Urease, fluoride inhibition

An alternative scenario was put forward based on the crystal structures of urease inhibited by either phosphate, diamidophosphate, or borate (4,5, 28). It gets some support from the kinetic findings for fluoride inhibition of urease (29), as well as from recent model calculations (30, 31). Boric acid, known to be a competitive inhibitor of urease, can be considered a good substrate analogue, since it is isoelectronic with urea and has the same shape and dimension. Bacillus pasteurii could be crystallized in the presence of boric acid. The structure reveals that a molecule of B(OH)3 is symmetrically spanning the nickel ions, replacing Wj, W2,... 

In addition, urease is inhibited by a variety of agents including fluoride and disulfldes, as observed recently in the acetohydroxamate-inhibited C319A variants of K. aero-genes urease. It is proposed that this mode of inhibition involves coordination of the inhibitor to only one nickel center, while another involves the inhibitor bridging the dinickel center. Urease is slowly inhibited by fluoride in both the presence and absence of substrate, and fluoride binding rates are directly proportional to inhibitor concentration. Fluoride inhibition is pH-dependent due to a protonation... 

The activity of the enzyme is also strongly affected by the presence of inhibitors. Fluoride ions inhibit urease (173) and oxalate ions inhibit lactate oxidase (174), but the major inhibitors are heavy-metal ions, such as Ag+, Hg +, Cu " ", organophosphates, and sulfhydryl reagents (/i-chloromercuribenzoate and phenylmercury(II) acetate), which block the free thiol groups of many enzyme active centers, especially oxidase (69). Inhibiting the enzyme electrodes makes it possible to quantify the inhibitors themselves (69), for example, H2S and HCN detection using a cytochrome oxidase immobilized electrode (176). 

When sodium fluoride is used alone for anticoagulation, three to five times greater concentrations than the usual 2 g/L are required. This high concentration and the inhibition of the glycolytic cycle are likely to cause fluid shifts and a change in the concentration of some analytes. Fluoride is also a potent inhibitor of many serum enzymes and in high concentrations also affects urease, used to measure urea nitrogen in many analytical systems. 

Sodium lodoacetate at a concentration of 2 g/L is an effective antiglycolytic agent and a substitute for sodium fluoride. Because it has no effect on urease, it can be used when glucose and urea tests are performed on a single specimen. It inhibits creatine kinase but appears to have no notable effects on other clinical tests. 

Tran-Minh and Beaux (1979) investigated the competitive inhibition by fluoride of urease bound to the silicone rubber membrane of a carbon... 

Fig. 116. Inhibition of urease as it depends on the concentration of fluoride and the enzyme loading of the sensor. (Redrawn from Tran-Minh and Beaux, 1979).

Sometimes a preservative is added to the sample, usually along with an anticoagulant. Sodium fluoride is widely used as a preservative for samples to be analyzed for glucose. This is an enzyme inhibitor that prevents the enzymatic breakdown of glucose, or glycolysis. One milligram sodium fluoride per milliliter blood is adequate. Since it also inhibits other enzymes, including urease, sodium fluoride should not be added to samples to be analyzed for enzymes or for urea based on urease catalysis. 

Heavy metals and fluoride ions have been reported to inhibit the activity of the enzyme urease. 

Blood for urea estimation may be preserved from coagulation by addition of oxalate or citrate. Fluoride must not be used as it has an inhibiting action on the urease. 

The determination of urea in blood is even possible when it contains inhibitors such as fluoride ions, which are often used to inhibit glycolysis in blood samples. The appropriate immobilization of urease on the sensor removes interference from fluoride ions [102] and enables direct determination of urea over the whole range of biological concentrations. This biosensor can also be coupled with a glucose oxidase electrode to measure urea and glucose concentrations simultaneously in serum samples [113]. 

Effect of substrate concentration — The influence of substrate concentration on the response of an enzyme electrode in the presence of an inhibitor varies according to the inhibition mode (see 4.2.4.a). The degree of inhibition for an uncompetitive inhibitor, like NaF on urease, increases with the substrate concentration for a given biosensor (Figure 4.19). The detection limit for fluoride ions is thus improved by using high urea concentrations [132]. 

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