Urease reaction

These large rate accelerations correspond to substantial changes in the free energy of activation for the reaction in question. The urease reaction, for example. 

The Enzyme Commission catalog (EC 3.5.1.5) lists the urease reaction as urea + 2 H20 = C02 + 2 NH3. Since two C-N bonds are broken it is evident that the stoichiometric relation above is the result of two component reactions. Any conjecture concerning the mechanisms of these reactions and the nature of the intermediates must encompass the action of inhibitors and the spectrum of substrates. Some of the organic inhibitors that have been reported are shown in Table I. The substrates that have been shown to be hydrolyzed are listed in Table II. 

Another urea electrode uses a carbon dioxide sensor covered with urease to measure the second product of the urea-urease reaction, HCOs. Na " and K" " had no influence on this electrode and the linear range was 0.1-10 mM (64). 

The urease reaction, in particular, is ideally suited to conductimetric quantitation.18 Urea is hydrolyzed by urease according to Eq. 3.31 ... 

Proton-consuming reactions such as the urease reaction are Hkely to show all the diffusion-related pH-shifts in the opposite direction. Obviously, they may be treated in an analogous manner. 

Besides these conventional reactors with spherical immobilizates, urease has also been immobilized inside nylon tubing and pipette tips ( enzyme pipette , Sundaram and Jayonne, 1979), on nylon fibers ( enzyme brush , Raghavan et al., 1986), and on the surface of a magnetic stirrer (Guilbault and Starklov, 1975). The urease reaction was in each case carried out at optimal pH after removal of the immobilized enzyme NH3 was assayed electrochemically or photometrically according to Berthelot s method. 

A further disadvantage of potentiometric gas sensors is the difference between the pH optima of the electrode and urease. Thus, NH3 electrodes are operated at a pH around 8 while the optimal pH of the urease reaction is at pH 7. 

Figure 22.4 Topical peaks registered from repetitive assays using an FI system with thermal detectors. The responses were obtained after injection of urea, penicillin V, and a mixture of the two into a system with an integrated thermal biosensor for simultaneous determinations of multiple analytes. Both signals were exothermic. The upper trace was registered with a thermistor pair called To-T. The lower trace was registered using another thermistor pair, T2-T3. In this case, the direction of the penicillin peaks was reversed by alternating the recorder polarity. A, response to 30 mmol T urea B, influence of the urease reaction on on the thermistor pair T2-T3 C, response to 30 mmol 1 penicillin V D, simultaneous responses for urea (5-60 mmol 1" ) in the mixed samples E, simultaneous responses for penicillin V (5-60 mmol I" ) in the mixed samples (from [47], with permission).

Urea is a diagnostic indicator for kidney function. Urea can be potentiometrically determined following the enzymatic reaction of the enzyme urease (reaction [I]) coupled with a variety of potentiometric transducers such as the pC02 electrode, the PNH3 electrode, the pH electrode, and the PNH4 electrode ... 

Urease-Nesslerization methods. The ammonia formed by the urease reaction reacts with mercuric and potassium iodides in alkaline solution to give a yellow colour. However, this method has several disadvantages which has led to its gradual disuse. The major disadvantages are that ... 

The urea-urease reaction is an enzyme-catalysed hydrolysis of urea, that produces ammonia and carbon dioxide according to the overall stoichiometry (1). This reaction occurs in numerous cellular systems, for example it is used by bacteria H. pylori in order to raise the local pH to protect itself from the harsh acidic environment of the stomach [6]. 

The urea-urease reaction follows Michaelis-Menten kinetics and has a bellshaped rate-pH curve with maximum at pH 7 (see Fig. 1). In non-buffered condition, the rate-pH curve can be exploited to obtain feedback-driven behaviour, for instance, through external stimuli, e.g. by delivering an acid or a base to the solution a reaction acceleration or inhibition can be obtained. Therefore, the conditions for spontaneous oscillations between two pH states can be achieved. This reaction has also the distinct advantages of high solubility and stability of substrate and enzyme in water making it suitable for experiments in vitro. 

The temporal dynamic of the urea-urease reaction in membranes in contact with a solution of urea (S) and acid (H+), as depicted in Fig. 2, can be captured with the model (2) ... 

Fig. 2. Lipid vesicle containing the urea-urease reaction and the fluorescence probe R = reaction rate, kn = exchange rate of the acid, ks = exchange rate of the urea, S, with the external solution of concentrations [H+]o and [S]o.

Thus, the first task was to obtain a pH switch from acid to base in the bulk urea-urease reaction in the presence of lipids and to ensure that no significant side reactions took place. Figured A and B shows the beginning and the end of the reaction in a stirred batch reactor, respectively. Panels C and D show the reaction in the presence of [POPC] = 0.5 x 10 M, the colour change indicates that the reaction took place and enough NH3 was produced to raise the pH. 

Fig. 5. Spectrofluorimetric trace of the kinetics of the urea-urease reaction. Experimental conditions are the same as in Fig. 4 with [urease] = 10.7 units/mL and [pyra-nine] = 1 X 10 M. Excitation A = 450nm, Emission A = 510nm...

Once established the chemical stability of the urea-urease reaction in the presence of lipids and of the fluorescent probe, the next step towards a successful oscillating system was the encapsulation of the enzyme into POPC liposomes through the droplet transfer method. This innovative method first takes advantage of the facile compartmentalization of water-soluble solutes (enzyme in this case) in water-in-oil (w/o) droplets, and then convert the solute-filled w/o droplets into vesicles that can be dispersed in an acidic solution of urea. 

The values of amino acid dehydrogenases for ammonia are in general very high (e.g., Bacillus sphaericus LeuDH 200 mM and AlaDH 28.2 mM, and Thermoactimyces PheDH 106 mM). In contrast, NADP-dependent GluDH from Proteus sp. exception ly exhibits a relatively low value (1.1 mM) for ammonia. Therefore, the GluDH is used for the determination of ammonia, as well as urea, by coupling of the urease reaction [104]. 

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