Eukaryotic cells such as liver cells enclose a assortment of different types of membrane edge structures called cell organs ( karyon, chondriosome ) every bit good as supermolecules ( ribosomes ) ( Padh, 1992 ) .
Subcellular fractional process is an priceless technique leting scientists and research workers likewise to successfully insulate and divide specific subcellular constituents within the cell ( Becker et al, 2009 ) . This allows research workers to analyze the different cell organs ( utilizing biochemical techniques ) in a greater grade of item therefore increasing our cognition about the many different types of cell organs and supermolecules, therefore taking to new scientific progresss in this of all time progressing epoch of scientific discipline and engineering ( Bonney, 1982 ; Berns, 1986 ) .
It is this really method which in the past allowed Christian de Duve to detect the lysosomes and peroxisomes for which he shared a Nobel Prize with Albert Claude and George Palade in 1974 ( Becker et al, 2009 ) .
Subcellular fractional process can be safely divided under 3 major headers: Homogenization proceeded by fractional process and eventually purification.
The purpose of the homogenization procedure is to efficaciously and expeditiously disrupt and interrupt the cells outer membrane thereby let go ofing their subcellular constituents ( nuclei, chondriosome ) . This break and breakage of the cells must be achieved in a mode that will go forth the delicate cell organs of involvement undamaged and morphologically integral ( Loewen, 2003 ) .
The cells to be homogenised are kept in an isosmotic buffer ( 0.25M sucrose, 1mM EDTA and 1mM of Tris at pH 7.0 ) . This is to protect the delicate cell organs from osmotic harm due to osmotic imbalance every bit good as environmental instability such as pH intervention ( Guteriezze, 2010 ) .
Many different homogenization techniques exist and are available, some such include mechanical crunching utilizing Potter-Elvehjen glass homogeniser, cutting methods utilizing warren liquidizer, supersonic quivers in a procedure called sonication and utilising high force per unit area such as in the Gallic Press ( Loewen, 2003 ) .
The Potter-Elvehjen glass homogenizer was used in this experiment. The Potter-Elvehjen glass homogeniser consists of a Teflon stamp which is closely fitted into a glass homogeniser. The homogenizing machine moves the Teflon stamp in a verticle up-down gesture while at the same time revolving within the glass homogeniser incorporating the cells to be homogenised ( Mangiapane, 2010 ) .
The infinite between the Teflon stamp and glass homogeniser is improbably little ( 0.004 ” -0.006 ” ) . Therefore as the Teflon stamp moves throught the glass homogeniser a shear force is generated which causes break of the cells. The cell organs which are released by this procedure base on balls undamaged, safetly through the spread between the stamp and glass homogeniser ( Loewn, 2003 ; Mangipane, 2010 ) .
The shear forces produced can sometime be destructive to the cell organs doing irreversible harm and hence shear forces need to be controlled. This can be controlled by seting the spread breadth between the stamp and glass homogeniser. A bigger breadth can protect cell organs from harm but the negative side consequence of this is that the generated shear forces will non be strong plenty to interrupt the cells and hence few or none cell organs will be present in the homogenate. Therefore a careful balance between cell break and organelle harm must be maintained.
Chemical, physical and structural harm can be caused to cell organs due to shear forces which can do mistakes when sublimating the cell organ utilizing biochemical techniques due to enzymes specific to the peculiar cell organ being harm or rendered inactive and these jobs must hence be overcome. Some such safeguards which when utilized can get the better of or understate unneeded harm includes the usage of different homogenization techniques which are more suited for the cells being homogenised ( osmotic break, chemical break may be considered ) . Carefull use of the homogenising equipment ( Lowen, 2003 ) .
Once the homogenate has been formed, it is ready to be placed in a extractor and undergo centrifugation which will divide the different fractions/organelles. Centrifugation generates a centrifugal force which separates the different types of cell organs based on their size and denseness every bit good as the denseness and viscousity of the solution the homogenate is in. Therefore the the higher the molecular weight of the cell organ the greater the distance I will go down the extractor tubing or the higher its deposit rate and accordingly the smaller the molecular weight of the cell organ the smaller the distance it will go down the extractor tubing or the lower its deposit rate ( Becker et al,2008 ; Mangipane, 2010 ) . The greater an organelles deposit rate is the greater the cell organs deposit coefficient ( in Svedberg units, named after Theodor Sveber who developed the ultracentrifuge ) will besides increase ( Becker et al, 2009 ) .
