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Urease

(Urea aminohydrolase; EC 3.5.1.5)

Urease catalyzes the hydrolysis of urea.

                UREASE
(NH2)2CO + 3H2O ------> CO2 + 2NH4OH
Urease activity has been reported in a variety of microorganisms and a number of higher plants. Commercially, it is produced from Jack beans. Urease exhibits absolute specificity for urea. Purified Jack bean urease has a molecular weight of 480,000. (Fishbein, W.N., Nagarajan, K and Scurzi, W., J. Biol. Chem., 245, 5985, 1790). Urease is widely used for determination of urea in biological fluids. Urease conjugates have also been proposed for use in enzyme immunoassays (EIA), a sensitive technique for detection and determination of antigens, antibodies and metabolites in biological fluids (Meyerhoff, M.E. and Rechnitz, G.A., Anal. Biochem., 95, 483, 1979).


1.Source: Jackbeans
   Form: Freeze-dried powder 
   Solubility: Soluble in distilled water or dilute buffer 
   Stability: -20 C; -4 F 
   Activity (approx.): 100 U/mg solid 
   Protein (approx.): 90% (biuret) 
   Contaminants (approx.): Substantially free from glucose and free ammonia 
   Catalog No.: 116A0100 
$.40/KU


The amount of enzyme which causes the oxidation of one micromole of NADH per minute at 25°C, pH 7.6 in a coupled reaction using glutamate dehydrogenase. Note: 1 Calzyme unit = 2.8 Ammonia units.


The assay for urease is carried out in a coupled enzyme reaction with glutamate dehydrogenase (GLDH). The amount of NADH oxidized is determined by measuring the change in absorbance at 340 nm (Kaltwasser, H. and Schlegel, H.G., Anal. Biochem., 16, 132, 1966).


  1. 0.10 M Potassium phosphate buffer, pH 7.6.
  2. 1.80 M Urea. Prepared in phosphate buffer.
  3. 0.025 M Adenosine-5'-diphosphate (ADP) (10.7 mg/ml) in buffer.
  4. 0.008 M NADH (5 mg/ml) in phosphate buffer.
  5. 0.025 M a-Ketoglutarate (3.7 mg/ml) in phosphate buffer.
  6. Glutamate dehydrogenase (GLDH) solution, free from ammonium ions. 50 U/ml phosphate buffer. Prepare fresh prior to assay.
  7. Urease solution. Dissolve in phosphate buffer to yield a concentration of 0.1-0.5 U/ml. Must be prepared fresh prior to assay.



  1. Adjust spectrophotometer (equipped with strip chart recorder and temperature control) to 340 nm and 25°C.
  2. In a cuvette place the following reagents as indicated:
    Phosphate buffer 2.40 ml
    Urea 0.10 ml
    ADP 0.10 ml
    NADH 0.10 ml
    a-Ketoglutarate 0.10 ml
    GLDH 0.10 ml

    Incubate cuvette in spectrophotometer at 25°C for 5 minutes to attain temperature equilibration and then establish blank rate, if any, at 340 nm.

  3. To initiate the reaction add 0.1 ml urease solution to the cuvette and record decrease in absorbance at 340 nm for 5-8 minutes.
  4. Calculate µE340 nm/min



Activity (U/mg) = (ΔE340nm/min)(Total Vol.)(Enz. Diln.)
(6.22)(2)(Enz. Vol.)(mg Enz./ml)
 

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