Biochemistry 1

BIOCHEMISTRY

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 Biochemistry 2

ABSTRACT.

Cellular compartments are separated based on their differences in sedimentation rate through a

process called subcellular fractionation. This separation helps in studying specific structures such

as protein or organelles in the cell. In subcellular fractionation, properties of the compartments

such as size, density, charge and shape are vital for differential centrifugation. As argued by

Amigo (2016) differential centrifugation employed in subcellular fractionation is done in a

highly viscous media such as mannitol, glycerol and sucrose. The fractionation yield and its

purity are assessed by detecting distinct markers in each collected fraction. The experiment

aimed at making a fraction that has been enriched in mitochondrion and in nucleus from a rat

liver homogenate using differential centrifugation process. Pereira (2015) argued that motor and

a pestle were used to obtain liver homogenates and isotonic buffer was used to prevent cell

damage by osmotic imbalance. I obtained mitochondrial fraction by centrifuging combined

supernatants obtained from nuclei fraction. I assessed mitochondrial presence in each collected

fraction by using succinate dehydrogenase enzyme for detection. SDH is mainly found in the

mitochondria therefore if its activity in the fraction is detected, mitochondria are present. The

samples were frozen at -20 Celsius between each of the sessions thus preventing SDH from

losing its catalytic activity

I obtained nuclear fractions by centrifugation of the liver homogenate at 15000 rpm to produce

nuclear pellets. In the measurement concentration of protein in the subcellular fraction I reacted

BSA with the Biuret agent and measured absorbance. I plotted a graph to determine protein’s

amount
 Biochemistry 3

Activity of succinate dehydrogenase was measured using FADH2 formed in the TCA cycle to

reduce tetrazolium to formazan (deep red). Formazan amount determined spectrophotometrically

at a wavelength of 490nm.

RESULTS

Session 1entailing Results for subcellular fractions of Differential centrifugation

Table 1 showing Cellular fractions’ total volumes

Fraction Total Volume (mL)

Homogenate (H) 13
Nuclei Fraction 11
Mitochondrial Fraction (MF) 9.5
Supernatant Fraction (SF) 30

The above results were obtained after differential centrifugation of the liver homogenates and the

volumes of each fraction was measured in milliliters. The Nucleic Fraction obtained had the

highest volume of 11 mL as compared to 9.5 mL of the mitochondrial fraction.

Session 2 covering Results for protein content measurement by Biuret assay

Part A illustrating Standard curve of Bovine Serum Albumin (BSA)

 Biochemistry 4

Table 2 showing Absorbance values for BSA standard curve

Protein amount (mg) 0 2 4 6 8 12 16

Absorbance at 550nm 0 0.077 0.079 0.108 0.1205 0.1625 0.195

(Blank)

Xiao (2016) argued that Bovine Serum Albumin was used in the preparation of the protein’s

standard curve against a blank which is obtained by the reaction of BSA and the Biuret reagent.

There is a direct proportionality between the absorbance and the protein amount which was

evidenced by the increasing absorbances with an increasing protein amount.

Table 3 showing the Amount of protein in homogenate and cellular fractions

Homogenate Nuclei F Mitochondria Supernatant

A The Average absorbance (550nm) 0.445 0.16275 0.331 0.3005
B The Protein amount in sample’s aliquot 6.180556 2.256944 4.597222 4.173611

(mg)
C The Protein concentration in fraction 30.905 5.6425 11.4925 21.9265

(mg/mL)
D The Protein amount in fraction’s Total 401.765 62.0675 109.17875 657.795

Volume

Protein amount in the cellular and homogenate fractions were determined by reading the

absorbance at 550nm. By comparing the absorbances read with the standard curve of BSA

graphically, the amount of proteins present in each fraction was easily determined. The results

show that protein amounts were much higher in mitochondrial fraction as compared to the

nucleic fraction. The calculated result in Table 3 were obtained using the same procedure for all

the fractions for example, in the calculation of amount of protein (mg) present in the
 Biochemistry 5

Mitochondrial fraction, the amount of protein present was determined by the use of an

absorbance of 0.331 in the standard curve.

Session 3 covering Succinate Dehydrogenase Activity using Red Formazan Assay

Table 4

Homogenate Actual concentratio

Volume of homogenate or Volume of diluent required (μL)
Or (mg/mL) *
fraction required (μL) to make up to 2000 µL total
Fraction
Homogenate:

to approx. 5 mg/mL 323.57 1,676.43 30.905

Nuclear fraction:

to approx. 2 mg/mL 708.90 1,291.1 5.6425

Mitochondrial fraction: 11.4925

to approx. 1 mg/mL 174.03 1,825.97

Supernatant fraction:

use undiluted N/A N/A Value from Table 3

 Biochemistry 6

Succinate dehydrogenase activity was determined indirectly by the reduction of Tetrazolium salt

with FADH2.

Table 5 showing Formazan content at Absorbance of 490nm

Fraction Control Test 1 Test 2

H 0.121 1.455 1.608

NF 0.003 0.517 0.6

MF 0.001 0.470 0.476

SF 0.053 0.622 0.789

The determination of Formazan was determined by the aspect of spectrophotometry where the

absorbances were read at a wavelength of 490nm. The amount of Formazan was higher in the

Nucleic fraction as compared to that of the mitochondrial fraction.