Centrifugal forces can be calculated utilizing
For illustration, if a homogenate containing karyon, chondriosome and ribosomes is subjected to a centrifugal force, logically the karyon will be near the underside of the tubing, the ribosomes at the top portion of the tubing and the chondriosome someplace in between the karyon and ribosomes.
There are 2 chief type of centrifugation methods: Differential centrifugation and denseness gradient centrifugation.
This type of centrifugation plants on the rules that big dense molecules ( nucei ) will hold a higher deposit rate compared to little and less heavy molecules ( ribosomes ) ( Becker et al, 2009 ) . During low extractor velocities and short times the heavy and heavy cell organs deposit and can be collected, while every bit high extractor velocities and longer timer the lighter and less heavy molecules will sediment and can besides be collected ( manipulative techniques ) .
Therefore in the homogenate used in the experiment, by utilizing appropriate extractor velocities and times the karyon and chondriosome can be separated utilizing 1500g for 10min and 20000g for 10min severally.
Density gradient centrifugation
The method used in denseness gradient centrifugation, besides known as rate-zonal centrifugation works on the rule of dividing molecules based on their densenesss and is achieved by utilizing a denseness gradient in the extractor tubing ( manipulative techniques ; Becker et Al, 2009 ) . The denseness gradient is usually provided by a concentrated saccharose solution which increases in denseness towards the underside of the extractor tubing. The sample necessitating fractional process is placed in a bed over the denseness gradient sucrose solution ( Becker et al, 2009 ) . As the centrigugation procedure returns, the different molecules or organelled of different densenesss are separated based on their densenesss and that of the increasing sucrose denseness. When the fractional process sets have been formed are are typical the fraction may be remover via a syringe or separation methods. This type of centrifugation can be used to further separate chondriosomes from lysosomes and peroxisomes since each of them has a different denseness.
Measurements of enzyme activity and macromolecular composing of fractions. “ pureness ” of fractions.
During the centrifugation processes, such as in the differential centrifugation the assorted types of cell organs and supermolecules form gelatinlike pellets at the terminal of each back-to-back extractor ( Dyson, 1979 ) . The different pellets produced incorporate a assortment of different fractions of subcellular cell organs and supermolecules and is non specific for merely a individual type of cell organ or supermolecule. As an illustration in the first centrifugation procedure to organize the nuclei fraction at 1500g for 10mins, the karyon is pelleted along with other molecules of similar size and molecular weight such as unbroken cells, cell dust and pieces of the cell membranes ( Bonney, 1982 ) . In the 2nd centrifugation to organize the mitochondrial fraction at 20000g for 10min the pellet contains chondriosomes, lysosomes and peroxisomes due to their similar sizes and molecular weight. In the concluding centrifugation procedure at 20000g for 10min a supernatant fraction was formed incorporating many little and low molecular weight molecules such as the endoplasmic Reticulum, microsomes and ribosomes ( Minorsky, 2009 ; Berns, 1986 ) .
As stated before fractions will non merely necessitate the cell organs of involvement but besides other cell organs and supermolecules. It is hence necessary to be able to measure the pureness of the fractions. This can be done in a assortment of ways.
Microscopic analysis via the light microscope or even electron microscope can be used to place the different supermolecules nowadays within the fraction, hence giving an indicant whether or non the fractional process process has been successful. A mitochondrian therefore can be differentiated from a peroxisome or lysosome basen on its construction ( Bonney, 1982 ) . Microscopic anaylsis can besides be used in measuring the biochemistry of the fraction by utilizing assorted cytochemical techniques.
Biochemical techniques are a really good manner of measuring the type of organelle nowadays every bit good as the pureness of a fraction. Measuring enzyme activity is an excellet method sine some enzymes are really specific and found in one peculiar cell organ.
Marker enzymes present in fractions and importance of the techniques involoved in the promotion of biochemistry and cell biological science.
