To standardize a procedure for the enzymatic assay of Urease, from Jack Beans.
The scope of this procedure is for all products that have a specification for the enzymatic determination of Urease, from Jack Beans. This assay is not to be used to determine the activity of Urease, from Bacillus pasteurii, U7127.
3.1 Purified Water – water from a deionizing system, resistivity ~18MΩ•cm @ 25ºC
3.2 Urease Unit Definition – One micromolar unit will liberate 1.0 μmole of NH3 from urea per min at pH 7.0 at 25 °C. It is equivalent to 1.0 I.U. or 0.054 Sumner unit (1.0 mg ammonia nitrogen in 5 minutes at pH 7.0 at 20 °C)
Urea + H2O Urease > CO2 + 2NH3
It is the responsibility of trained Analytical Services laboratory personnel to follow this procedure as written.
Refer to the Material Safety Data Sheet (MSDS) for hazards and appropriate handling precautions.
7.3.1 750 mM Sodium Phosphate Buffer, pH 7.0 at 25°C (Buffer)
Prepare a 90.0 mg/ml solution of Sodium Phosphate Monobasic in purified water with a Sigma-Aldrich Product such as S0751. Adjust to pH 7.0 at 25°C with 5N NaOH.
7.3.2 500 mM Urea with 0.05% (w/v) Bovine Serum Albumin Solution (Urea)
Prepare a 30.0mg/ml solution of Urea with a Sigma-Aldrich Product, such as U1250, and a 0.50 mg/ml solution of Bovine Serum Albumin, with a Sigma-Aldrich Product, such as A4503 in Reagent 7.3.1 (Buffer). Adjust to pH 7.0 at 25°C with 1N HCl or 1N NaOH, if necessary.
7.3.3 0.40% (w/v) p-Nitrophenol (Indicator)
Prepare a 4.0 mg/ml solution of p-Nitrophenol in purified water with a Sigma-Aldrich Product such as 1048. Sonication or vortexing may be necessary for complete dissolution.
7.3.4 100 mM Standardized Hydrochloric Acid (HCl)
Prepare a 1:10 dilution in purified water using 1N Hydrochloric Acid, such as Sigma-Aldrich Product Number 318949. Standardize according to Sigma-Aldrich Operating Procedure, PROC-DEK-OP-002884.
7.3.5 20 mM Sodium Phosphate Buffer, pH 7.0 at 25°C (Enzyme Diluent)
Prepare a 2.4 mg/ml solution of Sodium Phosphate Monobasic in purified water with a Sigma-Aldrich Product such as S0751. Adjust to pH 7.0 at 25°C with 1N NaOH.
7.3.6 Urease (Enzyme)
Immediately before use, prepare a solution containing 200-400 units/ml in cold Reagent 7.3.5 (Enzyme Diluent).
220.127.116.11 For Crude Urease products, it may be necessary to stir vigorously on ice to obtain a homogenous suspension. Samples may be centrifuged at 1000rpm for 1 minute if a homogenous sample is still not obtained through vigorous stirring. Proceed with testing of the supernatant if clear of large insoluble particulates.
7.4 TEST METHOD
7.4.1 Pipet (in milliliters) the following reagents into suitable containers:
|Reagent 7.3.2 (Urea)
7.4.2 Equilibrate to 25°C using a thermostatically suitable waterbath. Staggering each aliquot a minimum of two minutes add:
|Reagent 7.3.6 (Enzyme)
7.4.3 Mix by swirling and incubate at 25°C for exactly 5 minutes. Then add:
|Reagent 7.3.3 (Indicator)
|Reagent 7.3.6 (Enzyme)
7.4.4 Using a suitable magnetic stirrer, titrate immediately with Reagent 7.3.4 (HCl) by adding small amounts until the color of the indicator turns from yellow to colorless. This is the endpoint. Record the volume of Reagent 7.3.4 (HCl) used for both the Test and Blank solutions.
7.5.1 ΔVHCl = VTest - VBlank
||Units/ml enzyme =
ΔVHCl = Total Volume (in milliliters) of Reagent 7.3.4 (HCl) used in titration
1000 = Conversion factor from millimoles to micromoles as per unit definition
df = Dilution Factor, if applicable
5 = Time of assay (in minutes) as per unit definition
0.10 = Volume (in milliliters) of enzyme used
||Units/mg solid =
|mg solid/mL enzyme
7.5.4 Units/g solid = Units/mg solid x 1000
7.6 FINAL ASSAY CONTENTRATION
In a 1.10 ml reaction mix, the final concentrations are 684mM Sodium Phosphate, 455mM Urea, 0.05% (w/v) Albumin, Bovine and 20-40 units Urease.
8. REFERENCES & ATTACHMENTS
8.1 Worthington, C.E. (1972) Worthington Enzyme Manual, pp 146-148, Worthington Biochemical Corporation, Freehold NJ.
8.2 Replaces PEUREA01.
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