enzymes urease - sekisui diagnostics · enzymes urease catalogue no. 70-6734-01 e.c number 3.5.1.5...
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ENZYMES
UreaseCatalogue no. 70-6734-01E.C Number 3.5.1.5
ORIGINJack Bean
sekisuienzymes.com AP0197.4.0
UNIT DEFINITIONOne unit of activity is defined as the amount of enzyme that will convert 1.0 micromole of urea per minute at 37°C under the standard assay method conditions. Refer to Table 1 for guidance on factors to adjust units according to temperature of assay.
ASSAY PRINCIPLEUrease catalyses the following reaction:
Urea + H2O + 2H+ CO2 + 2NH4+
Urease
APPLICATIONUrease is used in combination with glutamate dehydrogenase to determine the level of urea in clinical analysis.
<0.0008 µmoles of ammonia per unit of Urease<150 CFU/mL
SPECIFICATIONSAppearance
Activity
pH
Contaminants: • Ammonia Content • Microbial Counts
Clear, slightly yellow solution, essentially free from particulate matter
8000 + 1000 U/ml at 37°C
7.10 + 0.20
ENZYMES
CHARACTERISTICS
sekisuienzymes.com AP0197.4.0
Native structure(1)
Isoelectric point(2)
Kmvalue (calculated using Eadie-Hofstee)
Optimum pH (Fig. 1)
Optimum temperature (Fig. 2)
pH stability (Fig. 3)
Thermal stability (Fig. 4)
91kD subunit arranged as a hexamer, 2 Ni ions per subunit
5.0 - 5.1
4 x 10-3 M (Urea)
pH 7.5
at least 50°C
pH 5.5 to 11.0 (25°C for 20 hours)
Stable at 70°C and below (pH 7.5 for 15 minutes)
TABLE 1 Temperature factors for unit conversion
ASSAY TEMPERATURE FACTOR RELATIVETO 37°C RESULT
25°C
30°C
37°C
45°C
0.54
0.70
1.00
1.35
Urease is a formulated as a liquid product with glycerol and so does not freeze at -20°C. Its main characteristics are as follows:
SODIUM ACETATE MES
POTASSIUM PHOSPHATE TRIS CHESBICINE
MOPS
FIG. 1 Optimum pH
120
100
80
60
40
20
0
RELA
T IV
E A
CTI
VIT
Y (%
)
pH
3 4 5 6 7 8 9 10 11
FIG. 2 Optimum Temperature
120
100
80
60
40
20
0
RELA
TIV
E A
CTI
VIT
Y (%
)
TEMPERATURE (°C)
25 30 35 40 45 50
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EMEASekisui Diagnostics (UK) Ltd50 Gibson Drive Kings Hill, West Malling Kent, ME19 4AF, UK
AMERICASSekisui Diagnostics, LLC4 Hartwell PlaceLexington MA 02421
JAPAN AND ASIA GROUPSekisui Medical Co., Ltd.13-5, Nihonbashi 3-Chome Chuo-ku, Tokyo 103-0027 JAPAN
ENZYMES
sekisuienzymes.com AP0197.4.0
FIG. 4 Thermal Stability (0.1M Potassium PhosphatepH 7.0 for 15 mins.)
120
100
80
60
40
20
0RE
LAT
IVE
AC
TIV
ITY
(%)
TEMPERATURE (°C)
30 35 40 45 50 55 60 65 70
FIG. 3 pH Stability (0.1M buffer at 25°C for 20 hours)
120
100
80
60
40
20
0
RESI
DUA
L A
CTI
VIT
Y (%
)
pH
3 4 5 6 7 8 9 10 11
MESSODIUM ACETATE
SODIUM BORATE
POTASSIUM PHOSPHATE TRIS
CHES
BICINE
MOPS
CAPS
(1) Takishima,K et al. (1988) The structure of jack bean urease. The complete amino acid sequence, limited proteolysis and reactive cysteine residues. Eur.J.Biochem., 175 (1), pp.151-65
(2) Sumner, J.B. and Hand, D.B (1929) The isoelectric point of crystalline urease. J.Am.Chem.Soc., 51, pp.1255-60