Download Word Count: 1390 An experiment to determine the amount of urea in

Word Count: 1390
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. The temperature was kept at
35oC as each enzyme has an optimum temperature at which it works best, so it was
important that the temperature remained constant for the duration of the reaction.
After 1 hour all the flasks were removed. A burette was washed first with
distilled water to remove any impurities. Then with HCl to prevent the acid from being
neutralised by the remaining water, as this would increase the pH of the acid and give a
less accurate titre. It will also remove any impurities not dealt with by the water. The
burette was then carefully filled to the top with HCl. A 10cm3 pippet was used to place
portions of the urea solution into a beaker, into which a few drops of methyl orange were
placed to act as an indicator. The beaker was arranged on top of a white tile so that the
end-point of the titration could be determined more accurately.
At the start of the titration the solution was yellow and at the end-point it turned red.
This process was repeated for each solution, and the volume needed to completely
neutralise 10cm3. Each time the procedure was repeated 3 times and the average titre
would be calculated.
Results.
(Fig 2.0)
Concentration of urea (g/100cm3) Volumes of 0.1M HCl
Required (cm3)
Mean volume of 0.1M HCl required (cm3) (1dp)
0.32 9.2, 9.3, 9.3 9.3
0.48 10.9, 11.0, 10.9
10.9
1.0
13.1, 13.5, 13.6
13.4
2.0
15.3, 15.2, 15.7
15.4
4.0
18.8, 18.4, 18.5
18.6
Unknown
11.4, 11.0, 11.1
11.2
Discussion.
My average titre for the unknown sample was 11.2cm3. When I applied this to the graph
I found the concentration of urea to be 0.58g/100 cm3.
Figure 2.2 clearly shows that as the concentration of urea increases, the volume of HCl
required for neutralisation also increases. This is to be expected as there are more moles
of urea being hydrolysed, which would mean more HCl would be required. However the
curve definitely indicates that the rate of increase decreases as the amount of urea present
initially rises. This means that the concentration of urea is not directly proportional to the
amount of ammonium carbonate produced, as the equations shown earlier suggested.
This leads me to believe that it is the activity of the enzyme, which has caused this result.
It may be due to availability of the active site on the enzyme, i.e. because the Urease
causes the release of CO2, which dissolves in the water forming carbonic acid. This in
turn reacts with ammonia forming ammonium carbonate, which is then titrated with HCl.
So the availability of the active site would dictate how much ammonium carbonate would
be produced, and hence the volume of HCl required to neutralise it. In which case I
would expect to see the graph level off if solutions of greater concentration were used.
Temperature could not have been a factor as this was kept constant through the
use of a water bath at 35oC.
The pH is a likely factor as throughout the reaction there are acids being
produced. The precise 3-dimensional shape of the enzyme is partly the result of hydrogen
bonding. However these bonds may be broken by the concentration of hydrogen ions
(H+) which are present when acids are in solution. The CO2 being produced formed
carbonic acid, which then went on to form ammonium carbonate. Both of which are
relatively weak acids, however it need not be strong to affect the enzyme as pH is a
logarithmic scale and a change of one pH point represents a tenfold change in the H+
concentration. By breaking the hydrogen bonds, which give shape to the enzyme, any pH
change can effectively denature it.
Every enzyme has an optimum pH at which it functions at its best and in this case the
enzyme is likely to have been affected by the change in pH that occurred. The enzyme
Urease has fallen victim to what could either be toxic accumulation, or a kind of feedback
inhibition, which resulted from its own waste products.
Limitations.
As described the fact that the pH changed during the course as a result of the
products formed, could have affected the activity of the enzyme. If I were to repeat the
experiment I would try using a buffer solution to overcome this problem. The time could
have been a factor as the enzyme may not have had sufficient time to catalyse the
reactions. Again if I were to repeat the experiment I would leave the reaction for 2 or
more hours.
Judging the end-point with the naked eye is not sufficiently accurate and human
error is more than likely, especially when the colour change is so gradual. The best way
to overcome this in future investigations would be to use a colorimeter. This would give
more accurate titration readings.
