Nuclei And Mitochondrial Fraction

Nuclei And Mitochondrial FractionThe objective of this experiment is to prepare a nuclei and mitochondrial portion utilise distinctial centrifugation, from a rat liver homogenate sample. The amount of drill of mitochondria in the fractions go off be noted utilise succinate dehydrogenase (SDH) as a marker. To measure the percentage recovery of the SDH of Mitochondrial, Nuclei and supernatant fractions in comparison to the Homogenate and to Calculate the specialized and relative body process of SDH in individually fraction.Figure 1 Shows a re indueative animal electric cell with the individual organelle comp iodinnts.Figure 2 Shows the typical features and functions of the organelles of wager in this report.Figure 1 + 2 Created on Microsoft paint with reference to Essential Biology (2004)Individual organelles differ in size besides are all usually around 10nm in diameter. There is a small surface area and size/density aims on the organelle, the littler organelles cosmos lysosomes and ribosomes.Mitochondria differs in cell type depending on the energy demand of that organ, the more ATP that is required in a particular organ the more mitochondria anchor. E.g. more mitochondria found in heart and liver cells than in a white blood cell like a lymphocyte. Smaller organelles include lysosomes and ribosomes.Metabolism no secondate be detected using dissimilar manners much(prenominal) as use of inhibitors. These whoremonger be both competitive and non-competitive, an example is seen with arsenic with inhibits pyruvate dehydrogenase. another(prenominal) method is with the use of radioisotopes to measure act aswell as histochemistry, immunocytochemistry and electromicroscopy.Preparation of the homogenate occurs in various stages. Firstly the homogenisation of liver cells. This usher out be done using a Potter Elvehjem homogeniser to extract organelles without damaging the actual cell. This is a simple and effective homogenisation method. A small g ap is made at heart the cell skirt which is then pressurised which forces the contents i.e organelles, cytoplasm etc. out of the cell. This occurs at a low temperature and mild pH, and to keep the isotonic solution a sucrose pilot burner is used, in that locationfore since there is the same water potential inside the cell and outside the cell there is no net movement of water (osmosis) and gum olibanum the cell remains the same size. Homogenized cells also must be kept at low temperatures to prevent autolysis (the degradation of a cell by its enzymes). (www.bookrags.com).Figure 3 shows a classic Potter Elvehjem homogeniser Image taken from (umwcellbiology.org)The second stage is fractionation of the homogenate sample. This process is called centrifugation and arse be just split into either a differential centrifugation or a density gradient centrifugation. The differential centrifugation splits the impure fraction into disjoined compartments due to the size of the various organelles in question and there density. The centrifuge applies a gravitational force onto the sample to separate components. The rate of centrifugation is find by the acceleration or pelt along applied to the homogenate and is usually measured in revolutions per minute (RPM) or g. Depending on the density of the organelles provide determine their isolation at a given speed. The high density organelles and the bigger organelles separate at a lower speed centrifugation. (K. Wilson 2005). The separation forms a pellet which is the precipitate proportion of the sample and the component of interest and a supernatant which is the liquid component. The supernatant readily decanted from the sample without removing the precipitate. Diferemces in centrifugation occur due to the techniques used, differential centrifugation is based upon the sedimentation rate of particles and thus the sedimentation rate separates them based on size and density. After initial sedimentation the largest pa rticles separate first into pellet and supernatant (K. Wilson 2005). minginess gradient centrifugation separates organelles using a media. Various media can be applied and depending on the particles willing be best for certain types and whitethorn not work well for others. (K. Wilson 2005).The 4 fractions we will obtain are nuclei, mitochondrial, supernatant and homogenate.Various tests can be carried out to distinguish between fractions and to determine their actual purity, test for specific enzymes can jurisprudence for the legal action occurring in the cell fractions therefore indicating the most abundant component.Some tests include Testing for DNA in both nuclei and mitochondrial fractions. This is because DNA is contained within the essence but also within the mitochondria. This is because relating to the endosymbiotic theory mitochondria was a separate aerobically respiring bacterial cell which was later engulfed by an early eukaryotic cell to merge into one aerobically respiring cell. Mitochondria is maternally inherited in the case of the majority of multicellular organisms, this is due to the high number of mtDNA molecules in the ooecyte and much fewer in a spermatozoan cell which