Urease enzymic activity measurement

Activity assays of enzymes bound to solid phases in EIA systems have previously been limited to fixed-time spectrophotometric methods following incubation of substrate and solid phase for extended periods of time. Kinetic assays of enzyme activity have not been used to date because of the difficulty in directly monitoring initial rates of enzyme reactions in a turbid solid phase suspension. With urease as the label, an ammonia gas sensing electrode can be used to directly quantitate the amount of urease-labeled antigen or hapten bound to a double-antibody solid phase by continuously measuring the initial rate of ammonia produced from urea as a substrate. 

With a gelatin membrane entrapped between two dialysis membranes and containing 46 U/cm of enzyme, the H2O2 formation corresponds to only 110 mU/cm, ie, less than 1% of the initial enzyme activity (Figure 14-19). This indicates a large excess of enzyme in the membrane. Consequently, the membrane is diffusion controlled. The low apparent activity may be attributed mainly to the diffusion resistance of the dialysis membrane for glucose. On the other hand, the measured activity of the membrane containing 46 U/cm is already about 70% of the activity used for immobilization. This value approaches that expected for pure kinetic control of the process [287], The apparent activity of urease immobilized in a cellulose triacetate membrane was found to be 66% of the initially applied enzyme activity [288]. 

The production of ammonia from urea gives rise to a shift of the C(V)-curve along the voltage cLxis as discussed above. The inhibition of the urease activity by Hg " was estimated from the slope of AV versus time plots, compared to the slope without added mercury(II) chloride. In Fig.5, we have shown the observed relative enzyme activity as a function of the Hg -concentration. The detection limit appears to be about 0.001 /xM Hg " " in this measurement system. One sample could be processed within 5 minutes. 

Hellerman (368) has found that crystalline urease in dilute pH 7.0 phosphate buffer loses its a type SH groups (32) upon treatment with pure mustard. This change in SH groups measured with the aid of iodoso benzoate solutions did not result in the loss of enzyme activity. 

Detection limits in EIA are ultimately determined by how low one can measure the label s concentration via an activity assay. Sensitivity in such a kinetic determination is dependent upon the turnover number of the enzyme molecule and the method employed to detect the product of the catalyzed reaction. Purified urease obtained from Sigma Chemical Co. has considerably higher activity on a molar basis (international units per mole of enzyme) than the best available commercial preparations of some other common enzyme labels such as alkaline phosphatase, /8-galactosi-dase, peroxidase, - and glucose oxidase. This is due to the high mo-... 

Enzyme-catalyzed reactions are used for the determination not only of substrates but also of the enzymes as well as activators and inhibitors of the enzymatic reaction. Substrate concentrations can be measured also by nonkinetic methods by allowing a reaction to proceed to completion before making measurements. Enzymatic methods of analysis have been reviewed by Guilbault. He listed over 150 enzymes with their sources many of these are now available in purified form with high specific activity. Urease was the first to be obtained in pure crystalline form. ... 

Nickel is required for the synthesis of active urease in plant and other cells. The enzyme catalyzes the hydrolysis of urea to carbon dioxide and ammonia, via the intermediate formation of carbamate ion (equation 46). The molecular weight has been redetermined recently as 590 000 30 000, with six subunits. Each subunit has two nickel centres and binds one mole of substrate. The activity of the enzyme is directly proportional to the nickel content, suggesting an essential role for nickel in the enzyme. Several approaches, including EXAFS measurements, suggest that histidine residues provide some ligands to nickel, and that the geometry is distorted octahedral. There appears to be a role for a unique cysteine residue in each subunit out of the 15 groups present. Covalent modification of this residue blocks the activity of the enzyme. 

A heterogeneous EIA coupled with a potentiometric electrode permitted the assay of BSA down to 10 ng/ml and cAMP down to 10 nmol/1 (Meyerhoffand Rechnitz, 1979). Urease was used as the marker enzyme and its activity was measured by means of an ammonia gas-sensing electrode. The equilibrium of the immunological reaction at the sensor was reached rather slowly. The advantage of the rapid response of biosensors could not therefore be exploited. 

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