OBJECTIVES

1. To study how to isolate sub-cellular compartments of chicken liver using cold isotonic
sucrose (0.25M) followed by differentiation centrifugation.

2. To find out how to confirm the mitochondria has been successfully isolated by using
malate dehydrogenase assay.

RESULTS
Table 1.0 Absorbance of soluble fraction and mitochondrial fraction at 340nm

Time(s) Soluble Fraction Mitochondrial Fraction

0.00(Initial) 0.184 0.030
15.00 0.194 0.085
30.00 0.200 0.085
45.00 0.209 0.087
60.00 0.217 0.088
75.00 0.226 0.090
90.00 0.230 0.084
105.00 0.241 0.086
120.00 0.250 0.086
135.00 0.258 0.088
150.00 0.265 0.088
165.00 0.275 0.089
180.00 0.281 0.089
195.00 0.291 0.090
210.00 0.301 0.091
225.00 0.312 0.092
240.00 0.317 0.093
255.00 0.322 0.094
270.00 0.335 0.095
285.00 0.345 0.096
300.00 0.352 0.097
 Graph of absorbance of soluble fraction versus time
0.4

0.3
Absorbance

0.2

0.1

0
0 50 100 150 200 250 300 350
Time(s)
Graph of absorbance of soluble fraction versus time

Graph of absorbance of mitochondrial fraction versus

time

0.12
Absorbance

0.09

0.06

0.03

0
0 50 100 150 200 250 300 350
Time(s)
Graph of absorbance of mitochondrial fraction versus time

QUESTIONS

1. What is the reason of adding Triton into the mitochondrial fraction and not into the
soluble fraction?

Triton is a type of non-ionic surfactants or also known as a mild detergent is used to lyse
the cells to draw out the cellular organelle .Therefore, Triton is added into the
mitochondria fraction and not the soluble fraction to break the cells and obtain the
mitochondria.
2. What is the purpose of using malate dehydrogenase assay in this experiment? Explain
the result of this assay.

Malate dehydrogenase is a mitochondrial enzyme that helps in the reversible reaction of

malate to oxaloacetate by the reduction of NAD+ to NADH. Malate dehydrogenase assay
is also used to test whether the mitochondria is successfully isolated or not. According to
the graph that is plotted based on the experiment, we found out that we did not succeed in
isolating the mitochondria. This is because the graph did not show an increase in the
amount of NADH at absorbance of 340nm. Moreover, the graph of absorbance versus
time for mitochondrial fraction is lower than the graph of absorbance versus time for
soluble fraction. Thus, mitochondria is not successfully isolated.

DISCUSSION

Liver is a convenient source for functional intact mitochondria for a number of reasons.
Animal tissue is more readily homogenized than plant tissue because there are no cell
walls, and liver in particular is a soft and fairly homogeneous tissue. Isolating
mitochondria from highly structured animal tissues such as muscle tissues. To remove
large cell and tissue fragments and cell nuclei (the 'nuclear pellet'), we centrifuge the
homogenate at 500 x g for 10 minutes. When the supernatant is poured off, the loose
upper part of the mitochondrial pellet may come off as well. Intact mitochondria tend to
sediment more quickly than damaged mitochondria. The loose part of the pellet most
likely contains a high proportion of damaged (uncoupled) mitochondria, and can be lost.
The white foamy material near the top of the tube consists of lipids, which must be kept
from contact with the mitochondria. We don't add buffer at all. Mitochondria keep best
when concentrated, to minimize exposure to oxygen. We keep the centrifuge tube on ice
while stirring, and try not to introduce air into the suspension. (David R. Caprette.1996)

Malate dehydrogenase (MDH) is a predominately periportal enzyme that is expressed

highly in the extra-mitochondrial cytoplasm of the liver, although 10% of MDH has been
reported in the mitochondria.( A.D. Aulbach, C.J. Amuzie.2016)It is an enzyme in the
citric acid cycle that catalyzes the reversible conversion of malate into oxaloacetate.
Oxidation of malate in the cytoplasm regenerates OAA and nicotinamide adenine
dinucleotide (NADH).( John W. 2006) Spectral scans from 200 nm to 600 nm of NAD+
and NADH solutions (1 mg/ml) demonstrate the differences between the oxidized and
reduced forms of the molecule. NADH in solution produces a significant absorbance
peak at 340 nm, while NAD+ has virtually no absorbance at this wavelength.( Held.2007).
Triton X-100 is a nonionic detergent that disrupts lipid interactions while leaving protein
interactions intact.( Orlando.G.2013)
CONCLUSION

Our result is not as expected, failed to isolate mitochondria so suppose not much NADH
present. As result got from graph shows its absorbance lower than the soluble one.
Results are indicating inaccuracy in both collecting the samples and also measuring the
absorbance, this could be due to error in homogenisation and centrifugation techniques
but could also be due to error in the reading of absorbance using the Spectrophotometric
assay since U.V wavelength has different absorbance levels if either oxidised or reduced
enzymes absorb light therefore giving inaccurate indication to enzyme present. This may
affect the absorbance levels in the fractions if specific enzymes are affected thus giving
an altered absorbance level and therefore undermined protein amount.

Some of the organelles which remain in the supernatant fraction are the smaller and less
dense proportions of the cell such as ribosomes and lysosomes. Further centrifugation at a
higher speed can be used to separate these smaller less dense organelles into pellets.

In conclusion we see different with what we predicted, the specific activity (NAD+
transferred to NADH) is highest in the soluble fraction and the total activity is highest in
the mitochondria fraction. The % recovery of each fraction and the relative specific
activity for each fraction calculated shows a higher proportion in the soluble
fraction .Thus,the mitochonria fraction did not properly be divided although differential
fractionation may be a method of dividing cells into their component parts.There are
many differences in types of centrifuges available and results depend on the speed of the
centrifugation and whether a vacuum is present and the type of rotor used.

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