Urease Assay

Method: Worthington has adopted an assay method where the hydrolysis of urea is measured by coupling ammonia production to a glutamate dehydrogenase reaction.


One unit results in the oxidation of one micromole of NADH per minute at 25°C and pH 7.6 under the specified conditions. In addition to increased sensitivity, the assay method possesses the advantage that it can be manipulated to permit quantitation of urea.


  • 0.1 M Potassium phosphate buffer, pH 7.6
  • 0.023 M Adenosine-5'-diphosphate (ADP) in phosphate buffer
  • 0.0072 M NADH in phosphate buffer
  • 0.026 M a-Ketoglutarate in phosphate buffer
  • 1.8 M Urea in phosphate buffer
  • Glutamate Dehydrogenase: Dilute to approximately 500 units/ml in 50% glycerol or phosphate buffer. Store cold during use.


Dissolve enzyme at one mg/ml in 0.1 M phosphate buffer, pH 7.6. Immediately prior to use, dilute further in buffer to obtain a rate of 0.02-0.04 ΔA/minute.


Adjust spectrophotometer to 340 nm and 25°C. Pipette into each cuvette as follows:

0.10 M Phosphate buffer, pH 7.6 2.4 ml
0.023 M ADP 0.1 ml
0.0072 M NADH 0.1 ml
0.026 M α-Ketoglutarate 0.1 ml
1.8 M Urea 0.1 ml
GLDH (500 units/ml) 0.1 ml

Incubate in spectrophotometer at 25°C for 5-10 minutes to achieve temperature equilibration and establish blank rate, if any. A slight change in absorbance may be observed due to trace ammonia in reagents. Upon obtaining a zero change in absorbance, add 0.1 ml appropriately diluted enzyme. Record decrease in A340 for 8-10 minutes. Determine ΔA340/minute from the linear portion of the curve. A slight lag may occur.



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