Urease method

Method, There are two standard methods for the estimation of urea, (i) the hypobromite method, (ii) the urease method (p. 519). The chief merit of the hypobromite method is the rapidity of the analysis the results obtained are considered sufficiently accurate for most medical requirements, e.g., for the estimation of urea in urine. For accurate metabolic work, however, the urease method should be employed. 

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). 

Urea ((NH2)2CO) is excreted by larger organisms, can be a product of bacterial organic matter decomposition, and is a highly labile form of N for plankton nutrition (Bronk, 2002). Reports of concentrations in oceanic waters are relatively scarce, but are quite low (<0.5 pM Antia et al, 1991). There are currently two methods commonly used to measure urea concentrations—the urease method (McCarthy, 1970) and the monoxime method (Mulvenna and Savidge, 1992 Price and Harrison, 1987). 

In the urease method, urea is enzymatically hydrolyzed to CO2 and NH3 (McCarthy, 1970). The released NH4+ is then measured using the phenol hypochlorite method described above. The urease method commonly suffers from incomplete hydrolysis, resulting in an underestimation of urea concentrations. 

Comparisons of the two methods found that the urease method tends to underestimate concentrations in seawater and so we recommend the monoxime method for analysis of marine samples (Price and Harrison, 1987 ReviUa et al, 2005). With respect to estuarine samples, the monoxime method is more accurate and less affected by salinity than the urease method (ReviUa et al, 2005). There has been some concern that freezing samples may result in lower urea concentrations (Mulvenna and Savidge, 1992). Other researchers, however, have shown that freezing does not alter the urea concentration measured (Cochlan and Bronk, 2001 Price and Harrison, 1987). 

McCarthy J.J. 1970. A urease method for urea in seawater. Umnol. Oceanogr. 15 309-313. Aminot A. and Kerouel R. 1982. Dosage automatique de I uree dans I eau de mer, une methode ties sensible a la diacetyhnonoxime. Can.. Fish. Aquat. Sci. 39 174-183. 

The method is based on the conversion of urea to amnionium carbonate and the estimation of the latter by titration with standard acid. For this purpose, two equal quantities of urea (or urine) are measured out into two flasks A and B. A is treated with 10 ml. of a strong urease preparation and some phenol-phthalein, warm water is added and the mixture is adjusted by the addition of V/io HCl from a burette A until the red colour is just discharged. This brings the mixture to about pH 8 (the optimum for urease) and also prevents loss of ammonia. 

Following this procedure urea can be determined with a linear calibration graph from 0.143 p.g-ml To 1.43 p.g-ml and a detection limit of 0.04 p.g-ml based on 3o criterion. Results show precision, as well as a satisfactory analytical recovery. The selectivity of the kinetic method itself is improved due to the great specificity that urease has for urea. There were no significant interferences in urea determination among the various substances tested. Method was applied for the determination of urea in semm. 

Results from an array of methods, including X-ray absorption, EXAFS, esr and magnetic circular dichroism, suggest that in all ureases the active sites are a pair of Ni" atoms. In at least one urease,these are 350 pm apart and are bridged by a carboxylate group. One nickel is attached to 2 N atoms with a fourth site probably used for binding to urea. The second nickel has a trigonal bipyramidal coordination sphere. 

Recently, the old alkaline phenol method has been revived, and is being widely used in clinical laboratories, without protein preclpltatlon(27). In this procedure, the serum is added to an alkaline phenol reagent, and the ammonia generated from urea is determined either after the action of urease or after strong alkaline treatment of the serum. The objection to this procedure is first, that all urease is rich in ammonia, and second, the color produced with alkaline phenol is not specific for ammonia. It will react with other compounds, especially for those that liberate ammonia. By this procedure one obtains a useful number from the point of view of determining whether the patient has nitrogen retention, but a value which is somewhere between a urea and an N.P.N. determination. 

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. 

Fig. 15.4 (A) Urease adsorption isotherms for (a) Zn2AI-CI, (b) Zn3AI-CI, (c) Zn4AI-CI (B) amount of immobilized urease in Zn3-AI by the coprecipitation method.

