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Uricase
Catalogue No. URIC-70-1701 and 70-1701-01
E.C. Number 1.7.3.3
ORIGIN
Bacillus fastidiosus
SPECIFICATIONS
Appearance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . White/off white free flowing powder
Powder activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . >10 U/mg powder at 37oC
Specific activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . >15 U/mg protein at 37C
Contaminants:
o Catalase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . <1%
o Cholesterol Oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . <0.005%
o Glucose Oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . <0.005%
Stability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stable for 1 year stored at -20C
ASSAY PRINCIPLE
Uricase catalyses the following reaction:
Uric Acid + 2H2O + O2
Uricase
Allantoin + H2O2 + CO2
APPLICATION
Uricase can be used (in test strips or other clinical chemistry formats) to determine the level of uric
acid in blood for clinical diagnosis.
UNIT DEFINITION
One unit of activity is defined as the amount of enzyme that will catalyse the oxidation of 1.0
micromole of uric acid per minute at 37C under the standard assay method conditions
(available on request).
TA B L E 1 Temperature factors for unit conversion
Assay Temp
Factor Realitve to 37C Result
25C
0.23
30C
0.48
37C
1.00
45C
1.16
Intended for use in the manufacture of IVDs
Note: Temperature can influence
the level of available oxygen in
the reaction mixture.
AP0199.3.0
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CHARACTERISTICS
Uricase is a highly purified product and is formulated with BSA as stabiliser. Its main characteristics
are as follows:
Molecular Weight (by SDS-Page) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38kD
Km value (calculated using Eadie-Hofstee) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 x 10-4 M (Uric Acid)
Optimum pH (Fig. 1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pH 7.0 (phosphate buffer)
Optimum temperature (Fig. 2). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45oC
Stable pH range (Fig. 3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pH 5.5 to 10.0 (25oC for 20 hours)
Thermal stability (Fig. 4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 min at 50oC and below
Lyophilised stablity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Up to 50oC (pH 7.0 for 15 mins)
Substrate Specificity
Substrate specificity was tested in-house by replacing uric acid with alternative substrates in
the assay i.e. at 2.3 mM concentration.
TA B L E 2 Substrate specificity
Substrate
% of Uric
Acid Activity
Uric acid
100
8-Azaxanthine
Oxonic acid
F I G . 2 Optimum Temperature
120
120
100
100
RELATIVE ACTIVITY (%)
RELATIVE ACTIVITY (%)
F I G . 1 Optimum pH
80
60
40
20
80
60
40
20
0
0
6
10
pH
30
35
40
45
50
TEMPERATURE (C)
POTASSIUM PHOSPHATE
MOPS
25
BICINE
CHES
TRIS
Note: Uric acid is particularly insoluble under acidic conditions and so determination of
uricase activity below a pH of 6.5 was not deemed practicable.
Intended for use in the manufacture of IVDs
AP0199.3.0
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F I G . 4 Thermal Stability
(0.1M buffer at 25C for 20 hours)
120
120
100
100
RELATIVE ACTIVITY (%)
RELATIVE ACTIVITY (%)
F I G . 3 pH Stability
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80
60
40
20
0
80
60
40
20
0
10
11
SODIUM ACETATE
POTASSIUM PHOSPHATE
MES
BICINE
30
35
40
45
50
55
60
65
70
TEMPERATURE (C)
pH
(1)
(0.1M Potassium Phosphate pH 7.0 for 15 min.)
MOPS
CHES
TRIS
CAPS
SODIUM BORATE
1. Bongaerts,G.P.A. et al. (1978) Uricase of Bacillus Fastidiosus: Properties and regulation of synthesis. Biochimica et Biophysica Acta Enzymology, 527 (2) pp.348-358
NOTES:
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