Assay Procedure for Xanthine Oxidase Microbial

Principle

Xanthine + O₂ + H₂O ^{xanthine oxidase} > Uric acid + H₂O₂

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. Tris-HCl buffer, pH 7.5	：0.1 M
B. Sodium hydroxide solution	：0.025 M
C. Xanthine solution	：10 mM［Dissolve 15.2 mg of xanthine (MW＝152.11) in 10 mL solution (B)］
D. Oxonic acid potassium salt solution	：1 mM (Dissolve 9.75 mg of oxonic acid・K salt in 50 mL H2O)
E. Enzyme diluent	：50 mM Tris-HCl buffer, pH 7.5

Procedure

1. Prepare the following reaction mixture in a cuvette (d＝1.0cm) and equilibrate at 37℃ for about 5 minutes.
   
   2.24 mL Tris-HCl buffer, pH 7.5 (A)
   0.08 mL Xanthine solution (C)
   0.08 mL Oxonic acid solution (D)

Concentration in assay mixture
Tris-HCl buffer	ca.89.6 mM
Xanthine	0.32 mM
Oxonic acid	32 μM

1. Add 0.1 mL 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 (E) is added instead of the enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (E), dilute to 0.1－0.2U/mL with the same buffer and store on ice.

Calculation

Activity can be calculated by using the following formula：

Volume activity (U/mL) ＝		Δ OD/min (Δ OD test - Δ OD blank) x Vt x df 12.5 x 1.0 x Vs
＝		Δ OD/min x 2.0 x df

Weight activity (U/mg)＝(U/mL)×1/C

Vt	：Total volume (2.5 mL)
Vs	：Sample volume (0.1 mL)
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 our quality control procedure, please contact our Technical Service Department.