Marker enzyems are routinely used in subcellular fractional process to distinguish between the many different types of cell organs and supermolecules present within the cell. Mitochondria for illustration can be detected indirectly by the presence of succinate dehydrogenase while lysosomes can be detected by Acid Phosphatase ( Bonner, 2007 ) .
The map of the chondriosome for illustration is to bring forth adenosine triphosphate ( ATP ) by a procedure called oxidative phosphorylation and an enzyme particular to the chondriosome called succinate dehydrogenase can be used as a marker enzyme to distinguish between the presence of chondriosomes and other cell organs and supermolecules present in the fraction ( Padh, 1992 ) . Succinate dehydrogenase ( SDH ) is specific to the interior chondriosomes membrane and is responsible for catalyzing the oxidization reaction of Succinate, which is a constituent of the citric acid rhythm, into fumarate which is another constituent of the citric acid rhythm. Since flavin A dinucleotide ( FAD ) is reduced bring forthing FADH2 ( Guterize, 2010 ; Padh, 1992 ; Girolamo, 2010 ) .
Succinate is the negatron doner while FAD is the negatron acceptor. The merchandises of the above reaction are so reacted with an unreal negatron acceptor called INT ( a tetrazolium salt ) to organize a ruddy coloured compound called formazan.
This reaction is required because both the fumarate and FADH2 produced in reaction are colorless and hence there is no certain manner of finding succinate dehydrogenase activity, hence the strength of the ruddy colored formazan produced during a specific timeframe in the 2nd reaction can be measured utilizing a spectrophotometer gives an indirect indicant of succinate dehydrogenase activity and hence an indicant of the presence of chondriosome every bit good as its pureness within the fraction ( guterize, 2010 ; padh, 1992 ) .
Electron microscopy of the stray cell organs is by and large the concluding measure in measuring the pureness of the fractions every bit good as analyzing their morphology ( padh, 1992 ) .
It is these methods and techniques used in subcellular fractional process which has allowed research workers such as George Palade and Christian de Duve analyzing to understand and detect the constructions, biochemistry and functions played by the assorted cell organs.
Table 1 shows the volumes of the homogenate, nuclei fraction, mitochondrial fraction and supernatant fraction. The Homogenate volume was obtained after rat liver homogenization ; NF volume was obtained after 2 back-to-back centrifugations at 1500g for 10min ; MF volume was besides obtained by 2 back-to-back centrifugations at 20000g for 10min ; SF volume was obtained from the supernatant of the MF centrifugation.
Entire Volume ( milliliter )
Homogenate ( H )
Nuclei Fraction ( NF )
Mitochondrial Fraction ( MF )
Supernatant Fraction ( SF )
Table 2 shows known sums of bovine serum albumen ( BSA ) which underwent the biuret reaction ; the optical density ‘s were step utilizing a spectrophotometer at 550nm. As protein sum additions so do the optical density ‘s. This information was used to plot a BSA criterion curve.
BSA protein sum ( milligram )
0 ( Blank )
Absorbance ( 550nm )
Figure 1 illustrates the BSA criterion curve which is a line of best tantrum. From this graph, the protein sum is determined by utilizing the optical density values for the different fraction shown in table 3 below. H, NF, MF and SF correspond to homogenate, nuclei fraction, mitochondrial fraction and supernatant fraction severally. The perpendicular and horizontal ruddy, bluish, green and black colored lines represent H, NF, MF and SF severally.
From the above graphical informations Protein concentration ( mg/ml ) , entire protein sum ( milligram ) and protein recovery for each fraction relation to the homogenate can be calculated.
From the above consequences the entire per centum of protein recovery relation to the homogenate can be determined:
The above calculated consequences are show together in table 3.
Table 3 shows the optical density values obtained from the spectrophotometer. Row B shows the sum of protein that was determined from the BSA criterion curve. Row C showed the sum of protein nowadays in 1ml of each fraction ; the homogenate had the highest protein concentration, followed by the SF and MF and eventually by the NF incorporating the lowest sum of protein concentration. Row D shows the entire sum of protein in each of the fraction and hence follows the same form as the values for Row C. Row E shows the sum of protein recovered comparative to the homogenate ; The per centum of protein recovery was as follows: SF & gt ; MF & gt ; NF.
