Lab

Aim : 1) To isolate various subcellular fractions (homogenate, mitochondria, lysosomes, microsomes and soluble fraction) by mechanical disruption of rat liver cells and differential centrifugation.

2) To investigate the biochemistry and physiology of organelles outside of the complex environment of the intact cell using cell fractionation.

3) To investigate the ‘purity’ of prepared fractions by determination of marker enzymes such as glucose-6-phosphatase, acid phosphatase, glutamate dehydrogenase and lactate dehydrogenase in the fractions.

4) To determine the amount of Protein in the fractions by the Lowry Assay and by plotting of a calibration curve.

5) To determine the amount of phosphate in the fractions by plotting of a calibration curve.

6) To determine the specific activity of each fraction in S.I. units.

7) To catalyze the hydrolysis of several phosphate esters at acid pH using p-nitrophenyl phosphate as the substrate.

8) To determine the amount of p-nitrophenol in the fractions by plotting of a calibration curve.

9) To determine the specific activities in S.I. units for free and total enzyme.

10) To determine the latent activity of the lysosomal fraction using the detergent Triton X-100.

11) To determine the subcellular location of Succinate dehydrogenase.

Theory:

The arrangement of macromolecules within a cell is as important to cellular function as their catalytic activities. Cellular compartmentalization provides efficiency by bringing together compounds related or by separating those compounds that can interfere with each other. As such, cellular compartmentalization is accomplished by various subcellular organelles (Alberts et al, 2006). Many studies in cell physiology and biochemistry require relatively pure preparations of subcellular organelles. General purification procedures require that the cells first be disrupted

mechanically, using a homogenizer or a Waring blender, before separation of the organelles. Organelles are separated from each other using a technique called differential centrifugation As such, in this lab exercise several subcellular organelle fractions are isolated from liver cells and their various properties are examined by the method differential centrifugation as seen in Figure 1. Subcellular fractionation allows access to low abundant and organelle specific proteins.

Figure 1: Schematic diagram for differential centrifugation (Baltimore et al, 2007)

With differential centrifugation, the heaviest or most dense organelles (i.e. nuclei) pellet in less time (and at less forces) than is required to pellet lighter organelles (such as mitochondria). At each step, more dense particles are separated from less dense particles, generating a pellet and a supernatant. The pellet is composed of the more dense particles that fall out of suspension and collect at the bottom of the tube. The supernatant is the liquid that is left, dissolved particles, and lower density particles that are suspended in it (Baltimore et al, 2007). In this experiment, the mixture of cellular materials (known as homogenate) is separated into different layers based on the molecular weight, size, and shape of each type of molecules. Subsequent rounds of centrifugation will pellet first the nuclei, followed by the mitochondria, then the lysozomes, and finally the microsomes. The soluble fraction of the homogenate is what remains after these materials have been removed. Successive centrifugations increase the speed of centrifugation until the target particle is isolated. At this point, further study or purification can be performed on it. Due to the heterogeneity in biological particles, differential centrifugation suffers from contamination and poor recoveries. Contamination by different particle types can be addressed by resuspension and repeating the centrifugation steps (i.e., washing the pellet). In this experiment the rat liver was used since this organ has one cell type that predominates, so was well suited for cell fractionation. A homogenizer was used to break open the cells without damaging the organelles. The homogenization buffer often includes sucrose to partially dehydrate the organelles keeping them intact.

In subcellular fractionation experiments, concentrations or activities of compounds are commonly expressed with respect to protein content in that fraction. This convention is recommended because tissue disruption and centrifugation techniques are not quantitative, with loss of material at each step of the procedure. Therefore, the protein concentration (mg/mL) and total protein (mg) must be determined for each subcellular fraction. This information can be used to calculate the protein distribution within the cell. In this experiment, the Lowry assay was used to determine protein content. The principle behind the Lowry method of determining protein concentrations lies in the reactivity of the peptide nitrogen[s] with the copper [II] ions under alkaline conditions and the subsequent reduction of the Folin-Ciocalteay phosphomolybdicphosphotungstic acid to heteropolymolybdenum blue by the copper-catalyzed oxidation of aromatic acids (Dunn, 1992). The Lowry method is sensitive to pH changes and therefore the pH of assay solution should be maintained at 10 - 10.5. The major disadvantage of the Lowry method is the narrow pH range within which it is accurate. However, in the experiment very small volumes of sample which will have little or no effect on pH of the reaction mixture are used. X100 and X200 dilutions of each fraction were made with distilled water. This was done so as to remove any compounds that will interfere with the Lowry procedure. These include some amino acid derivatives, certain buffers, drugs, lipids, sugars, salts, nucleic acids and sulphydryl reagents.

Some enzymes are located almost exclusively in a particular cell organelle or in the soluble cytoplasm as seen in Figure 2. This fact may be useful in ascertaining the main organelles present in a particular fraction obtained from differential centrifugation. Also, it may be used to check the purity of a particular preparation of cell organelles. Such enzymes serve as marker enzymes because their detection in any fraction implies that the relevant organelle containing them is also present in the sample.

Subcellular Fraction Relative Density Marker EnzymeNuclei 1 (most dense) DNA, histones

Mitochondria 2 Succinate dehydrogenase and Glutamate dehydrogenase

Lysosomes 3 Acid PhosphataseMicrosomes (E.R) 4 Glucose-6-Phosphatase

Cytosol (Soluble Fraction) 5 (least dense) Lactate dehydrogenase

Figure 2: Table showing the marker enzymes which are found in a particular cell organelle (Dunn, 1992).

In this experiment, glucose-6-phosphatase, acid phosphatase, lactate dehydrogenase, glutamate dehydrogenase and succinate dehydrogenase are assayed to study the distribution of microsomes, lysosomes, soluble fraction and mitochondria respectively.

Glucose-6-phosphatase is an enzyme that biocatalyzes the hydrolysis of glucose-6-phosphate to glucose and phosphate. The inorganic phosphate released is estimated to determine the enzyme activity (Champe et al, 2004). This enzyme is a marker enzyme for the cell fraction containing endoplasmic reticulum i.e. microsomes. 0.25M sucrose was used to dilute the fractions since at 0.25 M, sucrose iso-osmotic as such, not much damage is done to sub-cellular fractions. Also, the 10% TCA solution was used so as to terminate the reaction.

Enrichment of the organelle is determined with respect to its specific activity. Specific activity is expressed in units of enzyme per milligram protein (un/mg). During the differential centrifugation, proteins are separated. Some go into the pellet and some stay in the supernatant. Therefore, if the enzyme stays in the supernatant and many other proteins pellet out, the enzyme will represent a higher percentage of the protein in the supernatant than before it was centrifuged. However, if the enzyme activity is measured in units per ml of supernatant (unit/ml), the activity would stay the same, since the volume of the supernatant that enzyme is suspended in hasn't changed. Similarly, if the enzyme went to the pellet, its activity expressed in units/ml would be dependent on the volume that the pellet is resuspended in before doing the assay, and not on any enrichment created by the centrifugation (Hames and Hooper, 2005). For this reason, both the enzyme activity in a fraction and its protein concentration are measured and then the specific activity of enzyme is calculated by dividing by the concentration of protein in that fraction to give units/mg protein.

