Urease assay

Urease assay. When Proteus mirabilis grows in a urea-containing medium it hydrolyses the urea to ammonia and consequently raises the pH of the medium. This production of urease is inhibited by aminoglycoside antibiotics (inhibitors of protein synthesis Chapter 8). In practice, it is difficult to obtain reliable results by this method. 

Groves and Teng (1992) investigated the effect of compactional pressure on biologically active proteinaceous enzymes such as a-amylase, P-glucuronidase, lipase, and urease. Assaying the activity of these enzymes before and after the compaction... 

We prepared two samples, each containing an aqueous suspension (0.5 ml) of micro-capsules with urease (7.4 x 10 capsules/ml) in 0.01 M Tris-HCl (pH 8.0). A solution (0.5 ml) of proteinase K at 4 mg/ml in the same buffer was added to the first sample, and the same amount of the buffer was added to the second sample. Then, the solutions were incubated at 37°C. Every 10 min, 50-ml portions were taken and added to the urease assay mixture (1.95 ml) with 125 mM urea. Free urease at 20 pg/ml instead of microcapsules was used as a reference. 

The activity of enzymes in the film was estimated in the following way In order to test the activity of urease, we utilized a calorimetric assay based on urea hydrolysis the enzymatic reaction was followed at 590 nm, the suitable wavelength for bromcresol purple (Chandler 1982). Urea concentration was 1.67 ts 10 M. 

Wittekind E, Werner M, Reinicke A, Herbert A, Hansen P (1996) A microtiter-plate urease inhibition assay-sensitive rapid and cost-effective screening for heavy metals in water. Environ Technol 17 597-603... 

For reactions in which one or more reactants or products is a gas, manometry (the measurement of pressure differences) can provide a convenient means for monitoring the course and kinetics of the reaction Thus, enzymes that can be assayed with this method include oxidases, urease, carbonic anhydrase, hydrogenase, and decarboxylases. For example, bacterial glutamate decarboxylase is readily assayed by utilizing a Warburg flask and measuring the volume of gas evolved at different times using a constant-pressure respirometer. ... 

Urease. An enzyme of the hydrolase class that catalyzes the hydrolysis of urea to COj and ammonia. It is nickel protein found in micro-organisms and plant that is frequently used in clinical assays of plasma urea concentrations. 

Workers in several laboratories have noted that the activity of urease appears to increase upon standing at room temperature. Until this is understood assay procedures cannot be assumed to yield precise values. 

B.B. Rodriguez, J.A. Bolbot and I.E. Tothill, Development of urease and glutamic dehydrogenase amperometric assay for heavy metals screening in polluted samples, Biosens. Bioelectron., 19 (2004) 1157-1167. 

Urea is most commonly assayed by combined urease methods, in which the urea is first converted to two ammonium ions. The ammonium generated is then measured by either enzymatic or chemical methods. Urea nitrogen values determined by this method (mg/ml) are converted to urea values by the use of appropriate factors (2.14 for urea in mg/ml, 0.357 for urea in mmol/L) (Emeigh Hart and Kinter 2005). 

Recendy original all solid-state elearodes for NH4 were successfully combined with urease for the assay of urea (70,71). These electrodes consist of a conductive resin (epoxy -t- graphite) covered by a nonactin-PVC matrix. They offer interesting commercial advantages over membrane electrodes with internal solution and reference electrode. 

Ideally, the sensor used to sense the biocatalyzed reaction should not react with other substances in the sample. This requirement is not always met using either potentiometric or amperometric methods. For example, immobilized urease electrodes operating with a cation glass sensor measuring the NHj are inadequate for blood and urine assays because they also respond to Na+ and K+ (59, 60). However, a glass electrode sensor (165) or, better, a solid antibiotic nonactin electrode (61) gives more selective response. The latter has a selectivity of NHt/K+ of 6.5 and NHt/Na+ of 0.075. 

Such interference falls into two classes competitive substrates and substances that either aaivate or inhibit the enzyme. With some enzymes, such as urease, the only substrate that reacts at reasonable rate is urease hence, the urease-coated electrode is specific for use (59, 165). Likewise, uricase acts almost specifically on uric acid (167), and aspartase on aspartic acid (8, 168). Others, such as penicillinase and amino oxidase, are less specific (63,169,170). Alcohol oxidase responds to methanol, ethanol, and allyl alcohol (171, 172). Hence, in using electrodes of these enzymes, the analyte must be separated if two or more are present (172). Assaying L-amino acids by using either the decarboxylative or the deaminating enzymes, each of which acts specifically on a different amino... 

Bovine serum albumin (BSA) and cyclic AMP (cAMP) are determined by a competitive binding enzyme immunoassay (315). With urease as label, an ammonia gas-sensing electrode is used to measure the amount of urease-labeled antigen bound to a double-antibody solid phase by continuously measuring the rate of ammonia produced from urea as substrate. The method yields accurate and sensitive assays for proteins (BSA less than 10 ng/mL) and antigens (cAMP less than 10 nM), with fairly good selectivity over cGMP, AMP, and GMP. 

Figure 16 shows the long-term stability of the FET using a 1.7 mM urea solution. The response amplitude of the sensor decreases rapidly after 40 measurements without adding EDTA (ethylendiamine tetraacetic acid) to buffer solutions (Fig. 16, —EDTA). Adding EDTA to the buffer increases the life of the urea sensor. The urea sensor is usable for more than 2000 assays, showing no decrease in the response amplitude (Fig. 16, +EDTA). The rapid decrease in the response amplitude in the absence of EDTA suggests that heavy metal ions inactivate urease. The effect of EDTA on the durability of a urea sensor has been previously reported (48).

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