Uricase (UAO) from Candida sp., CAS No.9002-12-4

CAS: 9002-12-4 Cat. No.: U1493005 EC Number: 232-655-5 PubChem CID: 14228836

GRADE & PURITY Bioactive, ActiBioPure™, Native, High Performance, EnzymoPure™, ≥4U/mg enzyme powder

Accession #

G8ZWT4

Bioactivity

≥4U/mg enzyme powder

Overview

 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
Activity:	≥4U/mg enzyme powder (containing approx.20 % of stabilizers)
Contaminants:	Catalase ≤1.0 %
Stabilizers:	Borate, EDTA, nonionic detergents

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)

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
Uric acid:	40 μM
EDTA:	0.83 mM
Triton X-100:	0.00083 %

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(%)
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.

Specifications

Product Name

Uricase (UAO) from Candida sp., CAS No.9002-12-4

Synonyms

Urate: oxygen oxoreductase | factor independent urate hydroxylase | urate oxidase | uric acid oxidase | uricase | uricase II

Grade

ActiBioPure™, Bioactive, High Performance, Native, EnzymoPure™

Specifications & Purity

Bioactive, ActiBioPure™, Native, High Performance, EnzymoPure™, ≥4U/mg enzyme powder

Biochemical and Physiological Mechanisms

Catalyzes the oxidation of uric acid to 5-hydroxyisourate, which is further processed to form (S)-allantoin.

Bioactivity

≥4U/mg enzyme powder

Accession #

G8ZWT4

Predicted molecular weight

~120 kDa

CAS

9002-12-4

Molecule Type

Enzyme

Storage and Shipping

Concentration

≥4U/mg enzyme powder

Reconstitution

Reconstitute in 50 mM borate buffer containing 0.001 % Triton X-100 and 1.0 mM EDTA, pH 8.5.

Storage

Store at -20°C,Avoid repeated freezing and thawing

Shipped In

Ice chest + Ice pads

Stability And Storage

Store at -20℃ long term (12 months). Upon reconstitution, it is recommended to aliquot. Avoid freeze/thaw cycle.

Unit definition

One unit causes the oxidation of one micromole of uric acid per minute under the conditions detailed below.

References

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2. Fuad Abduro Bushira, Shimeles Addisu Kitte, Yong Wang, Haijuan Li, Ping Wang, Yongdong Jin.  (2021)  Plasmon-Boosted Cu-Doped TiO2 Oxygen Vacancy-Rich Luminol Electrochemiluminescence for Highly Sensitive Detection of Alkaline Phosphatase.  ANALYTICAL CHEMISTRY,      [PMID:34743510] [10.1021/acs.analchem.1c03842]

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