Homogenate ( 0.05ml )
Nuclei ( 0.2ml )
Mitochondria ( 0.2ml )
Supernatant ( 0.2ml )
Optical density values 1, 2
Average optical density ( 550nm )
Protein sum in samples aliquot ( milligram )
Protein concentration in fraction ( mg/ml )
Protein sum in fractions Entire Volume ( milligram )
Protein recovery relation to Homogenate ( % )
Table 4 shows the existent fraction concentrations used, obtained by thining the original fractions ( table 3 ) with phosphate buffer. The supernatant fraction was left undiluted.
Actual concentrations used ( mg/ml )
Table 5 shows optical density of each of the fractions ( 0.2ml ) which were diluted by the add-on of 4ml of ethyl ethanoate within formazan. The mean optical density minus the control gives the corrected mean optical density for each of the fractions. The control values for all 4 fractions were 0 because they were given as negative values by the spectrophotometer. The highest optical density was recorded for the SF followed by the homogenate, MF and NF.
Absorbance ( 490nm )
Trial 1 ( T1 )
Trial 2 ( T2 )
Average optical density ( T1+T2 ) /2
Average optical density – Control
By obtaining the informations collected from the antecedently computations in tabular arraies 1, 3 and 5 it is possible to cipher ; the entire activity of Succinate Dehydrogenase ( SDH ) , the per centum recovery of SDH relation to the homogenate, the specific activity of SDH and the comparative specific activity of SDH relation to the homogenate in all 4 fractions ( H, NF, MF and SF ) .
Below are the equations which will be used in the computations:
Beer-Lamberts Law: The computations below will do ( concentration ) the topic of the expression every bit good as prove that the units for = or.
a?? this can be rearranged to organize,
, since ever peers to, the equation can now be represented as,
, the units of this new expression can be calculated as follows,
the in the bottom fraction can be cancelled out with the at the top giving,
Which a?? gives which is Molarity or concentration.
The equation will be used throughout the remainder of the computations. The Formazan molar extinction coefficient= and the check volume used will be 0.004L ( 4ml ) .
The optical density for the homogenate in table 5 was 1.1385 hence,
a?? since this can be arranged to give,
The volume used was which gives hence,
a?? entire activity of in -1
The reply is required in a?? since
Therefore entire activity for Homogenate =
Therefore specific activity for Homogenate
Nuclei Fraction ( NF ) :
The optical density for the nuclei fraction in table 5 was 0.117 hence,
a?? since this can be arranged to give,
a?? entire activity of in
The reply is required in a?? since
Therefore entire activity for Nuclei Fraction =
Therefore specific activity for Nuclei Fraction
Mitochondrial Fraction ( MF ) :
The optical density for the mitochondrial fraction in table 5 was 0.398 hence,
a?? since this can be arranged to give,
a?? entire activity in in –
The reply is required in a?? since
Therefore entire activity for Mitochondrial Fraction =
Therefore specific activity for Mitochondrial Fraction
Supernatant Fraction ( SF ) :
The optical density for the supernatant fraction in table 5 was 1.485 hence,
a?? since this can be arranged to give,
a?? entire activity in in
The reply is required in a?? since
Therefore entire activity for Supernatant Fraction =
Therefore specific activity Supernatant Fraction
Calculations for the % SDH recovery and specific SDH activity relation to the homogenate ;
Since the % SDH recovery and specific SDH activity is to be calculated comparative to the homogenate, therefore the homogenate per centum for them both will be 100 %
The chief findings of these computations can be summarized in the tabular array below:
Table 6 shows that SDH activity is highest in the SF, followed by the homogenate, MF and eventually by NF. The % of SDH recovery ( comparative to the homogenate ) was greatest in the SF, followed by the MF and the NF. The specific SDH activity was greatest in the SF followed by the MF, NF and in conclusion by the homogenate. The % of specific SDH activity ( comparative to the homogenate ) was greatest in the SF, followed by the MF and NF.