Acid phosphatase acts upon organic phosphate esters, releasing inorganic phosphate at a pH between 4 and 6. The subcellular fraction containing lysosomes is rich in this enzyme. In this assay a colorless compound, para-nitrophenol phosphate (pNPP) is used as the substrate for acid phosphatase. When the phosphate group of pNPP is cleaved, para-nitrophenol is generated. Para-nitrophenol is a yellow compound that is easily measured in a spectrophotometer at 405nm (Champe et al, 2004). The membranes of intact lysosomes are normally impermeable to added substrates. If lysosomes are damaged, the enzymes are released and can then be assayed in the medium. The degree of damage can then be determined by measuring the soluble or free activity and comparing it with the activity determined after deliberately rupturing the membrane with a detergent (e.g. Triton X-100)(total activity). The difference between the two values is the latent activity, usually expressed as a percentage of the total. The Triton X-100 was used as a detergent in cell lysis (i.e. to release soluble proteins).

Glutamate dehydrogenase is a mitochondrial enzyme that catalyzes the reversible oxidative deamination of glutamate to -ketoglutarate (2-oxoglutarate) using NAD+ as coenzyme.

Glutamate + NAD+ H2O ⇌ NH4+ + -ketoglutarate + NADH + H+

The reaction can be readily measured by following the change in absorbance at 340nm. In this experiment, the reverse reaction is observed. In animals, the produced ammonia is usually used as a substrate in the urea cycle. Typically, the α-ketoglutarate to glutamate reaction does not occur in mammals, as glutamate dehydrogenase equilibrium favours the production of ammonia and α-ketoglutarate. Glutamate dehydrogenase also has a very low affinity for ammonia and therefore toxic levels of ammonia would have to be present in the body for the reverse reaction to proceed (Cox and Nelson, 2008).

Lactate dehydrogenase catalyzes the reversible reduction of pyruvate to lactate with NADH as the coenzyme as seen in Figure 3.

Figure 3: Reaction for Lactate Dehydrogenase (Champe et al, 2004)

The reaction mechanism of LDH follows an ordered sequence. In order for lactate to be oxidized NAD+ must bind to the enzyme first followed by lactate. Transfer of a hydride ion then happens quickly in either direction giving a mixture of the two teranary complexes, enzyme-NAD+-lactate and enzyme-NADH-pyruvate (Champe et al, 2004). Finally pyruvate dissociates from the enzyme followed by NADH. This enzyme is present in the soluble fraction (cytosol).

The enzyme succinate dehydrogenase (SDH) is an inner mitochondrial membrane protein involved in the Krebs cycle. It converts succinate to fumarate by oxidation, passing 2 electrons from succinate to FADH2 as seen in Figure 4. In the mitochondrion, FADH2 passes these two high-energy electrons to the electron transport chain, which uses them to produce 2 ATP

fumarate + FADH2 succinate + FAD (e– are transferred = redox)

Figure 4: Balanced Equation for Succinate Dehydrogenase (Cox and Nelson, 2008)

In this reaction, two hydrogen atoms are removed from succinate by flavin adenine dinucleotide (FAD), a prosthetic group that is tightly attached to succinate dehydrogenase. Two electrons from the reduced SDH-FADH2 complex are then transferred to ubiquinone, a soluble component of the electron transport system Complex II. The two half reactions are seen below in Figure 5.

(1) fumarate + 2H+ + 2 e– succinate (e– are gained = reduction)

(2 reversed) FADH2 FAD + 2H+ + 2 e– (e– are lost = oxidation)

Figure 5: Half reactions for succinate dehydrogenase (Cox and Nelson, 2008)

A suitable redox dye neotetrazolium (pale yellow) was used in this experiment as the hydrogen acceptor and a purple reduced form of the dye was produced. Hence the reduction of the dye indicates enzyme activity in the system.

2 Succinate + NT ⇌ Fumarate + NT diformazan

The subcellular fraction exhibiting this enzyme activity indicates the presence of mitochondria in the sample. SDH is a good marker enzyme for isolation of mitochondria because it remains attached to the inner membrane of the mitochondrion. As long as the mitochondria remain intact, the presence of SDH means that mitochondria are there (Cox and Nelson, 2008)

Procedure:

The materials and reagents used in this experiment were: test tubes, centrifuge tubes, acid-washed medium sized test tubes, beakers, distilled water, measuring cylinder, isolation medium consisting of sucrose 0.25M containing 0.1mM EDTA, 1mM 2-SH-ethanol and 5mM Tris-HCL buffer pH 7.4 (cold), Standard protein solution (200µg/mL BSA), Reagent A – 2% Na2CO3 in 0.1M NaOH, Reagent B – 0.5% CuSO4.5H2O in 1% sodium citrate, Reagent C –100mL of A and 2mL of B, Folin Ciocalteu’s Reagent – Diluted 1:1 with H2O, 2.5% ammonium molybdate in 2.5M Sulphuric Acid, Ascorbic Acid 1g/10mL (freshly prepared), Reagent D - water : ascorbic acid : acid-molybdate in the ratio 2 : 1 : 2, Phosphate standard – 2.18mg of KH2PO4 per 100mL, Maleate buffer 0.1M, pH 6.5, EDTA 10mM (in buffer pH 6.5), Glucose-6-phosphate 0.2M (in buffer, pH 6.5), 10% Trichloroacetic acid (cold), Acetate buffer 0.2M, pH 4.7, 8mM p-nitrophenol phosphate (freshly prepared), Tris-HCl 1M pH 9.0 containing 1M Na2CO3 and 0.4M K2HPO4, 100μM p-nitrophenol standard, Triton X-100, 10% (w/v), Sodium phosphate buffer 0.1M, pH 7.4, Sodium α-ketoglutarate 0.15M in buffer, adjusted to pH 7.4, EDTA 30mM in buffer, pH 7.4, Ammonium acetate 0.75M in buffer, pH 7.4, Triton X-100 10% (w/v) in buffer, pH 7.4, NADH 2.5mg/mL in buffer, pH 7.4 (freshly prepared), Sodium phosphate buffer 0.01M, pH 7.4, Sodium pyruvate 0.021M in buffer pH 7.4 (freshly prepared), NADH 3.5mM in buffer pH 7.4 (freshly prepared), 0.35% Na2S2O4, 0.1% NT – freshly prepared, 0.02M sodium succinate pH 7.4, 0.5M phosphate buffer.

A starved rat was killed, exsanguinated and the liver was rapidly removed and blotted lightly on filter paper. Approximately 8 -10g of liver was weighed and the tissue was rinsed twice with a small volume of cold sucrose to remove blood. The tissue was kept in a beaker on ice, cut into small pieces and enough sucrose was added to make a 20% homogenate (10g in 50mL). It was then transferred to a homogenizing tube and homogenized. An equal volume of medium was added to yield a 10% homogenate and poured into two centrifuge tubes and centrifuged. Each fraction was resuspended in sucrose and centrifuged, the second supernatant being added to the first. 6mL of the nuclei free homogenate was saved. All pellets were resuspended in 10mL of cold sucrose, by homogenization at a very low speed. The exact volume of each fraction was

measured and recorded. Exactly half of each fraction was poured into well-labeled capped containers and stored in the deep freeze until the next lab session.

