(Urea aminohydrolase; EC 220.127.116.11)
Urease catalyzes the hydrolysis of urea.
(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).
||Soluble in distilled water or dilute buffer
||-20° C; -4° F
||100 U/mg solid
||Substantially free from glucose and free ammonia
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).
- 0.10 M Potassium phosphate buffer, pH 7.6.
- 1.80 M Urea. Prepared in phosphate buffer.
- 0.025 M Adenosine-5'-diphosphate (ADP) (10.7 mg/ml) in buffer.
- 0.008 M NADH (5 mg/ml) in phosphate buffer.
- 0.025 M a-Ketoglutarate (3.7 mg/ml) in phosphate buffer.
- Glutamate dehydrogenase (GLDH) solution, free from ammonium ions. 50 U/ml phosphate buffer. Prepare fresh prior to assay.
- Urease solution. Dissolve in phosphate buffer to yield a concentration of 0.1-0.5 U/ml. Must be prepared fresh prior to assay.
- Adjust spectrophotometer (equipped with strip chart recorder and temperature control) to 340 nm and 25°C.
- 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.
- 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.
- Calculate µE340 nm/min
|Activity (U/mg) =
||(ΔE340nm/min)(Total Vol.)(Enz. Diln.)
|(6.22)(2)(Enz. Vol.)(mg Enz./ml)