Biological significance.
When patients undergo pregnancy and other urine tests they are asked for an early
morning sample. This is because no water has been drunk for a very long time and a
great deal of reabsorbtion has occurred in the kidneys. This is due to an increase in ADH
(antidiuretic hormone) secretion from the pituitary gland. Such reabsorbtion has caused
the urine to be more concentrated with urea than normal.
During the day water will taken in so the reabsorbtion of water in the kidneys will be
unnecessary. This will result in a decrease in the concentration of urea during the day.
Urease can also be used by plants, which take in urea from the soil and obtain the
nitrogen from its decomposition to CO2 and NH3. The plant then uses this to
make nucleic acids or amino acids.
Keywords:
word count experiment determine amount urea specimen urine introduction metabolism
produces number toxic products particularly nitrogenous wastes that result from
breakdown proteins nucleic acids amino groups result such metabolic reactions toxic
ammonia formed from them ammonia tends raise bodily fluids interfere with membrane
transport functions avoid this amino groups converted into urea which less toxic
transported stored released excretory system urea result amino groups being joined
carbonyl form process which called ornithine cycle takes place liver ornithine cycle
developed hans krebs similar krebs cycle through oxaloacetate steps breakdown arginine
into ornithine reaction catalysed enzyme arginase below arginine orthinine urease
enzyme which catalyses hydrolysis according following equation acidic ammonium
carbonate formed because carbon dioxide dissolves water produce carbonic acid
immediately reacts with ammonia form ammonium carbonate this shown following
equation resulting solution then titrated against hydrochloric acid with methyl orange
indicator order determine much present initially point neutralisation using methyl orange
indicator determined using following colour changes acid neutral yellow alkali orange
enzymes nearly made globular proteins structure enzymes divided into three categories
primary structure sequence acids secondary structure coiling protein alpha helix tertiary
shape protein folded this shape gives enzyme properties specificity shape held together
ionic bonds disulphide bridges weaker hydrogen bonds method solutions were prepared
placed conical flasks unknown concentration flasks were sealed prevent gases from
escaping then placed water bath hour temperature kept each optimum temperature works
best important that temperature remained constant duration reaction after hour flasks
were removed burette washed first distilled water remove impurities then prevent being
neutralised remaining would increase give less accurate titre will also remove impurities
dealt burette carefully filled pippet used place portions solution beaker drops methyl
placed indicator beaker arranged white tile that point titration could determined more
accurately start titration solution yellow point turned process repeated each volume
needed completely neutralise each time procedure repeated times average titre would
calculated results concentration volumes required mean volume required unknown
discussion average titre unknown sample when applied graph found concentration figure
clearly shows increases volume required neutralisation also increases expected there
more moles being hydrolysed would mean more however curve definitely indicates rate
increase decreases amount present initially rises means directly proportional amount
ammonium carbonate produced equations shown earlier suggested leads believe activity
caused availability active site because urease causes release dissolves forming carbonic
turn reacts forming titrated availability active site dictate much produced hence neutralise
case expect graph level solutions greater used could have been factor kept constant
through bath likely factor throughout reaction there acids produced precise dimensional
partly hydrogen bonding however these bonds broken hydrogen ions present when
formed carbonic went form both relatively weak however need strong affect logarithmic
scale change represents tenfold change breaking give change effectively denature every
optimum functions best case likely have been affected occurred urease fallen victim what
could either accumulation kind feedback inhibition resulted waste products limitations
described fact changed during course products have affected activity repeat experiment
using buffer overcome problem time been factor sufficient time catalyse reactions again
repeat experiment leave hours judging naked sufficiently accurate human error than
likely especially when colour gradual best overcome future investigations colorimeter
give accurate titration readings biological significance patients undergo pregnancy other
urine tests they asked early morning sample because drunk very long great deal
reabsorbtion occurred kidneys increase antidiuretic hormone secretion pituitary gland
such reabsorbtion caused urine concentrated than normal during will taken reabsorbtion
kidneys will unnecessary decrease during also used plants take soil obtain nitrogen
decomposition plant uses make nucleic
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