Tor [7] developed a new method for the preparation of thin, uniform, self-mounted enzyme membrane, directly coating the surface of glass pH electrodes. The enzyme was dissolved in a solution containing synthetic prepolymers. The electrode was dipped in the solution, dried, and drained carefully. The backbone polymer was then cross-linked under controlled conditions to generate a thin enzyme membrane. The method was demonstrated and characterized by the determination of acetylcholine by an acetylcholine esterase electrode, urea by a urease electrode, and penicillin G by a penicillinase electrode. Linear response in a wide range of substrate concentrations and high storage and operational stability were recorded for all the enzymes tested. 

Sahoo, B., Sahu, S.K., Pramanik, P., 2011. A novel method for the immobilization of urease on phosphonate grafted iron oxide nanoparticle. J. Mol. Catal. B Enzym. 69, 95-102. 

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. ... 

Methods based on the inhibitory effect of the analyte and the use of an enzyme thermistor have primarily been applied to environmental samples and typically involve measuring the inhibitory effect of a pollutant on an enzyme or on the metabolism of appropriate cells [162]. The inhibiting effect of urease was used to develop methods for the determination of heavy metals such as Hg(II), Cu(II) and Ag(I) by use of the enzyme immobilized on CPG. For this purpose, the response obtained for a 0.5-mL standard pulse of urea in phosphate buffer at a flow-rate of 1 mL/min was recorded, after which 0.5 mL of sample was injected. A new 0.5-mL pulse of urea was injected 30 s after the sample pulse (accurate timing was essential) and the response compared with that of the non-inhibited peak. After a sample was run, the initial response could be restored by washing the column with 0.1-0.3 M Nal plus 50 mM EDTA for 3 min. Under these conditions, 50% inhibition (half the initial response) was obtained for a 0.5-mL pulse of 0.04-0.05 mM Hg(II) or Ag(I), or 0.3 mM Cu(II). In some cases, the enzyme was inhibited irreversibly. In this situation, a reversible enzyme immobilization technique... 

Trichosporon cutaneum [89]. The principle of this method is based on the measurement of the rapid alteration (or acceleration) of respiration after addition of ammonium ions in the presence of glucose. The physiological background of this principle is probably the uptake of ammonium ions in connection with the respiration process. Finally, the combination of nitrifying bacteria with urease on a hybrid sensor also allows an amperometric determination of urea [93]. 

As Mamiya and Gorin (11) note, Sumner occasionally had difficulty in preparing crystalline urease from some jack bean meals and this is a technical problem that has received attention in each recent improved method of preparation (11-13). Modification of Sumner s extraction procedure by including / -mercaptoethanol (11, 14) to diminish aggregation and ethylenediaminetetraacetate (EDTA) (13,15) to maintain a low concentration of metal ions has been helpful. The procedure used in this laboratory with consistent success over a period of 4 years, and involving several students, is essentially that of Mamiya and Gorin (11) but with EDTA included in the buffers. [There is an error in Mamiya and Gorin (11). The concentration of acetone used was 32%, not 36%.]... 

A method for the isolation of urease from hydrated jack beans developed by Sehgal and Naylor (15) employs DEAE-cellulose as a final purification step. A method intended for purification of urease from mammalian sources (16) was developed using jack bean meal as a standard source. It employed no acetone but is a combination of (NH4)2S04 precipitation and Sephadex column separations. [Note Fish-... 

It is convenient to have a method for the detection and rough estimation of urease activity in gels. A simple staining procedure using cresol red has been described (27), but a more elaborate procedure (28) employing p-nitro blue tetrazolium results in sharper definition. 

A potentiometric method for the determination of EDTA that involves the use of a urease-based inhibition biosensor was described [19]. 

Roughly 30% of enzymes are metalloenzymes or require metal ions for activity and the present chapter will concentrate on the chemisty and structure of the plant metalloenzymes. As analytical methods have improved it has been possible to establish a metal ion requirement for a variety of enzymes which were initially considered to be pure proteins. A dramatic example is provided by the enzyme urease isolated from Jack beans and first crystallised by Sumner (1926) (the first enzyme to be crystallised). Sumner defined an enzyme as a pure protein with catalytic activity, however, Zerner and his coworkers (Dixon et al., 1975) established that urease is in fact a nickel metalloenzyme. Jack bean urease contains two moles of nickel(II) per mole of active sites and at least one of these metal ions is implicated in its mechanism of action. 

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