DISCUSSION:

This experiment involved the separating of cells from a liver into different organelles. Organelles

are membrane-enclosed vesicles in eukaryotic cells. Organelles have different tasks in the cell

that is catalyzed by specific enzymes. For this experiment the rat liver subcellular fractions was

first isolated by cell disruption after this was done , it underwent differential centrifugation.

Homogenization and centrifugation are two phases of subcellular fractionation.When cells

undergo homogenization the tissus ar disrupted and this causes cellular componnts to be

released .Isotonic sucrose which is part of the homogenization medium prevents the organelle

membranes from rupturing , the reason for this is that it is very important to to purify intact

organelles. (Alberts 2002).

(Graham, Rickwood 1997).

The first fraction is the cell homogenate

Made by rupturing of the cell membranes in the tissue

The homogenate is then centrifuged for a short period of time to remove cell debris

and nuclei

The order of sedimentation of components in cells is typically: nuclei,

mitochondria, lysosomes and microsomes and soluble fraction (cytoplasm)

Some errors may occur during this process of subcellular fractionation for instance there maybe

contamination because there is a possibility that an organelle may break open which would lead

to the contamination of other organelles. Due to this marker enzyme come in to play. These

enzymes show which organelles are present after each step. These enzymes are situated in a

particular cell organelle , it can also be found in the cytosol. . (Cooper 2000). Some of the marker

enzymes and the corresponding organelle are Glucose-6-Phosphatase in the Microsomes (E.R),

Acid Phosphatase in Lysosomes, Glutamate Dehydrogenase in the Mitochondria, Lactate

Dehydrogenase in the Soluble Fraction, and Succinate Dehydrogenase in the Inner Mitochondrial

membrane. The amount of marker enzyme present in a particular organelle is dependent on the

technique used to separate the cell. The technique used in this experiment was subcellular

fractionation, which separates the organelles based on their size, densities and shape (Graham,

Rickwood 1997). .An ideal marker is one that is distributed homogenously throughout one

particular subcellular fraction(Graham, Rickwood 1997). When unwanted marker enzyme are

present shows the level of contamination by other organelles, while the degree of enrichment for

the desired organelle is determined by the specific activity of the target marker enzyme. .( carter

1993)

Once cell fractionation is done , the Lowry Assay is carried out this is done in order to indicate

if the fraction contains protein . This will indicate the presence of specific enzymes.

(Lowry, 1951)

To obey Beer’s law, the equilibrium for reaction of the colorimetric reagent (Folin’s reagent) and

the protein to be measured must be more in favour of the coloured complex. In other words a

linear relationship should be obtained between the mass of the protein and the absorbance .From

the graph plotted it can be seen that this was achieved. A darker blue complex was observed as

the protein solution increased in volume The copper in Lowry reagent B acts as a catalyst and

exerts its effect upon the increased number of peptide bonds present. Since an increase in protein

also refers to an increase in the number of copper catalyzed tyrosine, tryptophan and cysteine

residues on the protein, on the addition of the Folin reagent there will be a darker coloured

Firstly it uses the “Biuret” reaction in which the cupric ions in alkaline solution react with the Nitrogen in the peptide bonds of the protein, forming protein copper complexes that are pink to purple in colour. The chromophore, also

known as the light absorbing centre, seems to be a complex between the peptide backbone of the protein and cupric

ions.

The second colour forming reaction depends upon the Folin-Ciocalteu phenol reagent, where colour development is based on the copper catalyzed reducing power of certain

residues on the protein such as tyrosine, tryptophan and cysteine.

An intense blue- green colour is formed due to the reduction of phosphomolybdicphosphotungstic acid to

heteropolymolybdenum blue.

complex being formed. This is due to a higher reducing power by the residues on the protein.

The reason why the tubes were left to stand for 10 minutes was to allow complete reduction of

the Folin Ciocalteu’s reagent by the protein.

According to Table 1.2, the total protein (mg) obtained for Fraction 1: the homogenate is by far

the highest i.e 11 056.2 mg. This is expected as the homogenate contains the most protein since

the only organelle removed from this fraction is the nuclei. As expected the sum of the other four

fractions was determined to have almost the same protein content as Fraction 1; i.e 10 490.48

mg. Throughout subcellular fractionation the total amount of protein from subsequent fractions is

supposed to add up to the amount of protein in Fraction 1 since each centrifugation step removes

a certain organelle (a certain amount of protein). However due to experimental error the value

would never be the same.(Lambowitz, 1979). From the results it is seen that the mitochondrial

fraction has the lowest amount of protein (875.33mg). This is because the majority of proteins is

located in the supernatant. Therefore there is more protein present in Fractions 3, 4 and 5.

Fraction 5 has a high protein content of 6572.12mg because it is the soluble fraction (cytosol)

which contains a lot of soluble proteins. From the % Recovery of protein in Table 1.2 the

percentage of protein in each fraction can be seen in relation to total protein which is in the

homogenate; fraction 1.

