Uricase (U-TE)

Enzymes for Clinical Chemistry

CD : 60199

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

Pipette the following reagent into a test tube.

2.0 ml Uric acid solution (Reagent B)

0.5 ml PDistilled water
Equilibrate at 25℃ for about 5 min.
Add 0.5 ml of sample and incubate for 5 min at 25℃.
Add 0.2 ml of KOH solution (Reagent A) to stop the reaction.
Read the absorbance at 290 nm in a cuvette (light path: 1 cm) (A_S).
The blank solution is prepared by reversing the sequence of addition of sample and KOH solution (Reagent A) (A₀).

2.0 ml	Uric acid solution	(Reagent B)
0.5 ml	PDistilled water

Calculation

Activity can be calculated by using the following formula:

EXPERIMENTAL DATA

FAQ

What precautions should be taken when handling the product after opening?: To avoid moisture absorption of the enzyme powder, please allow it to return to room temperature in a sealed state before use. (Recommended conditions: around 25°C, humidity below 55%)

How should U-TE be stored?: Please store at or below -20°C.

What stabilizers are used in U-TE?: Sucrose, citrate, and EDTA are used as stabilizers.

How thermostable is U-TE?: It is stable in liquid form below 60°C (Fig.4) and remains stable at 37°C for at least ten days (Fig.5). In powder form, it remains stable at 30°C for at least three weeks (Fig.6).

What are the optimal pH and temperature for U-TE?: The optimal pH is 7.0–8.0, and the optimal temperature is 37–45°C.

What is the molecular weight of U-TE?: The molecular weight is approximately 90 kDa (gel filtration), and it exhibits a dimeric structure composed of subunits of 35 kDa each.