UA-R (Uricase)
This enzyme is useful for enzymatic determination of uric acid in clinical analysis.
Specification
|
Appearance |
White amorphous powder, lyophilized |
|
Protein purity |
≥90% (SDS-PAGE) |
|
Activity |
≥20 U/mg solid |
|
Catalase |
≤0.03% |
|
NADH/NADPH oxidase |
≤0.01% |
Properties
|
EC number |
1.7.3.3 (Recombinant from microorganism) |
|
|
Molecular weight |
40 kDa (SDS-PAGE) |
|
|
Isoelectric point |
5. 1 |
|
|
Michaelis constants |
2. 1× 10-5 M (Urate) |
|
|
Inhibitors |
Fe3+, Co2+, Cu2+, Mn2+, Zn2+ |
|
|
Optimum pH |
8.5 |
Fig. 1 |
|
Optimum temperature |
37 ℃ |
Fig. 2 |
|
pH stability |
pH 5.5- 10 .0 (25 ℃, 16 h) |
Fig. 3 |
|
Thermal stability |
60 ℃ (pH 8.5, 30 min) |
Fig. 4 |
|
Stabilizer |
Borate, EDTA and nonionic surfactant |
|
Figures
Assay Principle
The decrement of uric acid is measured at 290nm by spectrophotometry.
Unit definition
One unit (U) is defined as the amount of enzyme which consumes 1 μmol of uric acid per min under the conditions described below.
Reagents preparation
Reagent I: 0.001% uric acid. Enzyme diluent: Add 3.09 g boric acid, 0.37 g EDTA-Na2·2H2O and 0.01g Triton X-100 in 800 ml pure water, adjust to pH 8.5 by NaOH and set to 1000 mL by pure water. Sample: The enzyme was diluted to 0.25-0.5U/mL with enzyme diluent.
Procedure
1. Preincubate the Reagent I at 37 ℃.
2. Add 2 mL of Reagent I, 0.5 mL of pure water, and then add the diluted enzyme to a cuvette.
3. Record the ΔAs at 290 nm in 1 minute in a spectrophotometer thermostated at 37℃.
At the same time, measure the blank rate ΔAb by using the same method as the test except that the enzyme diluent is added instead of the enzyme solution.
∆A = ∆As - ∆Ab
Calculation
3.0: Total volume (mL)
0.5: enzyme volume (mL)
1.0: Light path length (cm)
df: Dilution factor C: Enzyme concentration (mg/mL)
5.0: Conversion factor of activity
12.2: Millimolar extinction coefficient of uric acid under 290 nm (cm2/μmol)
References
1. Bongaerts, G. P. A., Uitzerter, J., Brouns, R. and Vogeis, G. D. (1978) Biochim. Biophys. Acta, 527, 348–358.
2. Bongaerts, G. P. A. and Vogeis, G. D. (1976) J. Bacteriol., 125, 689–697.