are mostly degraded before fertilization takes place.Test for histones which indicate nuclei fraction as well as testing for various enzymes such as ATPase found in cytoplasmic (supernatant) and mitochondrial fractions and phosphotase kinase indicating microsomes and golgi apparatus are present. Some enzymes are exclusive to the citric acid cycle which occurs in the mitochondria, therefore testing for these enzymes indicates the presence of mitochondria in a fraction. The enzyme marker to test for mitochondria which we use is succinate dehydrogenase which is exclusive to the inner mitochondrial membrane. Succinate dehydrogenase is formed widely during the citric acid cycle so is only given as an indication of mitochondria. However, since during the homogenisation process the mitochondria could potentially burst spilling their contents into the cytoplasm (supernatant fraction), this does not therefore give an accurate indication of mitochondria present in a fraction. Succinate dehydrogenase breaks down succinate into fumurate, therefore the measurement of formazan indicates presence of succinate dehydrogenase.Measuring Succinate Dehydrogenase Activity(Red Formazan assay)This occurs in 2 reactions1 succinate + rage fumarate + FADH2SDH breaks down succinate into fumarate. This is an oxidation reaction since the succinate loses 2 electrons, in addition a reduction of the enzyme flavin adenine dinucleotide occurs (FAD gains 2 electrons) (FAD + 2 electrons FADH2)Figure 4 Shows the redox reaction which occurs with succinate and FAD.Image taken from natuurlijkerwijs.comSDH activity is measured by the formation of formazan a deep red compound formed from the reduction on a tetrazolium salt. The reduced FADH2 reduces tetrazolium salt (INT).2 FADH2 + INT FAD + formazanCentrifugation and calculating the relative centrifugal field. (K. Wilson 2005)G = W2r = 4 II2 r (rPM)2 = 1.1110-5r (rPM)23600G= Relative centrifugal force (RFC)r = Radical distance from axis of whirlingw = Angular velocityrPM = Revolutions per minute.T = 9 (In Rt/Rb)2 W2rp2 (Pp -P) = Viscosity of mediumrp = Radius of particlePp = Density of particleP = Density of mediumRt = Radius to top of centrifuge tubeRb = Radius to bottom of centrifuge tube.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. (K. Wilson 2005)Analysis of marker enzymes in subfractions determines the recovery of subcellular organelles, with comparison to previous tests, quantative data can be used to assess contamination of fractions. Showing whether the subfractionation method has been successful or not.These tests also hold health benefits and implications e.g. micro some C causes cell death and can be found in mitochondrial fractions, only in cancer patients no microsome c is present, indicating no cell death will occur a honey oil feature of cancer cells.Enzyme measurement in subcellular fractions however does hold some implications such as the solubility of the environment which may cause differences in enzyme function. Another implication is latency of enzymes, this refers to whether proteins are bound to the enzyme which in turn activates them once bound signalling enzyme function. There may also be low recovery of enzymes in the fractions due to poor recovery of the organelles which they come from, in particular if the enzyme is confined only to a specific region.Over the 3 week period centrifugation will separate the fractions according to size/density and separating the sample into the pellet and supernatant fractions. The speed of the centrifuge determines whether the pellets will separate. A lower speed is needed to separate the nucl ei fraction due to the higher density, whereas the higher speed is needed to separate the supernatant due to the smaller density remaining organelles. (K. Wilson 2005). The protein content is also measure for each fraction using the biuret assay, absorbance look ons are given which determine the protein content of each fraction. Finally succinate dehydrogenase is measured. This causes a redox reaction and causes e- ions are released, using formazan as an indicator this changes the colour of solution red, showing a redox reaction has taken place.From this research I can predict that the mitochondrial fraction is expect to have the highest results in specific activity due to fewer proteins present in that fraction.ResultsCalculationsFormazans molar extinction coefficient (E490nm) = 20,100 M-1 cm-1The specific activity and relative activity of the fractions can be determined by measuring the niggardliness using Beer- Lamberts Law (www.chemguide.co.uk)A = x l x CA = Absorbance (no u nits) = Epsilon. The adsorbtion coefficient M -1 cm -1l= Cuvettes light path length, this is the length of solution a light passes through (always 1 cm)C= Concentration of substance in M (moles in 1 litre)Rearrange to give concentrationC = A / x lUnits M-1 x cm-1 = 1 / M x cmC = A / x l Gives units ( 1/ (1/M x cm) x cm). This can be simplified to give 1/ (1/M)And further simplified to give units M (moles per litre or dcm -3)Know values = the formazan adsorption coefficient is 20,100 M -1 cm -1A = refers to the absorbance at 490nm values for each fraction are found in the mean-control table section.Using the equation C = A / x lWe can work out the concentration of formazan formed in the reaction.The concentration value is for 1 litre, therefore we must calculate the actual concentration from the actual assay volume used.Concentration = amount/volume rearranged to give A = C x VThe final assay volume from week 3 is 6 ml* due to the addition of ethyl acetate.