URICASE from Candida sp.

UAO-201

PREPARATION and SPECIFICATION
Appearance:  White amorphous powder, lyophilized
Activity:  GradeⅡ 4.0U/mg-solid or more
(containing approx.20% of stabilizers)
Contaminant:  Catalase ≤1.0%
Stabilizers:  Borate, EDTA, nonionic detergents
PROPERTIES
Stability:  Stable at -20°C 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:  2.5×10⁻⁵M (Uric acid)
Inhibitors:  Heavy metal ions, cyanide,various urate analogs
Optimum pH :  8.5(Fig.4)
Optimum temperature :  40°C(Fig.5)
pH Stability :  pH 7.0-11.0 (25°C, 20hr)(Fig.6)
Thermal stability :  below 50°C (pH 8.5, 10min)(Fig.7)
Effect of various chemicals:  (Table 1)

APPLICATIONS

This enzyme is useful for enzymatic determination of uric acid in clinical analysis.

ASSAY

Principle:

uricase

Uricacid+O₂+2H₂O                                                  ► Allantoin+H₂O₂+CO₂
The disappearance of uric acid is measured at 290nm by spectrophotometry.

Unit definition:

One unit causes the oxidation of one micromole of uric acid per minute under the conditions described 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°C)
B. KOH solution:  20%
C. Enzyme diluent :  50mM borate buffer containing 0.001% Triton X-100 and 1.0mM EDTA, pH 8.5

Procedure

Concentration in assay mixture
Borate buffer 42 mM
Uric acid 40 µM
EDTA 0.83 mM
Triton X-100 0.00083%

1. Prepare the following reaction mixture in a test tube and equilibrate at 25°C for about 5 minutes.

2.0ml Uric acid solution (A)
0.5ml Distilled water

2. Add 0.5ml of the enzyme solution* and mix by gentle inversion.

3. After exactly 5 minutes at 25°C, add 0.2ml of 20% KOH solution (B) to stop the reaction and measure the optical density at 290nm against water (OD test).
At the same time, prepare the blank by first mixing the reaction mixture with 0.2ml of KOH solution after 5min-incubation at 25°C, followed by the addition of the enzyme solution (OD blank).

* Dissolve the enzyme preparation in ice-cold enzyme diluent (C) and dilute to 0.01−0.02U/ml with the same buffer and store on ice.

Calculation

Activity can be calculated by using the following formula :

ΔOD (OD blank-OD test)×Vt×df

Volume activity (U/ml) =                                                               = ΔOD×0.105×df

12.2×1.0× t ×Vs


Weight activity (U/mg)=(U/ml)×1/C

Vt
: Total volume (3.2ml)
Vs
: Sample volume (0.5ml)
12.2
: Millimolar extinction coefficient of uric acid (㎠/micromole)
t
: Reaction time (5 minutes)
1.0
: Light path length (cm)
df
: Dilution factor
C
: Enzyme concentration in dissolution (c mg/ml)

REFERENCES

  1. K.Itaya,T.Yamamoto and J.Fukumoto; Agric.Biol.Chem., 31, 1256 (1967)
  2. Y.Nakagiri and T.Yamamoto; Eisei Kensa(Japanese), 20, 751 (1971)
  3. N.Kageyama et al.; Eisei Kensa(Japanese), 19, 338 (1969)
  4. N.Kageyama et al.; Eisei Kensa(Japanese), 18, 59 (1969)
  5. N.Kageyama; Rirsho Kensa(Japanese), 16, 891 (1972)
  6. N.Kageyama; Clin.Chim.Acta, 31, 421 (1971)
  7. T.Kawashima et al.; Nihon Kagakukaishi(Japanese), 10, 1542 (1980)
  8. H.Nishimura et al.; J.Biochem., 91, 41-48 (1982)
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°C for 1hr.]
Chemical Concn.(mM) Residual
activity(%)		 Chemical Concn.(mM) Residual
activity(%)
None − 100 MIA 2.0 70
Metal salt
2.0   NEM 2.0 41
MgCl₂   95 IAA 2.0 94
CaCl₂
   91
 Hydroxylamine 2.0 95
Ba(OAc)₂   95 EDTA 5.0 90
FeCl₃   88 o-Phenanthroline 2.0 98
CoCl₂   88 α,α′-Dipyridyl 1.0 99
MnCl₂   84
 Borate 50 90
ZnCl₂   69 Naf 2.0 87
CdCl₂   64 NaN₃ 2.0 96
NiCl₂   79 Triton X-100 0.10% 111
CuSO₄   9.8 Brij 35 0.10% 94
Pb(OAc)₂   74 Tween 20 0.10% 85
AgNO₃   0 Span 20 0.10% 91
HgCl₂   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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