fresh)
C. FAD solution :0.2mM[19mg FAD・Na2/100ml or buffer solution (A)](Should be prepared fresh)
D. NADPH solution :3.0mM[272mg NADPH・Na4・4H2O/100ml of buffer solution (A)](Should be prepared fresh)
E. Enzyme diluent :50mM K-phosphate buffer, pH 6.0 containing 0.2% BSA

Procedure

  1. Prepare the following working solution (10 tests) in a brownish bottle and store on ice.

    21.0ml Buffer solution       (A)
    3.0ml   Substrate solution (B)
    3.0ml   FAD solution         (C)
    3.0ml   NADPH solution    (D)
Concentration in assay mixture
Tris-malate buffer 49 mM
p-Hydroxybenzoate 0.49mM
FAD 20 μM
NADPH 0.30mM
  1. Pipette 3.0ml of working solution into a cuvette (d=1.0cm) and equilibrate at 37℃ for about 5 minutes.
  2. Add 0.05ml of the enzyme solution* and mix by gentle inversion.
  3. Record the decrease in optical density at 340nm against water for 3 to 4 minutes in a spectrophotometer thermostated at 37℃ and calculate the ΔOD per minutes 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 (E) is added instead of enzyme solution.


* Dissolve the enzyme preparation in ice-cold enzyme diluent (E) (1.0mg/ml or more) and dilute to 0.2-0.6 U/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)
6.22 :Millimolar extinction coefficient of NADPH (F/micromole)
1.0 :Light path length (cm)
df :Dilution factor
C :Enzyme concentration in dissolution (c mg/ml)

 

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

Materials

     
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