* Note by erroneous bel ief 6ml of ethyl acetate was added instead of 4 ml giving a different final volume to the other groups.Converting 6ml into its litre value and x by the concentration gives the accurate mole product of formazan produced.Reaction time needs to be included to give the accurate units. Activity units can be determined using the following equation.Activity = Moles of formazan/reaction time (12 minutes)This gives the activity in M -1Calculating sum of money activity and specific activity of the fractions. put back 1 entireness volumes from each cellular fraction.FractionTotal Volume (ml)Homogenate12Nuclei Fraction12Mitochondrial Fraction12 supported Fraction26To do this we need to take into the accountThe total volumeThe total protein of the fraction.Dilution factorThe total volume values for each fraction can be found in table 1.The sample of each fraction used was 0.2ml, therefore the amount of moles of formazan calculated is in 0.2ml.(0.2 / total volume) x moles of formazan in 0.2mlX by the dilution factor of each fraction to give the total activity for each fraction, the values are given in table 4.To determine the specific activity we must consider the total protein of the fraction. Values are given in table 3. unique(predicate) activity = Total activity of fraction/ total protein of fractionTable 2 Bovine serum albumin (BSA) solution concentrationsVolume (ml)BSA (10mg/ml BSA00.20.40.60.81.21.60.1m NaOH2.0 (blank)1.8.1.61.41.20.80.4Table 3 Values for BSA standard curve.See represent 1 for the results from the corresponding fraction absorbance.Protein Amount (mg)0 (blank)24681216Absorbance at 550nm00.1050.1840.2750.3540.5110.531Table 4 Protein amount in homogenate and subcellular fractions.Homogenate 0.05mlNuclei 0.2mlMitochondria 0.2mlSupernatant 0.2mlAverage Absorbance (550nm)0.1690.0540.1740.199Protein amount in samples aliquot (mg)3.61.183.84.15Protein concentration in fraction (mg/ml)725.91920.75Protein amount in fractions total volume (mg)86470.8228539.5 Graph 2 Shows the difference in protein amount amongst cellular fractions.Table 5 factual concentration of fraction after dilution.Dilution FactorActual concentration (mg/ml)Homogenate203.6Nuclei32Mitochondrial200.95Supernatant120.75Table 6 Formazan content absorbance at 490nm.FractionControlTest 1Test 2Mean-ControlHomogenate0.1320.580.520.42Nuclei0.210.3520.3260.13Mitochondrial0.0570.3910.2650.27Supernatant0.1320.520.330.29Results for HomogenateFrom table 5, we have the absorbance of homogenate as 0.42 this divided by the adsorption co-efficient gives0.42/20,100 = 2.1 x 10 -5 MThe units for concentration are left wing as moles per litre (M). To get this into moles in the actual volume used (6ml not 1 litre) 2.1 x 10 -5 M x 0.006 lite = 1.3 x 10-7 MInclude the reaction time of 12 minutes to give moles per minute.1.3 x 10-7 M /12mins = 1.010-8 M -1To determine total activity and specific activity.The total volume from table 1 for the homogenate is 12ml, however the sample used was only 0.2ml we therefore divide actual volume / used volume x concentration of H x dilution factor (20 in the case of the homogenate from table 5 values)Total activity = (12/0.2) x1.0x10-8 M -1 x 20 = 1.2 x 10 -5 M -1specific activity = 1.2 x 10 -5 M -1/ total amount protein in homogenate from table 41.2 x 10 -5 M -1/864= 1.3 x 10-8 M min-1Results for nuclei fraction0.13/20,100 M-1 cm-1 = 6.5 x 10-6In 0.006 litre 6.5 x 10-6 x 0.006 = 3.9 x 10-8 M3.9 x 10-8 M / 12 = 3.2 x 10-9 M min-1Total activity = 3.2 x 10-9 M min-1 x (12/0.2) x 3 = 5.8 x 10 7M min-1 proper(postnominal) activity = 5.8 x 10 7/ 70.8 = 8.2 x 10 -9 M min-1Results for mitochondriaC = 0.27/20,100 m-1 cm -1 = 1.3 x 10-5M1.3 x 10-5M x 0.006 = 7.8 x 10-8 M7.8 x 10-8 M / 12 = 6.5 x 10-9 M min-1Total activity = 6.5 x 10-9 M min-1 x (12/0.2) x 20 = 7.8 x 10-6 M min-1Specific activity = 7.8 x 10-6 M min-1/228 = 3.4 x 10-8 M min -1Results for supernatantC = 0.29/20,100 m-1 cm -1 = 1.4 x 10-5M1.4 x 10-5M x 0.006 = 8.7 x 10-8 M = 8.7 x 10-8 M / 12 = = 7.3 x 10-9 M min-1Total activity = 7.3 x 10-9 M min-1 x (26/0.2) = 9.4 x 10-7 M min-1Specific activity = 9.4 x 10-7 M min-1/539.5 = 1.7 x 10-9 M min -1 destiny recovery of Succinate Dehydrogenase for the fractionsThis is done by dividing the amount of Succinate dehydrogenase in the individual fractions by the original homogenate and then multiplied by 100 to give a percentage.Table 7 Shows the total activity for each of the fractions.FractionTotal ActivityHomogenate1.2 x 10 -5 M -1Nuclei5.8 x 10 7M min-1Mitochondria7.8 x 10-6 M min-1Supernatant9.4 x 10-7 M min-1Nuclei fraction(5.8X10-7/1.210-5 ) x 100 = 4.8%Mitochondria fraction(7.810-6/1.210-5 ) x 100 = 65%Supernatant fraction(9.410-7/1.210-5) x 100 =7.8%Relative Specific Activity of Succinate