Assay Procedure for Nucleoside Phosphorylase Bacterial

Principle

The appearance of uric acid is measured at 293nm by spectrophotometry.

Unit definition

One unit causes the formation of one micromole of uric acid per minute under the conditions described below.

Method

Reagents

 

A. K-Phosphate buffer, pH7.7
:50mM
B. Inosine solution :32mM[Dissolve 85.8mg of inosine (MW=268.23) in 10ml of H2O with heating] (Stable for at least two weeks if stored at 4℃)
C. Xanthine oxidase solution :ca.6.6U/ml[Dissolve xanthine oxidase (XTO-212) to ca.6.6U/ml with ice-cold buffer A](Should be prepared fresh)
D. Enzyme diluent :buffer A

Procedure

  1. Prepare the following reaction mixture in a cuvette (d=1.0cm) and equilibrate at 37℃ for about 5 minutes.

    2.7ml K-Phosphate buffer, pH 7.7 (A)
    0.2ml Substrate solution                (B)
    0.1ml Xanthine oxidase solution  (C)
Concentration in assay mixture
K-Phosphate buffer ca.47 mM
Inosine 2.1 mM
Xanthine oxidase ca. 0.2U/ml
  1. Add 0.05ml of the enzyme solution* and mix by gentle inversion.
  2. Record the increase in optical density at 293nm 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 (D) is added instead of the enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (D) and dilute to 0.1-1.5U/ml with the same buffer and store on ice.

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)
12.5 :Millimolar extinction coefficient of uric acid under the assay condition (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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