The first enzyme to be assayed in this experiment was Glucose-6-Phosphatase. The results

showed that the Microsomal fraction (Fraction 4) had the highest total activity besides the

homogenate. The homogenate would have all the enzymes present, since it has all the organelles

and therefore can be omitted from the comparison of activity within the fractions. It can be used

thought to determine how much of the enzyme was recovered. This means that glucose-6-

phosphatase is most likely found in the microsomes and is therefore the marker enzyme for that

organelle. The experiment also showed results of activity for other fractions, although they were

low compared to that of the microsomes. This can be explained by contamination of fractions by

microsomes because they are formed by debris of the E.R. Therefore, if parts of the E.R. get

pelleted in another fraction, microsomes can form there and contain glucose-6-phosphatase

The phosphorous content is determined in each fraction by measuring the absorbance at 650nm

and reading the value off the calibration curve from which activity of the enzyme can be

determined.

For the assay, 5 tubes were used as zero time controls; that is 10% TCA was added to the tubes

before the fractions were added. The purpose of the TCA is to denature the enzyme when added,

resulting in no product being formed. From the results it is seen that the activity in fraction 1 was

fraction 4 were very close which indicates that most of the microsomes have remained intact

during centrifugation.Glucose-6-phosphatase is a marker enzyme for microsomes and thus a high

activity was expected in Fraction 4. The activity in fraction 4 was higher than activity of

fractions 2, 3 and 5. Enzyme activity is a measure of the quantity of active enzyme present.

(Bergmeyer H, 1974). Therefore this shows that microsomes were present in all fractions. This

should not occur as it takes centrifugation at 100,000xg for 60-90 minutes to pellet microsomes.

(BIOL 3361 Lab Manual). This can only be attributed to errors that occurred during the assaying

of the fractions. It is possible that the spectrophotometric readings were slightly erroneous since

the assay mixtures may not have been cooled causing condensation on the cuvettes. This may

have affected absorbance readings which represent the amount of enzyme present. Also dilutions

of fractions may not have been carried out accurately.

The specific activity is the number of enzyme units per milligram of total protein. Specific

activity is the measure of enzyme purity and indicates how much of the total protein is the

enzyme we try to isolate.(Lehninger 2005). Fraction 1 has a specific activity of 1.76 U/mg and

fraction 4 has a specific activity of 2.49 U/mg. Since Fraction 1 should represent the amount of

glucose 6 phosphatase in total protein, the value obtained for the microsomal fraction is fairly

accurate. The total enzyme activity refers to the amount of enzyme present in the entire stock

solution of a particular fraction. Theoretically fraction 1 reflects the total amount of enzyme

present and the sum of fractions 2-5 is supposed to equal the total activity of Fraction 1. In this

experiment however the sum of the total activities of Fractions 2-5 is equal to 16 351.57 which is

considerably less than the total activity of Fraction 1; 19 445.20. This indicates a loss of

organelles along the centrifugation process. Also from the results it is seen that the total activity

is higher for Fraction 5 than it is for Fraction 4 where fraction 5 should be totally devoid of

microsomes. The total recovery in fraction 1 is always 100% since it is the homogenate that

contains all organelles. Due to centrifugation, and hence loss of organelles it is always less than

100% for the remaining fractions. Differential subcellular compartments share similar physical

properties and cofractionate at least to some extent in conventional gradients(Huber, Pfaller,

Vietor. 2003).The fraction containing the marker enzyme is supposed to have the recovery

closest to 100% unlike this experiment.

Acid Phosphatase isused for the identification of lysosomes .This phosphataseis catalyses the

hydrolysis of several phosphate esters at acid pH. This can be seen in p-nitrophenyl phosphate

(pNPP) , this is converted to which is converted to p-nitrophenol.

From the results without detergent it is seen that acid phosphatase has a very high enzyme

activity in the lysosomal fraction. This is the marker enzyme found specifically in lysosomes.

The specific activity calculated is higher for the lysosomal fraction than it is for Fraction 1. This

is due to experimental error but it indicates that the majority of lysosmes in liver cells has

pelleted into Fraction 3. The sum of total enzyme activity for fractions 2-5 is 314.85U which is

close to the total enzyme activity value obtained for Fraction 1 ; 410.26U. This shows that not

too much enzyme was lost during subcellular fractionation.

Detergents are amphipathic molecules ,these detergents have a non polar tail and a polar head ,

this makes it aliphatic .Due to the non polar tail groups present this allows detergents to have

hydrophobic-associating properties . Their polar heads allow dispersion of water-insoluble,

hydrophobic compounds into aqueous media,

The detergent used for this experiment is called Triton X-100 .This is considered to be non

denaturing and also non ionic The reson this detergent is used its to cause the lysosomal embrane

to lyse, when this is done it will release most of the acid phosphatase enzyme which is present in

the lysosome , this will then go into buffer medium. When the medium is assayed more enzyme

will be convert the substrate into p- nitrophenol product .Due to this activity will be higher than

the free activity, i.e. when no detergent is added to the lysosomal fraction. The difference

between the two values is called the latent activity i.e (Total activity – Free activity) which is

168.91U.