DehydrogenaseThis is found by dividing the specific activity of the fractions (found above) by the specific activity of the homogenate (found above).Table 8 shows the specific activity for each of the fractionsFractio nSpecific ActivityHomogenate1.4 x 10-8 M min-1Nuclei8.2 x 10 -9 M min-1Mitochondria3.4 x 10-8 M min -1Supernatant1.7 x 10-9 M min -1Nuclei fraction8.2 x 10 -9 M min-1 /1.4 x 10-8 M min-1 = 0.586Mitochondrial fraction3.4 x 10 -8 M min-1 /1.4 x 10-8 M min-1 = 2.429Supernatant fraction1.7 x 10 -9 M min-1 /1.4 x 10-8 M min-1 = 0.121DiscussionNote There was very little protein found in the nuclei fractions total volume, this is abnormally low since we would expect this to be higher.From the results we can determine that the this supports our prediction thatThe mitochondrial fraction is judge to have the highest results in specific activity due to fewer proteins present in that fraction. Organelles have been isolated from each other as seen with the differing proportions of protein found in each fraction as well as the differing values for specific and total activity calculated.However the homogenate is expected to have the highest total activity due to the higher amount of protein since all fractions are present.However since protein was found in the cytoplasm or supernatant fraction, this indicates that there was an error in the separation of the fractions as SDH is present where it usually isnt found. Succinate dehydrogenase plant life by transferring 2 electrons from succinate which transfers it to fumerate, which blocks the rest of the reaction when it binds to FAD, from the measurement of formazan gives the value of activity.Results show that the relative specific activity is highest in the mitochondrial fraction, as well as the percentage recovery of the fractions. Therefore demonstrating that the fractions were purified and that the homogenisation and centrifugation has been relatively successful in separating fractions.However there were some inaccuracies from the results, this includes the very low protein amount found with in nuclei fraction, this was however predicted to contain a higher amount of protein due to the nature of the organelle and the enzy mes contained within it. Another inaccuracy in this experiment is that SDH was found within the supernatant. This is primarily a marker for mitochondria so would not usually be found within the cytoplasm, however due to mitochondria bursting and releasing its contents into the cytoplasm during the homogenisation stage and centrifugation the enzyme succinate dehydrogenase was present. Since the test was carried out under the same conditions in a neutral pH yellowish brown we can conclude that this was a fair test, however is it often found that the more molecules present in aThe separating of the homogenate could be improved by using another method of homogenisation, in this experiment we used a Potter Lethem homogeniser which is a glass and plastic hand homogeniser. This perhaps isnt the most accurate at pressurising cells with the force needed to accurately release cell content. Alternative homogenisers include ultrasonic and rotor based homogenisers which may provide more accura te. (www.proscientific.com)A different centrifugation method used. During this experiment differential centrifugation was used, however density gradients may provide more accurate at purifying a sample (www.coleparmer.co.uk). This method works by placing various layers after layer of gradient media such as sucrose in a tube with the heaviest layer at the bottom and the lightest at the top. The cell fraction to be separated is placed on top of the layer and centrifuged. Density gradient separation can be classified into 2 categories. Rate-tonal (size) separation. Isopycnic (density) in which organelles separate until their density matches the surroundings of the media in which they are. A very good medium for separating organelles is an iodinised media. (www.coleparmer.co.uk).Accuracy of the absorbance and accuracy of obtaining the protein amount. Results are slightly low indicating inaccuracy in both collecting the samples and also measuring the absorbance, this could be due to err or 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 innacurate indication to enzyme present (www.millipore.com) . 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. Another method to measure enzyme assay could be to use a caliometric method which measures heat radiance given off instead of the absorbance levels.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. This can also be used to further purify bacteria.In conclusion we see that as predic ted, the specific activity is highest in the mitochondrial fraction and the total activity is highest in the homogenate. The % recovery of each fraction and the relative specific activity for each fraction calculated shows a higher proportion in the mitochondrial fraction also. Overall the results indicate accurate laboratory skills and results conclude what was intended, however some slight changes to laboratory equipment would mean that some of the results such as SDH found in the supernatant may not come about in a future test.