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.

Reagents

• 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.

Enzyme

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.

Procedure

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.

Calculation