3mL of each fraction was collected and labeled accordingly. 7 test tubes were labeled 1-7. 1mL of distilled water was added to tube 1. 0.1mL of the standard protein solution and 0.9mL of water were added to tube 2. 0.3mL of the standard protein solution and 0.7mL of water were added to tube 3. 0.5mL of the standard protein solution and 0.5mL of water were added to tube 4. 0.5mL of the standard protein solution and 0.5mL of water were added to tube 5. 0.7mL of the standard protein solution and 0.3mL of water were added to tube 6. 1mL of the standard protein solution was added to tube 7. 5mL of Lowry reagent C were added to all tubes and left standing at room temperature for exactly 10 minutes. 0.5mL of Folin-Ciocalteu’s reagent (diluted) were added to all tubes and mixed well. After 30 minutes at room temperature, the absorbances were read against the blank at 750nm. For each fraction a x100 and x200 dilution of each fraction was made with distilled water and 1mL of the diluted fraction was used for protein determinations. A calibration curve of absorbance at 750nm vs. μg of protein was plotted.

7 acid-washed medium sized test tubes were labeled 1-7. 5mL of distilled water was added to tube 1. 0.5mL of the standard phosphate solution and 4.5mL of water were added to tube 2. 1mL of the standard phosphate solution and 4mL of water were added to tube 3. 2mL of the standard phosphate solution and 3mL of water were added to tube 4. 2mL of the standard phosphate solution and 3mL of water were added to tube 5. 3mL of the standard phosphate solution and 2mL of water were added to tube 6. 4mL of the standard phosphate solution and 1mL of water were added to tube 7. 4.0mL of Reagent D were added to each tube and mixed well. The tubes were placed in a water bath at 60ºC for 10 minutes. The tubes were cooled and the absorbances were read at 650nm. Absorbance at 650nm vs. micromoles of phosphorous was plotted.

For the homogenate fraction x10 dilution was made. For the mitochondria and lysosomes fractions x 5 dilutions were made. For the microsomes and soluble fractions x20 dilutions were made. All these fractions were diluted with 0.25M sucrose. 15 clean small acid washed test tubes were set up and labeled. Five test tubes were used as zero time controls (one for each fraction) and the other ten tubes were served as duplicates. To all 15 test tubes each of the following were added: 0.6mL Buffer, 0.1mL EDTA and 0.2mL glucose-6-phosphate. The test tubes were equilibrated at 35ºC for 5mins. The reaction was started by adding 0.1mL of the respective fraction and incubated at 35ºC for exactly 10min. For the zero-time tubes 1 mL of 10% TCA was added BEFORE adding the fraction. The reaction (in the non zero-time tubes) was terminated by adding 1mL of the cold 10% TCA solution at the end of 10 mins. The tubes were centrifuged at 3000rpm for 10 minutes in the bench centrifuge and 1mL of the supernatant was removed for phosphate determination. The specific activity of each fraction was calculated in S.I. units (µmoles of phosphorous produced/min/mg protein).

7 test tubes were labeled 1-7. 1mL of distilled water was added to tube 1. 0.2mL of 100µM p-nitrophenol and 0.8mL of water were added to tube 2. 0.4mL of 100µM p-nitrophenol and

0.6mL of water were added to tube 3. 0.4mL of 100µM p-nitrophenol and 0.6mL of water were added to tube 4. 0.6mL of 100µM p-nitrophenol and 0.4mL of water were added to tube 5. 0.8mL of 100µM p-nitrophenol and 0.2mL of water were added to tube 6. 1mL of 100µM p-nitrophenol was added to tube 7. 1.2mL of acetate buffer and 2mL of alkaline Tris buffer were added to each tube and the absorbencies at 405nm were read against the blank. A graph of absorbance at 405nm vs. micromoles of p-nitrophenol was plotted.

1 in 10 dilutions were made for all the fractions. For each of the 5 fractions, 2 tubes were required. One tube was the zero-time control while the other was the normal reaction tube. Five zero-time control tubes were labeled with their respective fraction numbers. To each tube 1.2mL of acetate buffer, 0.5mL of substrate solution, 0.1mL of water and 2mL of alkaline Tris buffer were added. Five other normal reaction tubes were labelled with their respective fraction numbers. To each tube 1.2mL of acetate buffer, 0.5mL of substrate solution and 0.1mL of water were added. To determine the latent activity of the lysosomal fraction only, the 0.1mL water was replaced with 0.1mL of 10% Triton X-100 in both the zero-time control and normal reaction tubes. All the tubes were incubated for 5 minutes at 35ºC. The reaction was started by adding 0.2mL of the diluted sample and incubated for 10 minutes. The reaction was terminated in the normal reaction tubes by adding 2mL of alkaline Tris buffer. The tubes were cooled and the absorbance read at 405nm.

For each assay 2.1mL phosphate buffer, 0.2mL fraction ( 1 in 50 dilution), 0.1mL NADH, 0.2mL Ammonium acetate, 0.2mL EDTA and 0.1mL Triton X-100 were prepared in small test tubes (in duplicates) and equilibrated at room temperature for 10 minutes. The reaction was started by adding 0.1mL of the substrate α-ketoglutarate. This was immediately transferred to a cuvette and the change in absorbance at 30-second intervals over a 4-minute period. Using the molar extinction coefficient of NADH (6220 cm-1 M-1), the specific activities of the different fractions were calculated in S.I. units.

For each assay 2.6mL phosphate buffer, 0.2mL fraction (1 in 50 dilution) and 0.1mL NADH were prepared (in duplicates) and equilibrated at 37ºC for 10 minutes. The reaction was started by adding 0.1mL of the substrate sodium pyruvate solution. This was immediately transferred to a cuvette and the change in absorbance at 30-second intervals over a 4-minute period at 340nm. The specific activities of the different fractions were calculated in S.I. units.

The 0.1% NT was diluted to 0.05% to make 5ml. 10 test tubes were set up and labeled. 1mL of distilled water was added to tube 1. 0.1mL of the 0.05%NT and 0.9mL of water were added to tube 2. 0.2mL of the 0.05%NT and 0.8mL of water were added to tube 3. 0.2mL of the 0.05%NT and 0.8mL of water were added to tube 4. 0.3mL of the 0.05%NT and 0.7mL of water were added to tube 5. 0.4mL of the 0.05%NT and 0.6mL of water were added to tube 6. 0.6mL of the 0.05%NT and 0.4mL of water were added to tube 7. 0.6mL of the 0.05%NT and 0.4mL of water were added to tube 8. 0.8mL of the 0.05%NT and 0.2mL of water were added to tube 9. 1mL of the 0.05%NT was added to tube 10. 2 mL of cold Na2S2O4 was added and mixed well. 7mL of

acetone was added to dissolve the coloured formazan and mixed well and allowed to stand for 10 minutes. The absorbance at 540nm was measured and the calibration curve (absorbance at 540nm vs. micromoles NT) was drawn. From the standard curve the absorbance equivalent to the reduction of 1 micromole of NT was determined.

0.5mL buffer, 1mL succinate, 0.5mL 0.1% NT and 0.5mL distilled water were pipetted into 20 test tubes and equilibrate for 5 minutes at 35 ºC. One enzyme blank and a substrate blank were included for each fraction. The reaction was started by adding 0.3mL of undiluted sample in the reaction tubes and these tubes were incubated for 20 minutes. The reaction was stopped by adding 7mL of acetone and shaking well. The solutions were transferred to centrifuge tubes, a small amount of ammonium sulphate was added and the tubes were centrifuged for 3 minutes at 3000rpm. The absorbance of the clear supernatant was recorded at 540nm and the specific activities of each fraction were calculated in S.I. Units.

