Uricase (U-TE)

  • Enzymes for Clinical Chemistry
Uricase

The enzyme is useful for the determination of uric acid in clinical analysis.

Origin recombinant E. coli
Systematic name

Urate : oxygen oxidoreductase
EC Number 1.7.3.3
Reaction formula

Urate + H2O + O2 →→→ Unidentified products + H2O2

　　　　　　　　　　　　　↓

　　　　　　　　　　 　　CO2 + Allantoin (racemic mixture)

SPECIFICATION

Appearance light brownish lyophilizate
Activity ≧4.0 U/mg
Contaminants Catalase　≦1.0 U/U%
Stabilizer sucrose, citrate, ethylenediaminetetraacetic acid (EDTA)
Storage condition below -20℃

PROPERTIES

Molecular weight ca. 90 kDa (gel filtration)
Structure 2 subunits of 35 kDa (SDS-PAGE)
Michaelis constant 1.1×10-5M (uric acid)
pH Optimum 8.5 (Fig. 1)
pH Stability 7.0–11.0 (Fig. 2)
Optimum temperature 45℃ (Fig. 3)
Thermal stability below 55℃ (Fig. 4)
Stability (liquid form) stable at 37℃ for at least ten days (Fig. 5)
Stability (powder form) stable at 30℃ at least three weeks (Fig. 6)
Inhibitors Hg2+，Ag+

APPLICATIONS

The enzyme is useful for the determination of uric acid in clinical analysis.

ASSAY PROCEDURE

Principle

The disappearance of uric acid is measured spectrophotometrically at 290 nm.

Definition of unit

One unit (U) is defined as the amount of enzyme which oxidizes 1 µmol of uric acid per min at 25℃ and pH 8.5 under the conditions described below.

Reagents

  • KOH solution, 20%: 20 g of KOH/100 ml of distilled water.
  • Uric acid solution, 0.001%: dilute the stock solution (0.01%) to 10-fold volume with enzyme dilution buffer (Reagent C) (prepare freshly). Stock solution: 10 mg of uric acid/100 ml of enzyme dilution buffer (Reagent C).
  • Enzyme dilution buffer: dissolve 3.09 g of boric acid, 0.37 g of EDTA-Na2-2H2O and 0.01 g of Triton X-100 in 800 ml of distilled water, adjust to pH 8.5 with 2 N NaOH and dilute with distilled water to 1000 ml.

Sample: dissolve the lyophilized enzyme to a volume activity of 0.01–0.02 U/ml with ice-cold enzyme dilution buffer (Reagent C) immediately before measurement.

Procedure

  1. Pipette the following reagent into a test tube.
    2.0 ml Uric acid solution (Reagent B)
    0.5 ml PDistilled water
  2. Equilibrate at 25℃ for about 5 min.
  3. Add 0.5 ml of sample and incubate for 5 min at 25℃.
  4. Add 0.2 ml of KOH solution (Reagent A) to stop the reaction.
  5. Read the absorbance at 290 nm in a cuvette (light path: 1 cm) (AS).
    The blank solution is prepared by reversing the sequence of addition of sample and KOH solution (Reagent A) (A0).

Calculation

Activity can be calculated by using the following formula:

EXPERIMENTAL DATA

REFERENCES

Koyama, Y. et al., J. Biochem., 120, 969–973 (1996).



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