In the continuous enzyme assay carried out the reverse reaction is measured by following a

change in absorbance of NADH over a 4 minute period at 340nm. Since glutamate

dehydrogenase is located in the matrix of the mitochondria, Triton X-100 was used in order to

lyse the mitochondrial membranes so that the enzyme would easily be released. To calculate the

activity of the enzyme the Beer Lambert’s Law was used. From the results, the enzyme activity

was found to be the highest in Fraction 2; 0.2875 µmol/min/mL which specifically contains the

marker enzyme glutamate dehydrogenase. The Specific activity was also found to be the highest

in Fraction 2: 0.0423U/mg which indicates that there was only slight contamination during

subcellular fractionation. The remaining fractions all had considerably lower specific activities.

The total enzyme activity which refers to the amount of enzyme present in the entire stock

solution of a particular fraction was highest for fraction 1. The total activity however was 37.07U

for fraction 2 which was extremely close to fraction 1 activity. Thus the total recovery of fraction

2 was 89.74% which indicates a high level of purity of the enzyme.

The bidirectional reaction is monitored spectrophotometrically by measuring the decrease in

NADH at 340 nm produced in the lactate to pyruvate reaction. (Vanderlinde R, 1985). From the

results it is seen that Fraction 5 (soluble fraction which contains the marker enzyme lactate

dehydrogenase) has a high total enzyme activity; 209.88U which is approximately half of the

value obtained for fraction 1. It is possible that some enzyme was lost during fractionation and

through transferring supernatant to new tubes to be spun down.

The specific activity which measures the purity of an enzyme shows that fraction 4 has the

highest amount of lactate dehydrogenase. This is inaccurate as the specific activity is shown to

be less in both fractions 1 and 5 which should contain most of the enzyme. It is probable that the

reason there is a higher specific activity in the mitochondrial fraction is that lactate is a good

oxidative substrate for the mitochondria. The explanation for this high specific activity is that

lactate enters the mitochondrial matrix and is turned into pyruvate by LDH. NADH formed from

this reaction can be used for the Glutamate Dehydrogenase reaction thus balancing the

mitochondrial redox. Pyruvate formed from lactate enters the Krebs cycle, producing α-the

substrate for glutamate formation.(Randall, Chew, Ip, 2001).The sum of the % recovery for

Fractions 2-5 was 84.89% in comparison to 100% recovery in Fraction 1. Fraction 1 contains all

the organelles and it is expected that the recovery for the remaining fractions would equal 100%.

This implies the loss of organelles along the centrifugation scheme.

A calibration curve for NT was plotted this was done in order to determine the absorbance

produced by reduction of one μmol of NT, which was used to determine the amount of formazan

produced.For the Succinate dehydrogenase reaction , for each fraction had one enzyme blank

and one sucrose blank .There was no enzyme present in the enzyme blank , i.e it contained n o

fraction and only the substrate succinate. Despite the fact that there is no substrate present to

form the product formazan, when absorbance is read a low reading maybe be obtained , this will

indicate that some product maybe present .When this occurs it is called the background

reaction.The values here are artificially higher the reason for this its because the enzyme was

obtained from a live animal . (Cleland, 1967). From the results obtained for this part of the

experiment the absorbance reading obtained was however, the absorbance reading was -0.019

for all fractions which indicates that absolutely no product was formed.As for the substrate blank

it contained no substrate , in other words it only contained succinate and the respective

fraction..The purpose of this its to act as a control , because there is a possibility that substrate

could spontaneously react with itself and some product can be formed .Another reason is that the

substrate can be absorbing at 540nm. Due to this it can be stated that a low absorbance reading

should be obtained .

According to the results obtained, the enzyme activity was highest for Fraction 2; 0.132

µmol/min/mL. This is the mitochondrial fraction which contains the marker enzyme succinate

dehydrogenase in the inner membrane. The total enzyme activity and the specific activity is also

highest for Fraction 2. This indicates that mostly all the enzyme was pelleted into the

mitochondria fraction during centrifugation. This assay is the most accurate experiment carried

out since there is a low specific activity of succinate dehydrogenase in Fraction 3 and none in

fractions 4 and 5. This indicates very little cross contamination between fractions.

ADDITIONAL DISCUSSION

From the results calculate the % contamination of the mitochondrial fraction and of the

lysosomal fraction by endoplasmic reticulum.

During subcellular fractionation by differential centrifugation, there is a lot of contamination

occurring .This is because differential centrifugation is based on separation according to size and

density. According to the results, it is obvious contamination of both the mitochondrial fraction

and lysosomal fractions has occurred. The specific marker enzyme for microsomes, glucose 6 –

phosphatase was found to have a % recovery of 5.01% and 6.20 % in the mitochondrial and

lysosomal fractions respectively. This shows that microsomes were present in both fractions.

In which fraction(s) peroxisomes are most likely to be found?

peroxisomes will most likely be found in Fractions 2 and 3. The reason for this is that their

density is similar to that of the mitochondria and lysosomes. (Masters, Crane. 1995)

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