DISCUSSION: This experiment dealt with the sub-cellular fractionation which is the

process by which various rat organelles were separated into different fractions. The purity
of these fractions was then determined by the use of marker enzymes and calculation the
specific activity, total activity, % total activity and total recovery for each.
The first part of this experiment dealt with the determination of the protein content in
each fraction. This was done by the use a Lowry Reagent. The expected results suppose
to show the highest protein content being in fraction1 Homogenate as it would contain
all the proteins present in the following fractions with the second highest being in fraction
5 Soluble Fraction as it would contain all proteins within the cytosol. This was reflected
in the obtained results with Fraction 1 containing total protein 22068.9mg and Fraction 5
containing 8000mg. The fraction with the smallest mg of proteins is suppose to be
fraction 3 lysosome which contain only digestive enzymes. This is also reflected in the
results with 1186.9mg. The mitochondria is the site of many metabolic processes such as
respiration and microsome would contain the ER which is the site of protein formation so
both would not contain to low of a protein concentration with it being 3343.6mg and
2662.4 mg respectively.
The second part of this experiment dealt with the enzyme glucose 6 Phosphatase
which is the marker enzyme for the microsome. It would be expected that Fraction 3
Microsome to have the highest activity/mL with low values in the lysosomal fraction.
This is because the lysosomal fractions suppose to contain digestive enzymes which
Glucose 6 Phosphatase is not part of. The results obtained did not completely reflect
this. The lysosomal fraction did have the lowest value of 1.45 U/mL, however the
microsome fraction did not contain the highest value, the homogenate did (5.36 U/mL
and 5.98 U/mL respectively.) Even though the values did not differ by much this
deviation from the theoretical results may be due to the fact that the homogenate which
contains the microsome has the enzyme present in it and there was no 100% separation of
microsome from the homogenate. The mitochondria has a higher than expected value
(3.66 U/mL), this may be due to contamination of the mitochondrial fraction by the
endoplasmic reticulum. The soluble fraction also has a high value (4.12 U/mL) and this
may be due to the fact that it would also contain some ER but not as much as the
microsome.

In the third part of this experiment the marker enzyme was Acid Phosphatase which was
the marker enzyme for lysosome. The lysosome, which usually contains digestive
enzyme has a very impenetrable membrane which incases these enzymes. Therefore
inorder for Acid Phosphatase enzyme to act upon the substrate provided the membrane
must first be lysed for disrupted so that it would be released. In this part we used both
intact lysosomes and lysed lysosomes which was facilitated by the Triton X detergent. It
would be expected that the lysed sample would have the highest activity/mL out of all the
fractions. This however was not observed; in actuality it had the lowest value of 0.0860
U/mL. This may be due to errors in the preparation of the sample. The soluble fraction
contained the highest value instead with 0.4390 U/mL and may be a result of the
lysosome be disrupted during the sub-cellular fractionation, the enzymes would remain in
the supernatant as it would be to light to pellet out. It can be noted that all values obtained
seem lower than the expected and this may be due to the enzyme starting to become
degraded, contamination or issues with the specimen.
The forth part of this experiment dealt with the enzyme glutamate dehydrogenase which
is the marker enzyme for the mitochondria which is more specifically located in the
mitochondrial matrix. It is to be expected that Fraction 2 Mitochondria would have the
highest activity/mL. This was observed in the obtained results with fraction 2 having
0.088 U/mL. The low results obtained for the homogenate fraction which was 0.040
U/mL is due to the fact that the substrate required for this enzyme cannot cross the
mitochondrial membrance. The high value present for the microsome (0.072 U/mL) may
be due to contamination by mitochondria
The next part of this experiment uses the marker enzyme Lactate Dehydrogenase which
is also for the soluble fraction. It would then be expected that fraction 5 would contain
have the highest activity U/mL, however this is not the case. The highest activity can be
seen in fraction 1 followed closely by fraction to ( 0.723 U/mL and 0.715 U/mL). This
may be a result of the fact in rats, lactate dehydrogenase is also found in the mitrocondria
which would account for the fraction 3 value. It should be noted that the enzyme does
show the highest specific activity in the soluble fraction i.e produces more product per
mg protein.
The last part of this experiment was to determine the subcellular location of succinate
dehydrogenase which is an intergral enzyme of the electron transport chain and and krebs

cycle. It is located on the inner mitochondrial membrane and so it would be expected that
fraction 2 would have the highest activity followed by fraction 1. This was reflected in
the results obtained with fraction 2 having an activity of 0.0275 U/mL and fraction 1
having 0.0028 U/mL. The other fractions showed no activity which showed there was no
contamination.
Errors which could have occurred in this experiment was that was we were working with
cell and enzymes certain conditions and procedures must be followed. In the dilutions of
the fractions sucrose must have been used instead of water. If water was used, it would
have entered the cells causing it to swell and burst. Also the enzyme would degrade after
a certain time and must be kept on ice.