The Amount of Urea in a Specimen of Urine

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An experiment to determine the amount of urea in a specimen of urine.

Introduction.

Metabolism produces a number of toxic by-products, particularly the nitrogenous wastes that result from the breakdown of proteins and nucleic acids. Amino (NH2) groups are the result of such metabolic reactions and can be toxic if ammonia (NH3) is formed from them. Ammonia tends to raise the pH of bodily fluids and interfere with membrane transport functions. To avoid this the amino groups are converted into urea, which is less toxic and can be transported and stored to be released by the excretory system.
Urea is the result of two amino groups being joined to a carbonyl (C=O) to form CO(NH2)2, the process of which is called the ornithine cycle and takes place in the liver. The ornithine cycle was developed by Hans Krebs in 1932 and is similar to the Krebs cycle through the use of oxaloacetate. One of the steps in the cycle the breakdown of arginine into ornithine and urea, a reaction catalysed by the enzyme arginase. (See below)

(Fig 1.0)

Arginine Orthinine Urea

Urease is the enzyme which catalyses the hydrolysis of urea according to the following equation:

(NH2)2CO(aq) + 3H2O(l)  CO2(g) + 2NH3(g)

The acidic ammonium carbonate is formed because the carbon dioxide dissolves in water to produce carbonic acid (H2CO3), which immediately reacts with ammonia to form the ammonium carbonate. This is shown by the following equation:

2NH3(g) + H2CO3(aq)  (NH4+)2CO3(aq)

The resulting solution can then be titrated against hydrochloric acid with methyl orange as the indicator in order to determine how much urea was present initially.
The point of neutralisation using a methyl orange indicator is determined using the following colour changes.

 Acid  Red.
 Neutral  Yellow.
 Alkali  Orange.

Enzymes are nearly all made up of globular proteins. The structure of enzymes can be divided into three categories:

1. The primary structure, which is the sequence of amino acids.
2. The secondary structure, which is the coiling of the protein into an alpha helix
3. The tertiary structure, which is the 3D shape into which the protein is folded. This shape gives the enzyme its properties and specificity. The shape is held together by ionic bonds, disulphide bridges and the weaker hydrogen bonds.

Method.

Six urea solutions were prepared an placed in conical flasks one of which was of unknown concentration. The flasks were sealed to prevent CO2 and NH3 gases from escaping and then placed in a water bath at 35oC for 1 hour.

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