Results :

Table 1: Results for Calibration Curve for Proteins

Volume of distilled water added (mL)

Volume of standard protein sol’n added

(mL)

Absorbance at 750nm

Amount of protein (µg)

1.0 0.0 0.000 00.9 0.1 0.052 200.7 0.3 0.147 600.5 0.5 0.283

0.282 1000.5 0.5 0.2810.3 0.7 0.372 1400.0 1.0 0.508 200

Table 2: Absorbance at 750nm for the Determination of Protein in Fractions

Fraction Absorbance at 750nm (X 100

dilution)

Amount of Protein (µg)

Absorbance at 750nm (X 200

dilution)

Amount of Protein (µg)

Homogenate 0.502 193.08 0.360 138.46

Mitochondria 0.653 251.15 0.441 169.62Lysosomes 0.252 96.92 0.120 46.15

Microsomes 0.541 208.08 0.375 144.23Soluble Fraction 0.437 168.08 0.303 116.54

Table 3: Average Protein Concentration % Recovery in Each Fraction

FractionX 100 dilution

(mg/ml)X 200

dilution(mg/ml)

Average (mg/ml)

Total Protein

(mg)

% Recovery

Homogenate 19.31 27.69 23.50 17860.0 100.00Mitochondria 25.12 33.92 29.52 3542.4 19.83

Lysosomes 9.69 9.23 9.46 1135.2 6.36Microsomes 20.81 28.85 24.83 2979.6 16.68

Soluble Fraction 16.81 23.31 20.06 10732.1 60.09

Table 4: Results for Calibration Curve for Inorganic Phosphorous

Volume of distilled water

added (mL)

Volume of standard

phosphate sol’n added (mL)

Absorbance at 650nm

Amount of phosphorous

(µg)

Amount of phosphorous

(µmol)

5.0 0.0 0.000 0.00 0.004.5 0.5 0.101 2.48 0.084.0 1.0 0.185 4.96 0.163.0 2.0 0.386

0.378 9.91 0.323.0 2.0 0.3702.0 3.0 0.543 14.87 0.481.0 4.0 0.704 19.82 0.64

Table 5: Absorbance at 650nm for the Assay of glucose-6-phosphatase

Fraction Absorbance at 650 nm (zero time control

tubes)

Absorbance at 650 nm (normal reaction

tubes)(A)

Absorbance at 650 nm (normal

reaction tubes)(B)

Average of A and B

Homogenate 1 0.022 6 0.137 11 0.116 0.127Mitochondria 2 0.023 7 0.118 12 0.115 0.117

Lysosomes 3 0.021 8 0.137 13 0.144 0.141Microsomes 4 0.021 9 0.151 14 0.160 0.156

Soluble Fraction

5 0.022 10 0.096 15 0.085 0.091

Table 6: Assay of Glucose-6-Phosphatase (cont’d)

FractionCorrected absorbance at 650nm

(average of A and B – zero time abs.)mol phosphate

Homogenate 0.105 0.093Mitochondria 0.094 0.084

Lysosomes 0.120 0.107Microsomes 0.135 0.120

Soluble Fraction 0.069 0.061

Table 7: Activity Results for Glucose - 6- Phosphatase

FractionActivity (U/ml)

Specific Activity (U/mg)

Total Activity (U)

% Total Activity

% Total Recovery

Homogenate 1.86 0.079 1413.6 101.00 100.00Mitochondria 1.68 0.057 201.6 14.41 14.26


Soluble Fraction

1.22 0.061 652.7 46.65 46.17

Table 8: Results for Calibration Curve for p-nitrophenol

Volume of distilled water

added (mL)

Volume of p-nitrophenol (mL)

Absorbance at 405 nm

Amount of p-nitrophenol

(µmol)1.0 0.0 0.000 0.000.8 0.2 0.099 0.020.6 0.4 0.203

0.205 0.040.6 0.4 0.2060.4 0.6 0.240 0.06

0.2 0.8 0.334 0.080.0 1.0 0.410 0.10

Table 9: Absorbance at 405nm of Acid Phosphatase Enzyme Assay

Fraction Absorbance at 405nm(zero time controls)

Absorbance at405 nm

(normal reaction tubes)

Corrected Absorbance at

405nm

mol p-nitrophenol

Homogenate 0.077 0.517 0.440 0.97Mitochondria 0.089 0.399 0.310 0.68

Lysosomes 0.093 0.185 0.092 0.20Microsomes 0.109 0.396 0.287 0.63

Soluble Fraction 0.002 0.224 0.222 0.49Lysosome

(Triton X) (total)0.051 0.100 0.049 0.11

Table 10: Activity Results for Acid Phosphatase

FractionActivity (U/ml)


Total Activity

(U)

% Total Activity

% Total Recovery

Latent Activity of Lysosomes

Homogenate 4.85 0.21 3686 161.5 100.0Mitochondria 3.40 0.12 408 17.9 11.1

Lysosomes (free)

1.00 0.04 120 5.3 3.3

Microsomes 3.15 0.13 378 16.6 10.3Soluble Fraction

2.45 0.12 1311 57.4 35.6

Lysosome (Triton X)

(total)0.55 0.06 66 2.9 1.79 -2.4

Table 11: Results for Glutamate Dehydrogenase Activity in Different Fractions

TimeFraction 1 Fraction 2 Fraction 3 Fraction 4 Fraction 5

30 0.713 0.655 0.653 0.714 0.691 0.715 0.704 0.704 0.668 0.65460 0.704 0.645 0.639 0.681 0.687 00.71

20.700 0.699 0.667 0.652

90 0.693 0.636 0.627 0.650 0.683 0.708 0.695 0.693 0.666 0.651120 0.685 0.626 0.614 0.615 0.680 0.705 0.691 0.688 0.665 0.650150 0.676 0.618 0.602 0.580 0.677 0.703 0.686 0.683 0.664 0.648180 0.666 0.613 0.590 0.547 0.674 0.699 0.681 0.679 0.662 0.647210 0.659 0.611 0.578 0.516 0.670 0.696 0.675 0.672 0.661 0.645240 0.650 0.607 0.567 0.481 0.667 0.692 0.670 0.668 0.658 0.644

Table 12: Results for Lactate Dehydrogenase Activity in Different Fractions

Time Fraction 1 Fraction 2 Fraction 3 Fraction 4 Fraction 530 0.522 0.548 0.495 0.492 0.544 0.549 0.521 0.437 0.472 0.46560 0.421 0.448 0.452 0.450 0.522 0.523 0.436 0.363 0.397 0.39590 0.327 0.357 0.412 0.408 0.500 0.496 0.348 0.289 0.329 0.327120 0.243 0.276 0.370 0.366 0.479 0.474 0.275 0.228 0.261 0.254150 0.190 0.214 0.329 0.326 0.455 0.446 0.224 0.178 0.203 0.196180 0.163 0.177 0.295 0.290 0.431 0.422 0.199 0.149 0.146 0.144210 0.153 0.160 0.257 0.255 0.409 0.397 0.192 0.136 0.104 0.103240 0.151 0.156 0.225 0.222 0.387 0.375 0.189 0.131 0.073 0.072

Table 13: Change in Absorbance/ min for Enzymes Shown

FractionΔ Abs/min

Glutamate Dehydrogenase Lactate DehydrogenaseHomogenate (F1) 0.014 0.095Mitochondria (F2) 0.040 0.068

Lysosomes (F3) 0.006 0.042Microsomes (F4) 0.009 0.080

Soluble Fraction (F5) 0.003 0.099

Table 14: Activity Results for Glutamate Dehydrogenase

Fraction Activity (U/ml)


Total Activity (U)

% Total Activity

% Total Recovery

Homogenate 0.113 0.0048 85.88 130.4 100Mitochondria 0.322 0.0109 38.64 58.7 45


Soluble Fraction

0.024 0.0012 12.84 19.5 15

Table 15: Activity Results for Lactate Dehydrogenase



Total Activity (U)

% Total Activity

% Total Recovery

Homogenate 0.765 0.033 581.4 95.6 100Mitochondria 0.545 0.018 65.4 10.8 11.2

Lysosomes 0.338 0.036 40.6 6.7 7Microsomes 0.640 0.026 76.8 12.6 13.2

Soluble Fraction

0.795 0.040 425.3 69.9 73.2

Table 16: NT Calibration Curve Results

0.05% NT (ml) NT (μmol) [email protected] 0.000 0.0000.1 0.076 0.0770.2

0.1520.160

0.1580.2 0.1550.3 0.228 0.2780.4 0.304 0.3180.6

0.4560.552

0.5580.6 0.5630.8 0.608 0.6211.0 0.760 0.770

Table 17: Succinate Dehydrogenase Results

Fraction

Abs@540nmSubstrate

BlankEnzyme Blank

Average of two Samples

Corrected Sample

Abs.

