This is the Biochemistry Protocols section post. Here we are providing the “Assay of Urease enzyme activity” protocol. before going to the protocol we need to know a few points about “Urease Enzyme”.

Table of Contents

What is Urease?

Urease is a hydrolytic enzyme that catalyzes Urea into Carbon dioxide and ammonia. The Enzyme Commission Number is 3.5.1.5. This reaction follows

(NH₂)2CO + H₂O →CO₂ + 2NH₃

In 1926, James B. Sumner, an assistant professor at Cornell University, showed that urease is a protein by examining its crystallized form. Sumner’s work was the first demonstration that a pure protein can function as an enzyme, and led eventually to the recognition that most enzymes are in fact proteins and the award of the Nobel Prize in chemistry to Sumner in 1946.

Special characteristic features of Urease Enzyme

An active site requiring nickel in jack-beans and several bacteria. However, in vitro activation also has been achieved with manganese and cobalt
Molecular weight: 480 kDa or 545 kDa for jack-bean Urease (calculated mass from the amino acid sequence). 840 amino acids per molecule, of which 90 are cysteines.
Optimum pH: 7.4
Optimum Temperature: 60 degrees Celsius
Enzymatic specificity: Urea and Hydroxyurea
Inhibitors: Heavy metals (Pb⁻& Pb²⁺)

Assay of Urease Enzyme Activity (UPDATED)

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Assay of Urease Enzyme Activity

Aim:

To assay the urease activity

Reaction:

The enzyme splits urea liberating NH₃ and CO₂ as per the following reaction.

Principle:

The reaction is stoichiometric the enzyme activity is easily determined by measuring the amount formed this can be observed colorimetrically. Since NH₃ forms a brown complex in the presence of Nessler’s reagent (K₂ Hg I).

Reagents:

66N H2SO4 (N=36) [calculated value is 1.83ml / 100ml distilled water]
3% Urea
1M Na tungstate [16.493 gms/50ml]
2M Phosphate buffer (pH = 7)
Stock standard: Dissolve 20mgs of NH₄ (SO₄)₂ in 100ml of distilled water
Nessler’s reagent

Standard Graph:

A pure ammonium sulfate solution (20mg/100ml) is prepared and different aliquots of it are taken and the volume is made up to 3ml with distilled water. To this 1ml of Nessler’s reagent is added. After mixing the color intensity is measured at 500nm. A standard graph is drowning in the usual way.

Enzyme source:

Horse gram seeds contain a high concentration of the enzyme. The seeds are powdered in a pestle and motor and about 1gm of finely grind powder is suspended in 100ml of distilled water and stirred well. This suspension is filtered through a coarse cloth and filtrate is used as the enzyme source.

Enzyme Assay:

Pipette out 1ml of substrate solution i.e., 3% Urea solution buffered with 1ml of 0.2M phosphate buffer (pH 7). Add 1 ml of enzyme extract and incubate at 55⁰C for 15 minutes. At the end of incubation time quickly place the tubes on the ice. Add 1 ml of 0.66N H₂SO₄ to stop the reaction and 1 ml of 1M sodium tungstate solution to precipitate the protein. Filter or centrifuge to remove the precipitate and aliquots of supernatant are assayed for NH₃ and the enzyme activity is calculated.

Result:

The amount of Ammonia present in the given unknown sample is _____________mg of Ammonium (NH₃) formed / 1 ml of enzyme / 15 minutes.

Assay of Urease Enzyme Activity (UPDATED)

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Few other Protocols

Assay of Urease Enzyme Activity (Enzymology Practical Protocol)

What is Urease?

Assay of Urease Enzyme Activity