Enzymatic Assay of Urease from Jack Beans

1. OBJECTIVE
To standardize a procedure for the enzymatic assay of Urease, from Jack Beans.

2. SCOPE
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. DEFINITIONS

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)

4. DISCUSSION
Urea + H2O    Urease   > CO2 + 2NH3

5. RESPONSIBILITIES
It is the responsibility of trained Analytical Services laboratory personnel to follow this procedure as written.

6. SAFETY
Refer to the Material Safety Data Sheet (MSDS) for hazards and appropriate handling precautions.

7. PROCEDURE

7.1 CONDITIONS
T=25°C, pH=7.0

7.2 METHOD
Titrimetric

7.3 REAGENTS

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).

7.3.6.1    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:

  Blank Test1 Test2 Test3
Reagent 7.3.2 (Urea) 1.00 1.00 1.00 1.00

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) --- 0.10 0.10 0.10

7.4.3   Mix by swirling and incubate at 25°C for exactly 5 minutes. Then add:

Reagent 7.3.3 (Indicator) 0.20 0.20 0.20 0.20
Reagent 7.3.6 (Enzyme) 0.10 --- --- ---

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 CALCULATIONS

7.5.1   ΔVHCl = VTest - VBlank

7.5.2 Units/ml enzyme = (HCl Molarity)(ΔVHCl)(1000)(df)
(5)(0.01)  

Where:
   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

7.5.3 Units/mg solid = Units/mL enzyme
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.

9. APPROVAL

Review, approvals and signatures for this document will be generated electronically using the EDMS. Print a “For Use” copy if hardcopy with signature verification is required.

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