Micromoles of NT

Homogenate 0.010 0.000 0.050 0.040 0.038Mitochondria 0.020 - 0.264 0.244 0.230

Lysosomes 0.000 - 0.062 0.062 0.059Microsomes 0.020 - 0.040 0.020 0.019

Soluble Fraction

0.010 - 0.080 0.070 0.066

Table 18: Activity Results for Succinate Dehydrogenase



Total Activity (U)

% Total Activity

% Total Recovery

Homogenate 0.013 5.5 x 10-4 9.88 40.9 100Mitochondria 0.077 2.6 x 10-3 9.24 38.3 93.5

Lysosomes 0.020 2.1 x 10-3 2.40 9.9 24.3Microsomes 0.006 2.4 x 10-4 0.72 3.0 7.3

Soluble Fraction

0.022 1.1 x 10-3 11.77 48.8 119.1

Calculations:

Determination for µg of protein

Using Table 1 and tube 2:

Concentration of standard protein solution = 200µg/mL

Volume of standard solution in tube 2 = 0.1mL

1mL of standard protein solution = 200µg

0.1mL of standard protein solution = (200 x 0.1)/1 = 20µg

Average Absorbance

Using tubes 4&5 of Table 1:

= (0.283+0.281)/2

= 0.282

Determination of Protein Content in Diluted Fractions

The absorbance reading (X100 dilution) for Fraction 1 (Homogenate) at 750nm was 0.502. Using the equation of the calibration curve: y = 0.0026x

0.502 = 0.0026x

x = 0.502/0.0026 = 193.08

Therefore in Fraction 1, X100 dilution, there was 193.08µg of protein as seen in Table 2.

Determination of Protein Concentration in X100 dilution (mg/mL)

In X100 dilution of Fraction 1 there is 193.08µg of protein

Therefore, 1mL of undiluted fraction contains 193.08 x 100 = 19308µg protein

Since 1000µg = 1mg

19308µg = (1/1000) x 19308

= 19.31mg

Determination of Protein Concentration in X200 dilution (mg/mL)

In X200 dilution of Fraction 1 there is 138.46µg of protein

Therefore, 1mL of undiluted fraction contains 138.46 x 200 = 27692µg protein

Since 1000µg = 1mg

27692µg = (1/1000) x 27692

= 27.69mg

Determination of Average = (27.69 + 19.31)/2

= 23.5mg/mL

Determination of Total Protein (mg)= Average x Total volume of Homogenate (Fraction 1)

= 23.5mg/mL x 760mL

= 17860mg

Determination of % Recovery= ((Total protein of mitochondrial fraction/ Total protein of homogenate (F1) )x 100

= (17860/17860) x 100

= 100%

Determination for µmol of phosphorous

Concentrated stock KH2PO4= 2.18mg/100mL

136mg KH2PO4 = 1mmol2.18mg KH2PO4 = (1/136) x 2.18 = 0.016mmol = 16µmol

Using tube 2 of Table 4,

100mL of phosphate solution = 16µmol

0.5mL of phosphate solution = (16/100) x 0.5 = 0.08µmol KH2PO4

Determination for µg of phosphorous

Molar Ratio 1:1 KH2PO4 : P

1µmol KH2PO4 = 30.97µg P

Using tube 2 of Table 4,

0.08µmol KH2PO4 = (30.97 x 0.08)/1

= 2.48µg of P

Determination of Phosphate Content (µmol) in Fractions

The absorbance reading for Fraction 1 (Homogenate) at 650nm was 0.105. Using the equation of the calibration curve: y = 1.1232x

0.105 = 1.1232x

x = 0.105/1.1232= 0.093

Therefore in Fraction 1, there was 0.093 of phosphate as seen in Table 6.

Determination of Activity for Glucose-6-Phosphatase

Using Table 7, Fraction 1:

0.093µmol of P from 1mL of supernatant,

This was taken from 2mL of solution therefore:

2mL of solution contained: 0.093 x 2 = 0.186 µmol of P

This 2mL solution contained 0.1mL of diluted sample therefore:

0.1mL of diluted sample contains 0.186 µmol of P;

1mL of diluted sample would contain: (0.186/0.1)x1 = 1.86µmol of P

This reaction occurred in 10 mins hence:

In 1 minute 1mL of sample would contain: (1.86/10) x1 = 0.186 µmol of P/min/mL;

This was obtained from sample diluted 10x therefore:

1mL of undiluted sample would have: 0.186 x 10 = 1.86 µmol of P/min/mL;

Since 1 µmol of P/min = U

Undiluted Sample activity = 1.86 U/mL

Specific activity:

From Table 3: 1mL of sample contained 23.50 mg of protein

Therefore in 23.50 mg of protein there was 1.86 U of activity;

1mg would contain: (1.86/23.5) x 1 = 0.079 U/mg

Total Activity:

Total volume of Fraction (mL) x Activity (U/mL);

760 x 1.86 = 1413.6 U of activity.

% Total Activity:

(Total activity of F1/Sum of Total Activity of F2,F3,F4,F5) x 100;

(1413.6/1399.1) x 100 = 101%

Determination for µmol of p-nitrophenol:

1000mL contains 100µmol of p-nitrophenol;

0.2mL would contain: (100/1000) x 0.2 = 0.02µmol of p-nitrophenol

Determination of p-nitrophenol Content (µmol) in Fractions


0.440 = 0.4558x

x = 0.440/0.4558= 0.97

Therefore in Fraction 1, there was 0.97 of p-nitrophenol as seen in Table 9.

