Uricase participates in purine catabolism. It catalyzes the conversion of highly insoluble uric acid into 5-hydroxyisourate. Accumulation of uric acid causes liver/kidney damage or chronically causes gout. In mice, a mutation in the gene encoding uricase causes a sudden increase in uric acid. Mice, deficient in this gene, exhibit hyperuricemia, hyperuricosuria, and uric acid crystalline obstructive nephropathy.
PREPARATION and SPECIFICATION
| Appearance: | White amorphous powder, lyophilized |
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| Activity: | ≥4U/mg enzyme powder (containing approx.20 % of stabilizers) |
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| Contaminants: | Catalase ≤1.0 % |
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| Stabilizers: | Borate, EDTA, nonionic detergents |
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PROPERTIES
| Stability | Stable at −20℃ for at least one year | (Fig.1) | | Molecular weight | approx. 120,000 | | | Structure | 4 subunits per enzyme molecule (Reactive SH groups are present in the enzyme molecule) | | | Isoelectric point | 5.4 | | | Michaelis constant | 2.5×10⁻³M (Uric acid) | | | Inhibitors | Heavy metal ions, cyanide, various urate analogs | | | Optimum pH | 8.5 | (Fig.4) | | Optimum temperature | 40℃ | (Fig.5) | | pH Stability | pH7.0−11.0 (25℃, 20hr) | (Fig.6)
| | Thermal stability | below 50℃ (pH 8.5, 10min) | (Fig.7)
| | Effect of various chemicals | (Table.1) |
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APPLICATIONS
This enzyme is useful for enzymatic determination of uric acid in clinical analysis.
ASSAY
Principle:
The elimination of uric acid is measured at 290 nm by spectrophotometry.
Unit definition
One unit causes the oxidation of one micromole of uric acid per minute under the conditions detailed below.
Method:
Reagents
A. Uric acid solution: 0.001% [Dilute the stock solution (0.01%) to 10-fold volume with 50mM borate buffer containing 0.001% Triton X-100 and 1.0mM EDTA, pH 8.5] (Should be prepared fresh). Stock solution: 10mg uric and/100ml of above buffer (store at 0−5℃)
B. KOH solution: 20%
C. Enzyme diluent: 50mM borate buffer containing 0.001% Triton X-100 and 1.0mM EDTA, pH 8.5
Procedure
1. Prepare the following reaction mixture in a test tube and equilibrate at 25 ℃ for approximately 5 minutes.
2.0 mL Uric acid solution (A)
0.5 mL Distilled water
Concentration in assay mixtureBorate buffer
| Borate buffer: | 42 mM |
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| Uric acid: | 40 μM |
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| EDTA: | 0.83 mM |
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| Triton X-100: | 0.00083 % |
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2. Add 0.5 mL of the enzyme solution* and mix by gentle inversion.
3.After exactly 5 minutes at 25 ℃, add 0.2 mL of 20 % KOH solution (B) to stop the reaction and measure the optical density at 290 nm against water (OD test).
At the same time, prepare the blank by mixing the reaction mixture with 0.2 mL of KOH solution after incubation for 5 minutes at 25 ℃, and then adding the enzyme solution (OD blank).
*Dissolve the enzyme preparation in ice-cold enzyme diluent (C) and dilute to 0.01−0.02 U/mL with the same buffer and store on ice.
Calculation
Activity can be calculated by using the following formula:
| Vt | : Total volume (3.2 mL) |
| Vs | : Sample volume (0.5 mL) |
| 12.2 | : Millimolar extinction coefficient of uric acid (cm²/micromole) |
| t | : Reaction time (5 minutes) |
| 1.0 | : Light path length (cm) |
| df | : Dilution factor |
| C | : Enzyme concentration in dissolution (c mg/mL) |
Table 1 Effect of Various Chemicals on Uricase
[The enzyme dissolved in 50mM borate buffer, pH 8.5 containing 0.001% Triton X-100 and 1.0mM EDTA (10U/ml) was incubated with each chemical at 25℃ for 1hr.］
| Chemical | Concn.(mM) | Residual activity(%) | Chemical | Concn.(mM) | Residual activity(%) |
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| None | — | 100 | MIA | 2.0 | 70 | | Metal salt | | | NEM | 2.0 | 41 | | MgCl₂ | 2.0 | 95 | IAA | 2.0 | 94 | | CaCl₂ | 2.0 | 91 | Hydroxylamine | 2.0 | 95 | | Ba(OAc)₂ | 2.0 | 95 | EDTA | 5.0 | 90 | | FeCl₃ | 2.0 | 88 | o-Phenanthroline | 2.0 | 98 | | CoCl₂ | 2.0 | 88 | α,α'-Dipyridyl | 1.0 | 99 | | MnCl₂ | 2.0 | 84 | Borate | 50 | 90 | | ZnCl₂ | 2.0 | 69 | NaF | 2.0 | 87 | | CdCl₂ | 2.0 | 64 | NaN₃ | 2.0 | 96 | | NiCl₂ | 2.0 | 79 | Triton X-100 | 0.10% | 111 | | CuSO₄ | 2.0 | 9.8 | Brij 35 | 0.10% | 94 | | Pb(OAc)₂ | 2.0 | 74 | Tween 20 | 0.10% | 85 | | AgNO₃ | 2.0 | 0 | Span 20 | 0.10% | 91 | | HgCl₂ | 2.0 | 0 | Na-cholate | 0.10% | 96 | | 2-Mercaptoethanol | 2.0 | 101 | SDS | 0.05% | 91 | | PCMB | 1.0 | 34 | DAC | 0.05% | 89 |
Ac, CH₃CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; EDTA, Ethylenediaminetetraacetate; IAA, Iodoacetamide; NEM, N-Ethylmaleimide; SDS, Sodium dodecyl sulfate; DAC, Dimethylbenzylalkylammonium chloride.
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