Experiment 04Biochemical Cytology • B.Sc. / M.Sc.
Subcellular Fractionation & Differential Centrifugation of Liver/Plant Tissue
🎯 Aim: To isolate crude nuclear, mitochondrial, and post-mitochondrial cytosolic fractions from mammalian liver or cauliflower inflorescence homogenate by differential centrifugation, and verify fraction purity through the biochemical spectrophotometric assay of the mitochondrial marker enzyme Succinate Dehydrogenase (SDH).
🔬 Theoretical Principle & Centrifugal Mechanics
Subcellular fractionation enables isolation of intact, functionally active organelles based on differences in their physical size, mass, shape, and buoyant density. In a centrifugal gravitational field, particles sediment through a liquid medium at a sedimentation velocity ($v$) governed by Svedberg’s sedimentation law:
v = frac{d^2 (rho_p – rho_m) cdot g}{18,eta}Where $d = text{particle diameter}$, $rho_p = text{particle density}$, $rho_m = text{medium density}$, $eta = text{medium viscosity}$, and $g = text{centrifugal acceleration}$.
Because sedimentation velocity scales with the square of particle diameter ($d^2$), larger, denser organelles pellet at lower centrifugal fields ($RCF$), while smaller organelles remain in the supernatant:
- Nuclear & Cellular Debris Fraction: Pelleted at $600 – 1,000 times g$ for 10 minutes.
- Mitochondrial (& Lysosomal) Fraction: Pelleted at $10,000 – 15,000 times g$ for 15 minutes.
- Microsomal (ER & Golgi Vesicles) Fraction: Pelleted at $100,000 times g$ for 60 minutes.
- Cytosol Supernatant: Soluble cytoplasmic enzymes and proteins.
Succinate Dehydrogenase (SDH, Complex II) is an integral protein exclusively localized to the inner mitochondrial membrane. In this assay, SDH oxidizes sodium succinate to fumarate, coupling electron transfer via flavin adenine dinucleotide (FAD) to artificial electron acceptors (Phenazine Methosulfate, PMS, and 2,6-Dichlorophenolindophenol, DCPIP). Reduced DCPIP turns from deep blue to colorless ($Delta A_{600}$ measured spectrophotometrically), providing a quantitative readout of mitochondrial recovery and enzyme specific activity.
Figure 4.1: Differential Centrifugation Flowchart & SDH Purity Assay
🧪 Reagents, Buffers & Equipment
Homogenization & Assay Buffers
- Isolation Buffer (pH 7.4): 0.25 M Sucrose (isotonic), 10 mM Tris-HCl, 1 mM EDTA (chelator), 0.1 mM PMSF (protease inhibitor).
- Sodium Succinate Substrate: 0.2 M in $50,text{mM}$ sodium phosphate buffer (pH 7.4).
- DCPIP Solution: 0.1 mM 2,6-dichlorophenolindophenol in water.
- PMS Solution: 1 mM Phenazine methosulfate (electron mediator).
- Sodium Azide ($text{NaN}_3$): 0.1 M (inhibits cytochrome c oxidase / Complex IV).
Instruments & Consumables
- Refrigerated high-speed centrifuge with fixed-angle rotor ($4^circtext{C}$)
- Motorized Potter-Elvehjem Teflon-glass tissue homogenizer
- UV-Visible double-beam spectrophotometer ($600,text{nm}$) & 1 cm quartz cuvettes
- Fresh goat/rat liver or cauliflower inflorescence, crushed ice bath
📋 Step-by-Step Fractionation Protocol
- Pre-Chilling & Dissection: Pre-chill all buffers, glassware, centrifuge tubes, and homogenizer barrels on ice ($0–4^circtext{C}$). Excise 5.0 g fresh liver, wash repeatedly in ice-cold isolation buffer to remove blood, blot dry, and finely mince with scissors.
- Homogenization: Suspend 5.0 g tissue in 20 mL ice-cold isolation buffer (1:4 w/v). Transfer to Potter-Elvehjem homogenizer. Perform 6–8 smooth vertical strokes at 1000 RPM while keeping the vessel immersed in ice bath to prevent heat denaturation of enzymes.
- Filtering: Filter crude homogenate through two layers of sterile cheesecloth into an ice-cold beaker to remove connective tissue and unbroken tissue blocks. Save 1.0 mL aliquot as ‘Crude Homogenate’.