Determination of Activity of Acid Phosphatase Assay:

Using Fraction 1 of Table 10,

0.97 µmol of p-Nitrophenol was present in 4 mL of solution;

This solution contained 0.2mL of diluted sample hence;

0.2mL of diluted sample reacts to form: 0.97µmol of p-Nitrophenol;

In 1mL of diluted sample would contain: (0.97/0.2) x 1 = 4.85µmol of p-Nitrophenol;

This reaction took place over 10 minutes, therefore:

In 1 minute: (4.85/10) x 1 = 0.485 µmol of p-Nitrophenol/min

This was taken from a 1 in 10 sample hence;

In 1mL of undiluted sample would react to form: 0.485 x 10 = 4.85 µmol of p-Nitrophenol/min/mL = 4.85 U/mL

Specific Activity:

1mL of undiluted homogenate contains: 23.5mg of protein/mL

If 1mL has an activity of 4.85U then 23.5mg of protein has 4.85U of activity;

1mg would have: (4.85/23.5) x 1 = 0.21U/mg

Total Activity:

1mL of homogenate contains: 4.85U/mL;

In 760mL of homogenate would have: 4.85 x 760 = 3686U

% Total Activity:

(3686/2283) x 100 = 161.5%

Latent Activity (lysosomes):

Total activity (Triton X) – free activity (no Triton X) = 2.9-5.3= -2.4

Change in Absorbance/min for Glutamate Dehydrogenase in homogenate:

Average Abs at 30 sec = (0.713+0.655)/2 = 0.684


Change in Abs/min = Abs @ 30 sec – Abs @ 240 sec/ 4 = (0.684-0.629)/4 = 0.014

Lactate Dehydrogenase in homogenate:



Change in Abs/min = Abs @ 30 sec – Abs @ 240 sec/ 4 = (0.535-0.154)/4 = 0.095

Determination for Activity of Glutamate Dehydrogenase:

For Homogenate:

From equation A = C x el where:

C refers to concentration in moles, A refers to Absorbance, el refers to the molar extinction coefficient of NADH (6220 cm-1 M-1 ).

Converted to:

C = A/el; 0.014/6220 = 2.25 x 10-6

To µmoles:

Since 1mol= 1000000µmol

2.25 x 10-6mol= (1000000/1) x 2.25 x 106 = 2.25 µmoles/min/litre

To µmoles/min/mL:

2.25 /1000 = 2.25 x 10-3µmoles/min/mL

This was in a 1 in 50 diluted solution hence:

In undiluted sample: 2.25x 10-3x 50 = 0.113 µmoles/min/mL = 0.113U/mL

Specific Activity:

In 1mL of homogenate contains 23.5mg of protein.

Therefore the activity in 1g of protein = 0.113 / 23.5 = 0.0048 U/mg or 4.8 x10-3 U/mg

Determination of µmol of NT:

0.05%NT

0.05g in 100ml

Now 662g = 1mol NT

0.05g = (1/662) x 0.05

= 7.6 x 10-5 mol NT

= 76µmol NT

100ml = 76µmol NT

Therefore, using tube 2 of Table 16,

0.1ml = (76/100) x 0.1

= 0.076µmol NT

Determination of NT Content (µmol) in Fractions


0.040 = 1.0592x

x = 0.040/1.0592= 0.038

Therefore in Fraction 1, there was 0.038µmol of NT as seen in Table 17.

Determination of Activity for Succinate Dehydrogenase:

Using Fraction 1 of Table 18,

0.3mL of diluted sample = 0.038 µmol of NT

The ratio of Succinate : NT is 2:1 so 0.038 x 2 = 0.076µmolNT

As such, 1mL of diluted sample = (0.076/0.3) x 1

= 0.253µmolNT

This reaction took place over 20 minutes, therefore:

In 1 minute: (0.253/20) x 1 = 0.013 µmol of NT/min

This sample was not diluted therefore:

1mL of undiluted sample = 0.013µmol of NT/min/mL;

Since 1 µmol of P/min = U

Undiluted Sample activity = 0.013 U/mL

Specific Activity:

1mL of undiluted homogenate contains: 23.5mg of protein/mL

If 1mL has an activity of 0.013U then 23.5mg of protein has 0.013U of activity;

1mg would have: (0.013/23.5) x 1 = 5.53 x 10-4U/mg

Total Activity:

1mL of homogenate contains: 0.013U/mL;

In 760mL of homogenate would have: 0.013 x 760 = 9.88U

Discussion:

In this experiment, various subcellular fractions were isolated from rat liver cells by differential centrifugation. The rat liver was used since this organ had one cell type that predominates, so it was well suited for cell fractionation. A homogenizer was used to break open the cells without damaging the organelles. The homogenization buffer included sucrose so as to partially dehydrate the organelles keeping them intact. As can be seen in Table 1, as the volume of standard protein solution increased, the amount of protein increased thus leading to the increase in the absorbance values at 750nm from tubes 1-7. This occurred since the electrons in the chemical bonds only absorb certain wavelengths of light thus, the more concentration of protein, more bonds and more electrons to absorb the light. However, tubes 4 &5 had similar absorbance values since the exact amount of protein were placed in those tubes.

Using Table 1, a calibration curve of absorbance at 750nm against µg of protein was plotted as seen in Graph 1. This was used to find the unknown concentration in the various fractions. Beer-Lambert’s law states that there is a logarithmic dependence between the transmission, T, of light through a substance and the product of the absorption coefficient of the substance, α, and the distance the light travels through the material (i.e., the path length), ℓ (Cox and Nelson, 2008). Thus, if the path length and the molar absorptivity (or the absorption cross section) is known and the absorbance is measured, the concentration of the substance can be deduced as seen in Table 2.

In this experiment, the Lowry assay was used to determine protein content. The principle behind the Lowry method of determining protein concentrations lies in the reactivity of the peptide nitrogen[s] with the copper [II] ions under alkaline conditions and the subsequent reduction of the Folin-Ciocalteay phosphomolybdicphosphotungstic acid to heteropolymolybdenum blue by the copper-catalyzed oxidation of aromatic acids. The Lowry method is sensitive to pH changes and therefore the pH of assay solution should be maintained at 10 - 10.5 (Dunn, 1992). The major disadvantage of the Lowry method is the narrow pH range within which it is accurate. However, in the experiment very small volumes of sample which will have little or no effect on pH of the reaction mixture were used. X100 and X200 dilutions of each fraction were made with distilled water. This was done so as to remove any compounds that would have interfered with the Lowry procedure. These include some amino acid derivatives, certain buffers, drugs, lipids, sugars, salts, nucleic acids and sulphydryl reagents.

The results from Table 2 were also included in Table 3. So, for the X100 and X200 dilution, the highest amount of protein was found in Fraction 2 (mitochondria) followed by Fraction 4 (microsomes), Fraction 1 (homogenate), Fraction 5 (soluble fraction) then Fraction 3 (lysosomes). As such, fraction 2 had the highest average protein concentration with a value of 29.52mg/mL, followed by fraction 4 with a value of 24.83mg/mL, then fraction 1 with a value of 23.5mg/mL, then fraction 5 with a value of 20.06mg/mL, then finally fraction 3 with a value of 9.46mg/mL. The fraction containing mitochondria had the highest amount of protein as this oganelle is sometimes called the “power house” of the cell because of their role in energy production. Mitcohondria also have their own DNA unique from that which is found in the nucleus. Proteins of interest found in the mitochondria would be those involved in the conversion of organic materials into ATP through oxidative phosphorylation (Bradfield et al, 2001). Nevertheless, when the total amount of protein was calculated F1 contained the highest, followed by F5, F2, F4 and F3. The total amount of protein is the quantity of protein present in a fraction which is obtained by determining the protein concentration of a part of each fraction and multiplying by the fraction's total volume. The total volumes for the homogenate and soluble fraction were very high (760mL and 535mL respectively) hence the total amount of protein was the highest in these fractions. The % total recovery was obtained by taking the total protein and dividing by the homogenate (F1). Hence, F1 had the highest % recovery of 100% followed by F5, F2, F4 and F3. This simply meant that F1 (homogenate) recovered all of the proteins from the original extract whereas F3 (lysosomes) recovered the least amount of proteins from the original extract.

