Following the progress of an enzyme-controlled rea

ction.

Aim: To follow the progress of an enzyme-controlled reaction using starch
solution and amylase.

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Hypothesis: It may be assumed that the longer the starch will be in contact
with the enzyme the more starch will be converted into maltose. It can also
be expected that the more substrate is digested into the break down product
the lower color readings will be obtained with the colorimeter confirming
the hypothesis that as time goes by the amylase breaks down more starch. It
is highly probable that the rate of reaction will be very high at first due
to the great abundance of starch and gradually slower as the substrate
reduces in quantity and the product increases.

Concerning the starch depletion/enzyme activity rate it is
likely to slow down towards the end as the amylase is running out of starch
to break down.

Regarding the final Benedicts’ test on the remainder of the solution,
it can be confidently stated that it will reveal to be positive due to its
high concentration of maltose.


Variables:
|Aspects modified|Aspects maintained the|Uncontrolled aspects |
|purposely |same purposely ||
|The amount of time the|The amount of iodine |Amount of distilled|
|starch was left to|in each test tube.|water used to dilute |
|interact with the|The amount of|each test tube. |
|amylase. |starch/amylase |Each colorimeter|
||solution in each test |sample holder might|
||tube|have varied slightly |
||The ratio of starch|in transparency |
||solution mixed with|although they were not|
||amylase (1:2)|apparent at first|
|||sight.|
|||The exact size of the |
|||drops of iodine,|
|||amylase/starch |
|||solution placed in the|
|||sample holders |
Apparatus:
. 11 test tubes
. Amylase
. Starch solution
. Pipette
. Syringe
. Beaker
. Colorimeter
. 10 colorimeter sample holders
. Iodine
. Benedict’s solution
. Bunsen burner
. Test tube holder
Diagram:
Method:
1. Place one drop of iodine in 10 colorimeter sample holders
2. Mix amylase and starch solution to a ratio of 1:2 and place two drops
of the mixture in the first sample holder at once.

3. Wait a minute before placing to drops into the next sample holder
4. While waiting fill the first sample holder with distilled water to the
brim.

5. Repeat steps 3-4 for the remnant sample holders.

6. Measure the color absorption with the colorimeter.

7. Graph the results.

8. With the remaining amylase/starch solution ad benedict’s solution and
boil under flame.

9. Note changes of color.


Results and Observations:
When Benedicts’ solution was added to the remaining amylase/starch and
boiled an unmistakable change of color occurred. The presence of sugar – in
this case maltose – can be confirmed with this test. The solution became a
bright orange/yellow.

Absorption readings for iodine tests (colorimeter – 5% uncertainty)
||Absorption readings (colorimeter – 5%|
||uncertainty)|
|Mins |Group 1 ?|Group 2 +|Group 3 ?|Group 4 ?|
|1|1.42|0.80|0.96|1.16|
|2|– |0.58|1.00|0.88|
|3|– |0.56|0.71|– |
|4|1.39|0.43|0.50|0.66|
|5|1.31|0.31|0.48|– |
|6|1.24|0.31|0.44|0.66|
|7|1.09|0.25|0.37|– |
|8|0.44|0.21|0.22|0.43|
|9|0.42|0.21|0.28|– |
|10|– |– |– |0.27|
|11|– |– |– |0.41|
|12|– |– |– |0.73|
|13|– |– |– |0.25|
Transmission Readings for iodine tests (Colorimeter – 5% uncertainty)
||Transmission readings|
||(Colorimeter – 5% |
||uncertainty) |
|Mins |Group 2 +|Group 4 ?|
|1|0.49 |0.27 |
|2|0.56 |0.34 |
|3|1.00 |0.44 |
|4|1.19 |0.41 |
|5|1.63 |0.53 |
|6|1.44 |0.67 |
|7|1.29 |0.96 |
|8|1.92 |1.16 |
|9|1.98 |–|
|10|2.08 |–|
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Absorption readings for Benedict’s test (Colorimeter – 5% uncertainty)
||Absorption Readings for |
||benedicts’ test.|
||(Colorimeter – 5% uncertainty) |
|Mins|Group 3|
|1 |0.97|
|2 |0.84|
|3 |1.19|
|4 |1.6|
|5 |1.38|
|6 |0.89|
|7 |1.38|
|8 |1.04|
|9 |1.09|
|10 |1.18|
|Remainder|0.99|
Absorption rate of Starch/Amylase
|Mins |Absorption Rate|
|||
||of |
||Starch/Amylase |
|1|0.275|
|2|0.115|
|3|0.125|
|4|0.118|
|5|0.055|
|6|0.040|
|7|0.010|
|8|0.050|
|9|0.025|
|10|0.010|
|11|0.015|
|12|0.010|
|Standard Curve|
|Absorption |Starch Concentration|
||(%)|
|1|1.20|
|0.5 |0.70|
|0.25 |0.30|
|0.10 |0.10|
The standard curve numbers were obtained by Mr. Moore
Reaction Rate (Rate of Starch Depletion) (Assumed enzyme Activity Rate) –
results found thanks to the standard curve + absorption results.