- Nuclear Fractionation ($1000 times g$): Centrifuge homogenate at $1,000 times g$ ($3,000,text{RPM}$) for 10 minutes at 4°C. Decant the supernatant ($S_1$) into a fresh chilled tube. Resuspend the pellet ($P_1$) in 5 mL isolation buffer as the ‘Nuclear Fraction’.
- Mitochondrial Fractionation ($12,000 times g$): Centrifuge the supernatant ($S_1$) at $12,000 times g$ ($11,000,text{RPM}$) for 15 minutes at 4°C. Decant the supernatant ($S_2$) as the ‘Post-Mitochondrial Cytosolic Fraction’. The tan-brown pellet ($P_2$) represents the ‘Mitochondrial Fraction’.
- Mitochondrial Washing & Resuspension: Gently wash the surface of pellet $P_2$ with 1 mL buffer to dislodge fluffy microsomal debris. Resuspend pellet in 3.0 mL isolation buffer on ice.
- SDH Enzymatic Assay: Set up spectrophotometer at 600 nm. Prepare assay cuvette: 2.3 mL phosphate buffer (50 mM, pH 7.4), 0.2 mL 0.1 M $text{NaN}_3$, 0.2 mL 0.1 mM DCPIP, 0.1 mL 1 mM PMS, and 0.1 mL enzyme fraction. Add 0.1 mL 0.2 M sodium succinate to start reaction. Record absorbance at 600 nm every 30 seconds for 5 minutes.
📊 Representative Enzyme Activity & Subcellular Distribution Table
| Subcellular Fraction | Total Volume (mL) | Protein Conc. (mg/mL) | $Delta A_{600}$ / min | Total SDH Units ($mutext{mol/min}$) | Specific Activity ($text{Units/mg}$) | Enrichment (Fold) |
|---|---|---|---|---|---|---|
| Crude Homogenate | 20.0 | 18.5 | 0.082 | 154.2 | 0.417 | 1.0× (Ref) |
| Nuclear Pellet ($P_1$) | 5.0 | 9.2 | 0.018 | 8.4 | 0.183 | 0.44× |
| Mitochondrial Pellet ($P_2$) | 3.0 | 6.4 | 0.315 | 88.6 | 4.614 | 11.1× (Enriched) |
| Cytosolic Supernatant ($S_2$) | 16.5 | 8.8 | 0.003 | 4.6 | 0.032 | 0.08× |
⚠️ Good Laboratory Practice & Subcellular Integrity
- Maintain strict ice-bath conditions ($0–4^circtext{C}$) at every step: lysosomal hydrolases released during homogenization rapidly degrade mitochondrial membrane enzymes at higher temperatures.
- 0.25 M sucrose must be maintained; replacing it with pure water causes hypotonic osmotic shock, bursting mitochondria and releasing cytochrome c.
- Sodium azide ($text{NaN}_3$) is essential in the assay to poison terminal cytochrome c oxidase (Complex IV); otherwise, electrons from succinate bypass DCPIP and reduce oxygen to water.
💬 Viva-Voce Questions & Model Answers
Q1: Why is 0.25 M sucrose buffer used for subcellular fractionation instead of isotonic 0.9% NaCl?
0.25 M sucrose is non-ionic and provides an isotonic osmolarity ($approx 290 text{mOsm/L}$) that preserves organelle membrane integrity without causing charge-mediated organelle aggregation or chromatin precipitation, which occurs in high ionic strength salts like NaCl.
Q2: What is the biological function of marker enzymes in cell fractionation?
Marker enzymes are catalytically active proteins strictly localized to a specific organellar compartment. Assaying them quantitatively demonstrates recovery and purity of the target fraction (e.g. Succinate Dehydrogenase for mitochondria, Galactosyltransferase for Golgi, Acid Phosphatase for lysosomes, Lactate Dehydrogenase for cytoplasm).
Q3: How does relative centrifugal force (RCF) differ from revolutions per minute (RPM)?
RPM is the rotational speed of the rotor motor. RCF (expressed in units of $g$) represents the actual centrifugal acceleration experienced by the sample, which depends directly on rotor radius ($r$): $RCF = 1.118 times 10^{-5} times r times text{RPM}^2$. Two centrifuges operating at identical RPM will generate completely different $g$-forces if their rotor radii differ.