As can be seen in Table 4, as the volume of standard phosphate solution increased, the amount of phosphate increased thus leading to the increase in the absorbance values at 650nm from tubes 1-7. This occurred since the electrons in the chemical bonds only absorb certain wavelengths of light thus, the more concentration of protein, more bonds and more electrons to absorb the light. However, tubes 4 &5 had similar absorbance values since the exact amount of protein were placed in those tubes. Using Table 4, a calibration curve of absorbance at 650nm against µmol of phosphate was plotted as seen in Graph 2. This was used to find the unknown concentration in the various fractions.

As can be seen in Table 5, the average absorbance at 650nm for the assay of glucose-6-phosphatase was the highest in F4, followed by F3, F1, F2 then F5. As such when the corrected absorbance was calculated as seen in Table 6, the same trend was seen for both the absorbances as well as the phosphate content. This was because Glucose-6-phosphatase is an enzyme that biocatalyzes the hydrolysis of glucose-6-phosphate to glucose and phosphate. The inorganic phosphate released is estimated to determine the enzyme activity (Baltimore et al, 2007). As such, this enzyme is a marker enzyme for the cell fraction containing endoplasmic reticulum i.e. microsomes. As seen in Table 7, the same trend occurred for the enzyme activity of glucose-6-phosphatase i.e. F4 having the highest value of 2.4U/mL and F5 having the least value of 1.22U/mL. The specific activity for each of the cell fractions was calculated. Specific activity is expressed in units of enzyme per milligram protein (un/mg). During the differential centrifugation, proteins are separated. Some go into the pellet and some stay in the supernatant. Therefore, if the enzyme stays in the supernatant and many other proteins pellet out, the enzyme will represent a higher percentage of the protein in the supernatant than before it was centrifuged. However, if the enzyme activity is measured in units per ml of supernatant (unit/ml), the activity would stay the same, since the volume of the supernatant that enzyme is suspended in hasn't changed. Similarly, if the enzyme went to the pellet, its activity expressed in units/ml would be dependent on the volume that the pellet is resuspended in before doing the assay, and not on any enrichment created by the centrifugation (Baltimore et al, 2007). For this reason, both the enzyme activity in a fraction and its protein concentration are measured and then the specific activity of enzyme is calculated by dividing by the concentration of protein in that fraction to give units/mg protein. Specific activity is defined in terms of enzyme activity per mg protein in the sample (i.e. specific activity relates the enzyme units to the amount of protein in the sample). Purification results when the ratio of the amount of a particular protein to the total amount of protein present in the sample is increased. Thus, the higher the proportion of a particular enzyme in a sample, the higher the specific activity, and the more ‘pure’ the fraction is. From Table 7 it can be seen that F3 had the highest value for specific activity, followed by F4, F1, F5 then F2. This meant that F3 had the highest proportion of glucose-6-phosphatase and was the most ‘pure’ fraction whereas F2 had the lowest proportion of glucose-6-phosphatase and was the least ‘pure’ fraction. This was due to the low protein content found in F3 and the high protein content found in F2, thus affecting the specific activity values. Theoretically, the homogenate fraction should have the highest rate of enzyme activity but the lowest specific activity because the homogenate contains all the components of the cell thus more enzymatic activity occurs in this fraction (Dunn, 1992).

The total activity of the enzyme was found to be the highest in F1 followed by F5, F4, F3 then F2 due to the different volumes of each fraction. However, the %total activity of the enzyme was found to be the highest in F1 with a value of 101% which was taken to be 100% and the lowest in F2 with a value of 14.41%. The same trend was seen for the %total recovery i.e. the highest in F1 followed by F5, F4, F3 then F2. This simply meant that F1 had the highest enzyme activity and recovered all of the activity of the enzyme whereas F2 had the least enzyme activity and recovered least of the activity of the enzyme.

As can be seen in Table 8, as the volume of the p-nitrophenol solution increased, the absorbance values at 405nm also increased from tubes 1-7. This occurred since the electrons in the chemical bonds only absorb certain wavelengths of light thus, the more concentration of protein, more bonds and more electrons to absorb the light. However, tubes 4 &5 had similar absorbance values since the exact amount of protein were placed in those tubes. Using Table 8, a calibration curve of absorbance at 405nm against µmol of p-nitrophenol was plotted as seen in Graph 3. This was used to find the unknown concentration in the various fractions as seen in Table 9.

Acid phosphatase acts upon organic phosphate esters, releasing inorganic phosphate at a pH between 4 and 6. The subcellular fraction containing lysosomes is rich in this enzyme. In this assay a colorless compound, para-nitrophenol phosphate (pNPP) is used as the substrate for acid phosphatase. When the phosphate group of pNPP is cleaved, para-nitrophenol is generated (Cox and Nelson, 2008). Para-nitrophenol is a yellow compound that is easily measured in a spectrophotometer at 405nm. As can be seen in Table 9, the content of p-nitrophenol was found to be the highest in F1, followed by F2, F4, F5 then F3. This meant that the homogenate (F1) contained the highest amount of the lysosomal enzyme acid phosphatase and the lysosomal fraction contained the least. This was not to occur as the subcellular fraction containing lysosomes is rich in this enzyme. This high value in the homogenate and the other fractions could have been due to contamination of the homogenate and other fractions with lysosomes (or lysis of lysosomes and release of their enzymes into the homogenate and other fractions) had occurred. As such, the enzyme activity took the same trend as seen in Table 10. The activity of each organelle fraction can be influenced by a variety of factors: diet, sex, age and extent of fasting of the host animal, and also by the experimental method used to obtain the fraction (Hames and Hooper, 2005).

However, the specific activity of the enzyme was found to be the highest in F1, followed by F4, the same in F2&F5, followed by the fraction contain lysosomes (with the detergent) then F3 (lysosomal fraction without detergent). This meant that F1 had the highest proportion of glucose-6-phosphatase and was the most ‘pure’ fraction whereas F3 had the lowest proportion of glucose-6-phosphatase and was the least ‘pure’ fraction. This could have been due to contamination as mentioned above.

The total activity of the enzyme was found to be the highest in F1 followed by F5, F2, F4, F3 then the lysosomal fraction with the detergent due to the different volumes of each fraction. As such, the %total activity of the enzyme was found to follow the same trend i.e. highest in F1 with a value of 161.5% which was taken to be 100%. The % total recovery also followed this same trend. This simply meant that F1 had the highest enzyme activity and recovered all of the activity of the enzyme whereas the fraction with the detergent had the least enzyme activity and recovered least of the activity of the enzyme.

If lysosomes are damaged, the enzymes are released and can then be assayed in the medium. The degree of damage can then be determined by measuring the soluble or free activity and comparing it with the activity determined after deliberately rupturing the membrane with a detergent (e.g. Triton X-100)(total activity). The difference between the two values is the latent

activity which was found to be -2.4. This very low value was due to contamination errors as mentioned above and also experimental errors such as the tubes were left longer than the stated incubated time thus altering the latent activity of the lysosomal fraction. The Triton X-100 was used as a detergent in cell lysis (i.e. to release soluble proteins).

As can be seen in Tables 11 & 12, the absorbance values for glutamate and lactate dehydrogenase activity in different fractions were seen to gradually decrease over a 4 minute time frame. One explanation of this observation is that the settling of the substrate to the bottom of the test tube caused the enzyme to become less efficient since it could not attack the substrate as well. The settling reduced the surface area of the substrate that could be attacked by the enzyme thus the gradual decrease in absorbance values.