Entire SDH activity ( Aµmoles/min )
% of SDH recovery ( rel. to Homogenate )
Specific SDH activity ( Aµmoles/mgxmin )
% of specific SDH activity ( rel. to Homogenate )
Figure 2 illustrates the chief findings from table 6. It can be seen that % SDH recovery additions from the Nuclei fraction to the supernatant fraction. The % of specific SDH activity steadily falls from the supernatant fraction to the nuclei fraction.
Harmonizing to the consequences obtained in table 3, it was seen that 99.25 % of the protein relation to the homogenate was still present within all the fractions. This high per centum recovery indicates that really small protein was lost during the formations of the karyon, mitochondrial and supernatant fractions by centrifugation. The 0.75 % of protein that was lost is most likely to hold been lost while homogenising the pellets formed during each back-to-back centrifugation procedure. During the use of manus homogenizers little measures of the pellet incorporating the proteins are stuck to the homogenising vas or the stamp. These little measures of proteins being lost during each manus homogenising procedure hence contributes to the loss of proteins recovery. From this high protein recovery it can be said that the overall homogenization procedure was really efficient.
During each consecutive centrifugation at different velocities a distinguishable pellet was formed, therefore bespeaking the separation of cell organs. In table 3, different sums of proteins were present within the pellets. Since these proteins are associated with the different cell organs present, this indicates that since different sums of proteins were found in the fractions therefore assorted different types of cell organs must besides be present. But this is non ever the instance since proteins from other fractions could hold been damaged due to the homogenisation and centrifugation procedures. Therefore the computations performed on Succinate Dehydrogenase activity, recovery and specificity ( table 6, figure 2 ) showed that that the entire SDH activity was highest in the supernatant fraction. Since SDH is a specific marker enzyme to the chondriosome cell organ as explained earlier, the information suggests that the separation of chondriosome during centrifugation to be present within the suspected mitochondrial dfraction was non optimum. The supernatant besides had a really high protein content of 885mg ( table3 ) which indicated therefore that most of the cell organs have separated into this freaction, therefore bespeaking the hight sum of SDH activity within the supernatant fraction.
In a differential centrifugation procedure the consecutive additions in the centrifugal forces applied should make a gradient of the presence of different cell organs, with the heaviest molecules in the extractor tubing with lowed centrifugal forces, the mediam molecular weighted cell organs such as chondriosomes in a centriguge in the extractor with a medium centrifugal force is applied and little molecular weight cell organs such as ribosomes in the centrigue tubing where the highest centrifugal forces are applies.
Therefore the separation of cell organs has occurred but non to a great extent as seen by the reults in table 3 and table 6. Seperation of cell organs could hold been greatly improved by perchance polishing the lab protocol. To ahieve better mitochondrial separation and hence more accurate SDH activity measurments the centrifugation procedure should be done at 20000g but for 20min and non 10min as stated by Loewen ( 2003 ) and Becker et Al ( 2009 ) . This will assist divide the chondriosome out better.
Different centrifugation methods such as denseness gradient centrifugation can be utilized after the intital differential centrifugation to better separate cell organs of similar sizes such as chondriosomes, lysosomes and peroxisomes. The new fractions produced can by the densiy gradient centrifugation can be recovered with the usage of a syringe. Many other techniques such as the initail homogenization phase could besides hold been changed and other techniques could hold been used as described earlier.
It was found by this experiment that subcellular fractional process is non a perfect method and therefore inaccuracies must be expected. But it is a procedure that has revolutionaised our apprehension of cell construction and map.
It was found in the experiment that differential centrifugation can divide cell organs to an extent to organize a nucleic fraction, mitochondrial fraction and supernatant fraction. Marker enzymes which are present in specific cell organs can be used to assist separate between different cell organs every bit good as the fractions relative pureness. SDH was used in this experiment and was found to be present higher in the supernatant, perchance due to experimental mistake. SDH was specific to the supernatant fraction therore once more bespeaking the presence of chondriosome in the supernatant.
The use of such techniques in this of all time progressing epoch of scientific discipline and engineering has set the phase for future surveies and techniques involved in farther analyzing the cells and increasing our cognition of life as each twenty-four hours passes.