|Tim|Group |Group |Group |Group |
|e |1|2|3|4|
|1 |1.55 |0.87 |1.05 |1.27 |
|2 ||0.63 |1.1|0.96 |
|3 ||0.61 |0.78 ||
|4 |1.53 |0.47 |0.55 |0.72 |
|5 |1.44 |0.34 |0.52 ||
|6 |1.36 |0.34 |0.48 |0.72 |
|7 |1.2|0.27 |0.4||
|8 |0.98 |0.23 |0.35 |0.47 |
|9 |0.48 |0.23 |0.24 ||
|10 |0.46 |0.23 |0.31 |0.29 |
|11 ||||0.45 |
|12 ||||0.8|
|13 ||||0.27 |



Conclusion: The most fulfilling results were the absorption readings for
iodine. The reasons behind this were that first of all there were more
results gathered by all four groups and the numbers were rather advancing
in a logical progression. Thanks to the absorption readings for iodine we
were able to determine that the longer the amylase is left in contact with
the starch the more maltose is produced. This can be calculated by
observing the progressive drop of absorption figures in every group. There
seemed to be an anomaly in the results of group one (clearly seen in the
second graph). The reason for these results is obscure but the figures
descend nonetheless suggesting hat the irregularity still demonstrates a
progressive decrease of substrate and increase of product.

The transmission results were less satisfying. First of all only two
groups made this type of readings and the graph formed was unproductive (at
least compared to the absorption readings). But although the data is quite
set apart when in comparison with both groups a slow ascent can be noted
which confirms the conclusion that starch is slowly being broken down, the
fact that the transmission graph ascends and the absorption graph descends
is quite helpful in confirming this deduction – transmission is the
opposite of absorption.

The Benedicts’ test was valuable as it clearly confirmed that sugar
was present in the starch/amylase solution after 13 minutes. This verifies
that maltose has indeed been produced – Maltose occurs mainly as a
breakdown product during digestion of starch by enzymes called amylases.

This commonly occurs in animals and germinating seeds. 1
However the Benedicts’ distilled colorimeter readings gave strange
incoherent results that unabled us to takeanyreliableresults.

Nevertheless the graph that may have given slightly better readings would
be the XY scatter one due to the fact that it is easier to plot a straight
line. The reason why the colorimeter results were unsatisfactory may have
been due to the fact that the benedicts’ test dissolved badly with
distilled water and made the orange particles sink to the bottom, making it
extremely difficult for the colorimeter to take accurate readings. Another
reason or at least influencing factor may have been that the colorimeter
has difficulty in distinguishing green and yellow.

After calculating the absorption rate it is possible to establish
that in the beginning the breaking down of the substrate into the product
is rapid and effective, this is probably due to the abundant quantities of
starch. As the amount of unbroken down starch decreases so does the rate of
absorption. – The rate of an enzyme reaction is measured by the amount of
substrate changed, or amount of product formed, during a period of time.2
Thanks to the standard curve provided by Mr. Moore it was possible to
enforce the preceding conclusion about the absorption rate. As time goes by
the starch depletion rate starts from very high and slopes down o very low
demonstrating that as the amount of starch left decreases so does the
reaction rate.


Evaluation:
The first problem encountered during this experiment was that the
experiment wouldn’t work unless it was diluted with distilled water because
the iodine seemed to have an extremely high concentration. The readings
obtained without diluting the solutions were unfruitful.

Furthermore two types of iodine were available and none of the groups
recorded which iodine was used. One type may have produced different
results or the concentration level may have been slightly higher or lower.

Therefore, group 1-s absorption results should be treated completely on
their own as a completely different variable.

Although the ratio was kept the same when amylase was mixed with
starch higher quantities could have a higher reaction rate than smaller
quantities – as demonstrated with the absorption of starch/amylase, the
higher the amount of starch present the higher the reaction rate.

The exact amount of distilled water in each sample holder was not
measured. They were simply filled to the brim with no measurement involved.

This may have caused a slight misreading of results as the concentration of
the starch/amylase solution may have varied from sample holder to sample
holder.

The most probable reason why no profitable results could be taken
from the Benedicts’ test colorimeter readings is due to its precipitation
to the bottom. This may have been remedied if the colorimeter were equipped
with a system that would agitate the sample holder preventing any particles
from depositing in the bottom.

If more results had been provided by more groups on the transmission
readings of the starch/amylase solution and the benedict’s absorption
readings then perhaps more attenuate conclusions may have been obtained and
discussed. This is reinforced when one looks at the second graph for the
starch/amylase absorption readings, which demonstrates that although three
groups out of four acquired similar and complementary results group 1 had
very distinctive curve and set of data. If one were to base their results
solely on one set of readings the conclusions may be erroneous.

The more graphs and readings obtained the more accurate and abundant
readings and results obtained which gives a consistent and precise write-
up.



———————–
1 D. J. Taylor, N. P. O. Green, G. W. Stout, Biological Science, 1997,
University Press, Cambridge, UK, page 85
2 D. J. Taylor, N. P. O. Green, G. W. Stout, Biological Science, 1997,
University Press, Cambridge, UK, page 118
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