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.
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:
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.