URICASE from Bacillus sp

UAO-211

PREPARATION and SPECIFICATION
Appearance:	White amorphous powder, lyophilized
Activity:	GradeⅡ 1.5U/mg-solid or more
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.150,000
Structure:	4 subunits per molecule
Isoelectric point :	4.7
Michaelis constant:	1.36×10⁻⁵M (Uric acid)
Inhibitors:	Ag⁺,Hg⁺⁺
Optimum pH :	8.5(Fig.4)
Optimum temperature :	45°C(Fig.5)
pH Stability :	pH 6.0-9.5 (25°C, 20hr)(Fig.6)
Thermal stability :	below 60°C (pH 8.0, 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

Uric acid+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 8.0](Should be prepared fresh) Stock solution : 10.0mg uric acid/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 8.0

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 37°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 37°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 37°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

S.Asano, K.Yamamoto, S.Teshima, T.Kikuchi and Y.Kawamura; Rinsho Kagaku, 23 (3), 214 (1994)
K.Yamamoto, Y.Kojima, T.Kikuchi, T.Shigyo, K.Sugihara, M.Takashio and S.Emi; J.Biochem. 119, 80 (1996)

**Table 1. Effect of Various Chemicals on Uricase**
[This enzyme dissolved in 50mM borate buffer, pH 8.0 (5U/ml) was incubated with each chemical at 25°C for 1hr.]
Chemical	Concn.(mM)	Residual activity(%)	Chemical	Concn.(mM)	Residual activity(%)
None	−	100	EDTA	5.0	104
Metal salt	2.0		o-Phenanthroline	2.0	91
MgCl₂		89	α,α′-Dipyridyl	1.0	89
CaCl₂		91	Borate	50	102
Ba(OAc)₂		118	IAA	2.0	93
FeCl₃		95	NEM	2.0	107
CoCl₂		91	Hydroxylamine	2.0	91
MnCl₂		93	2-Mercaptoethanol	2.0	104
ZnSO₄		91	Triton X-100	0.10%	93
Cd(OAc)₂		87	Brij 35	0.10%	93
NiCl₂		89	Tween 20	0.10%	98
CuSO₂		78	Span 20	0.10%	103
Pb(OAc)₂		91	Na-cholate	0.10%	98
AgNO₃		44	SDS	0.05%	91
HgCl₂		46	DAC	0.05%	89
PCMB	2.0	96
MIA	2.0	91
NaF	2.0	91
NaN₃	2.0	91

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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