 Biochemistry 7

A graph showing BSA protein amount (mg) against

absorbance at 550nm.
0.25

0.2
0.2
0.16
ABS at 550nm

0.15
0.12
0.11
0.1 0.08 0.08

0.05

0
0
0 2 4 6 8 10 12 14 16 18

Protein amount (mg)

The above graph is a standard curve obtained by plotting the absorbances read at 550nm against

the BSA protein amount. This is a linear graph obtained by the estimation of line of best fit and

obeys the Beer-Lamberts Law.

Calculations

i) Total enzyme activity

Average absorbance of the homogenate = 0.445 obtained from table 3.

Applying Beer Lamberts Law Aλ = ελ c L

0.445 = 20100 × c × 1

0.445
C=
20100

= 0.000022139 moles/l

= 2.2139 micromole/l/min
 Biochemistry 8

ii) Percentage recovery

Percentage recovery = activity in MF, NF/ HF × 100%

a) Percentage recovery in mitochondrial fraction = Activity in MF/HF x 100%

= 11.4925/30.905 x 100%

= 38.31%

a) Percentage recovery in Nucleic fraction = Activity in NF/HF x 100%

= 5.6425/30.905 x 100%

= 18.26%

iii) Specific activity

Specific activity = total enzyme activity/total protein amount in the Mitochondrial fraction

= 2.2139/109.17875

= 0.0203X micromoles / min / mg protein

Discussion

From the results, subcellular fractionation of the liver homogenate was consistent with standard

values hence the homogenization process was efficient. Nannipieri (2018) said that the graphical

representation of absorbance of the fractions falling within the range of standard values was

clearly evident. In addition, the process was efficient as protein amount and enzyme activities

could easily be determined. Yang (2016) said that succinate dehydrogenase activity is higher in

fraction collected from the mitochondria than other fractions because this enzyme is mainly

found in mitochondria and catalyzes conversion of succinate to fumarate. However, in other

 Biochemistry 9

fractions there was little activity of SDH indicating that fractions obtained from the mitochondria

well separated from the nuclear fraction. Varying amounts of proteins in each fraction also

indicated an effective separation. Therefore, a good separation of the organelles via differential

centrifugation occurred and was thus the process efficient.

The process of differential centrifugation can be improved considering the new technologies and

a great increase in expertise in the world’s molecular study sector. Gobrecht (2015) brought the

approach I can give in improving the method is by implementation of appropriate use of buffers

and also to reduce the time spent using the buffers which have been shown to be affecting the

quality of the mitochondrial separations. Development of proprietary buffers is encouraged as

this provides specific use and correct application of buffers. It is therefore with great concern in

the recent studies which focuses mainly in trying to maintain the viability, yield and quality of

yielded mitochondria.

As argued by Mortelmans (2019), the component of cells which remain in the supernatant after

centrifugation is the microsomal portion. It can be recovered by centrifugation at 100,000 g for

ten minutes of the previous supernatant. Evaluations have been carried out on carbazeran 4-

oxidation among other markers of liver microsomes.

Conclusion

In conclusion, subcellular fractionation of liver homogenate is stepwise process that separates

cellular organelles based on their sedimentation rate. The biuret assay is also usefully in

determining the amount of proteins available in the nucleus and mitochondrion. From the results

obtained it can easily be deduced that specific location of the succinate dehydrogenase enzyme is

mitochondria.
 Biochemistry 10

Reference

Amigo, I., Traba, J. and Rueda, C.B. (2016) Isolating Liver Mitochondria by Differential

Centrifugation.

Araújo, O., Pereira, P., Cesário, R., Pacheco, M. and Raimundo, J. (2015) Marine pollution

bulletin.

Liu, Z. and Pan, J. (2017) A practical method for extending the biuret assay to protein

determination of corn-based products. Food chemistry.

Xiao, W., Sarsour, E.H., Wagner, B.A., Doskey, C.M., Buettner, G.R., Domann, F.E. and

Goswami, P.C. (2016) Archives of toxicology.

Nannipieri, P., Trasar-Cepeda, C. and Dick, R.P. (2018) Biology and fertility of soils.

Yang, X., Liu, J., McGrouther, K., Huang, H., Lu, K., Guo, X., He, L., Lin, X., Che, L., Ye, Z.

and Wang, H. (2016) Environmental Science and Pollution Research.

Gobrecht, A., Bendoula, R., Roger, J.M. and Bellon-Maurel, V. (2015) Analytica chimica acta.

Saleem, M. (2017) The Journal of Solid Waste Technology and Management.

Ayed, M.R., Jumaa, F.H. and Ali, A.A. (2016) kirkuk university journal for scientific studies.

Shenkman, R., Gunzelmann, D., Yang, T., Traylor, M. and Manning, K. (2016) Bioreactor

perfusion via single-use centrifugation has fewer product quality implications than tangential

flow filtration.