Glutamate dehydrogenase is a mitochondrial enzyme that catalyzes the reversible oxidative deamination of glutamate to -ketoglutarate (2-oxoglutarate) using NAD+ as coenzyme (Baltimore et al, 2007). As such, this enzyme is a marker enzyme for the fraction containing mitochondria. As seen in Table 13, the change in absorbance/min for glutamate dehydrogenase was found to be the highest in F2, followed by F1, F4, F3 then F5. This meant that the mitochondria (F2) contained the highest amount of the mitochondrial enzyme glutamate dehydrogenase and the soluble fraction (F5) contained the least. This was to occur as the subcellular fraction containing mitochondria is rich in this enzyme. The enzyme activity took the same trend as seen in Table 14. However, the specific activity of the enzyme was found to be the highest in F2, followed by F3, F1, F4 then F5. This meant that F2 (mitochondria) had the highest proportion of glutamate dehydrogenase and was the most ‘pure’ fraction whereas F5 had the lowest proportion of glutamate dehydrogenase and was the least ‘pure’ fraction. The total activity of the enzyme was found to be the highest in F1 followed by F2, F5, F4 and F3. As such, the %total activity of the enzyme was found to follow the same trend i.e. highest in F1 with a value of 130.4% which was taken to be 100%. The % total recovery also followed this same trend. This simply meant that F1 had the highest enzyme activity and recovered all of the activity of the enzyme whereas the fraction with the lysosomes (F3) had the least enzyme activity and recovered least of the activity of the enzyme.

Lactate dehydrogenase catalyzes the reversible reduction of pyruvate to lactate with NADH as the coenzyme (Baltimore et al, 2007). This enzyme is present in the soluble fraction (cytosol). As seen in Table 13, the change in absorbance/min for lactate dehydrogenase was found to be the highest in F5, followed by F1, F4, F2 then F3. This meant that the soluble fraction (F5) contained the highest amount of the cytosolic enzyme lactate dehydrogenase and the lysosomes (F3) contained the least. This was to occur as the soluble fraction is rich in this enzyme. The enzyme activity took the same trend as seen in Table 15. However, the specific activity of the enzyme was found to be the highest in F5, followed by F1, F3, F4 then F2. This meant that F5 (soluble fraction) had the highest proportion of lactate dehydrogenase and was the most ‘pure’ fraction whereas F2 had the lowest proportion of lactate dehydrogenase and was the least ‘pure’ fraction. The total activity of the enzyme was found to be the highest in F1 followed

by F5, F4, F2 and F3 due to the different volumes used (i.e. 760ml and 535ml for F1 and F5 respectively). As such, the %total activity of the enzyme was found to follow the same trend i.e. highest in F1 with a value of 95.6%. The % total recovery also followed this same trend. This simply meant that F1 had the highest enzyme activity and recovered most of the activity of the enzyme whereas the fraction with the lysosomes (F3) had the least enzyme activity and recovered least of the activity of the enzyme.

As can be seen in Table 16, as the volume of NT solution increased, the amount of NT increased thus leading to the increase in the absorbance values at 540nm from tubes 1-10. This occurred since the electrons in the chemical bonds only absorb certain wavelengths of light thus, the more concentration of NT, more bonds and more electrons to absorb the light. However, tubes 3&4 and tubes 7&8 had similar absorbance values since the exact amount of NT were placed in those tubes. Using Table 16, a calibration curve of absorbance at 540nm against µmol of NT was plotted as seen in Graph 4. This was used to find the unknown concentration in the various fractions.The enzyme succinate dehydrogenase (SDH) is an inner mitochondrial membrane protein involved in the Krebs cycle. It converts succinate to fumarate by oxidation, passing 2 electrons from succinate to FADH2 (Cox and Nelson, 2008). The subcellular fraction exhibiting this enzyme activity indicates the presence of mitochondria in the sample. As can be seen in Table 17, the highest absorbance values and NT content was found in F2, followed by F5, F3, F1 then F4. This meant that the mitochondrial fraction (F2) contained the highest amount of the mitochondrial enzyme succinate dehydrogenase and the microsomal fraction (F4) contained the least. This was to occur as the soluble fraction is rich in this enzyme. The enzyme activity took the same trend as seen in Table 18. However, the specific activity of the enzyme was found to be the highest in F2, followed by F3, F5, F4 then F1. This meant that F2 (mitochondria) had the highest proportion of succinate dehydrogenase and was the most ‘pure’ fraction whereas F1 had the lowest proportion of succinate dehydrogenase and was the least ‘pure’ fraction. The total activity of the enzyme was found to be the highest in F5 followed by F1, F2, F3 and F4 due to the different volumes used (i.e. 760ml and 535ml for F1 and F5 respectively). As such, the %total activity of the enzyme was found to follow the same trend i.e. highest in F5 with a value of 40.9%. The % total recovery also followed this same trend. This simply meant that F5 had the highest enzyme activity and recovered most of the activity of the enzyme whereas the fraction with the microsomes (F4) had the least enzyme activity and recovered least of the activity of the enzyme. As such it could be determined that succinate dehydrogenase was found in the mitochondria. Perioxisomes are likely to be found in the mitochondrial fraction as both the perioxisomes and mitochondria display certain morphological and functional similarities.

In this experiment, the tissue samples were kept on the ice so as to ensure the enzymes and proteins in the specific fractions do not become degraded as the ice inhibits nuclease or protease enzyme activity. Buffers were used in the assay to maintain the stable pH such as the Tris buffer used for removal of unwanted cellular components and precipitation (during cell lysis). The homogenization buffer included sucrose so as to partially dehydrate the organelles

keeping them intact. 0.25M sucrose was used to dilute the fractions since at 0.25 M, sucrose iso-osmotic as such, not much damage is done to sub-cellular fractions. Also, the 10% TCA solution was used so as to terminate the reaction.

Some precautions taken in this lab experiment were: 1) Ensure that readings were taken at eye level so as to avoid experimental errors. 2) Ensure that the tubes were rinsed properly with distilled water so as to avoid contamination. 3) Ensure that the entire fraction was not diluted but rather a predetermined aliquot based on the volume of diluted fraction needed for the assay. Some sources of error that may have occurred were: 1) Cuvettes may have been scratched, or have had fingerprints on them, despite efforts to keep them clean and so, this would affect the absorbance readings. 2) The enzyme assay was not incubated for the indicated time thus giving inaccurate absorbance readings.

References:

1) Alberts, B., Roberts, K. and Walter, P. 2006. Molecular Biology of the Cell. Garland Science, New York.

2) Baltimore, D., Berk, H., Darnell, J., Lodish, H., Matsudiara, P. and Zipersky, L. 2007. Molecular Cell Biology. W.H. Freeman, New York.

3) Bradfield, P. and Dodds, J. and Taylor, N. 2001. AS Level Biology. Pearson Educational Limited, London.

4) Champe, P., Ferrier, D. and Harvey, R. 2004. Lippincott’s Illustrated Reviews: Biochemistry. Lippincott Williams and Wilkins, Baltimore.

5) Cox, M. and Nelson, D. 2008. Lehninger Principles of Biochemistry. W.H. Freeman and Company, USA.

6) Dunn, M.J. 1992. Protein Determination of Total Protein Concentration. IRL Press, Oxford.

7) Elle, W. and Rolfes, S. 2008. Understanding Nutrition. 11th Edition. Wadsworth Publishing, California.

8) Hames, D. and Hooper, N. 2005. Instant Notes Biochemistry. 3rd Edition. Taylor and Francis Group, New York.

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