Lecture 2 - Muscle Lactate Dehydrogenase Isolation

Outline of the Experiment

• Day 1: Experiment 1A: Buffer and Column Preparation

  • Buffer preparation.

  • Pour a 10 mL column of Sephadex G-25 column.

  • Pour a 3-5 mL column of Cibacron-Blue Agarose column.

• Day 2: Experiment 1B: LDH Extraction and Precipitation

  • Homogenize fresh chicken breast tissue.

  • 40% Ammonium Sulfate Precipitation

  • A lot of “junk” goes into this pellet.

  • The LDH is in the supernatant.

  • 60% Ammonium Sulfate Precipitation

  • A lot of “junk” stays in the supernatant.

  • The LDH goes into this pellet.

• Day 3: Experiment 1C: Pseudo-Affinity Chromatography

  • Resuspend the ammonium sulfate pellet in buffer.

  • Desalt the sample using a G-25 column.

  • Chromatograph the protein on a Cibacron-Blue Agarose column.

Day 2: Homogenization and Precipitation of LDH from Chicken Breast Muscle

• Preparation of Extraction Buffer (100 mL)

  • Add phenylmethylsulfonyl fluoride (PMSF) and 1 mM 2-mercaptoethanol (BME).

  • Hormogenize and centrifuge to remove debris.

  • Filter through cheese cloth to remove fat.

  • Save aliquots in Eppendorf tubes for later PAGE and activity assays.

• Ammonium Sulfate Fractionation/Precipitation

  • Add AS to 40% of saturation.

  • Discard the pellet.

  • Add AS to supernatant to 60% of saturation.

  • Centrifuge and keep the pellet where the LDH is and flash freeze it.

  • Keep the other aliquots frozen for later assays.

Methods for Cell Disruption

• Various methods are available for the rapid disruption of cells and tissues, which is important for preventing degradation of proteins.

  • Hand-operated homogenizers

  • Waring blender (motor-driven)

  • Ultrasound

  • Vibrating bead mill

  • Manton-Gaulin homogenizer

Differential Centrifugation

• Separation Process

  • Filter homogenate to remove clumps of unbroken cells, connective tissue, etc.

  • Centrifuge at 600 g for 10 minutes, pour out the supernatant.

  • Centrifuge at 15,000 g for 5 minutes, pour out the supernatant.

  • Centrifuge at 100,000 g for 60 minutes, pour out the supernatant.

  • Centrifuge at 300,000 g for 2 hours, pour out the supernatant.

• Resulting Fractions: Separates insoluble, small, and larger molecules, including proteins and various cellular structures.

  • Nuclei, mitochondria, chloroplasts, plasma membrane, lysosomes, peroxisomes, ribosomal subunits, small polyribosomes, soluble cytoplasm (cytosol).

Role of Reducing Agents

• 2-Mercaptoethanol (BME)

  • Low concentrations stabilize native protein conformation by reducing S-S bonds.

  • High concentrations can denature proteins by breaking essential S-S bonds.

  • Utilization of low concentrations in buffer.

Use of PMSF in Protein Purification

• Phenylmethylsulfonyl Fluoride (PMSF)

  • Specific serine protease inhibitor.

  • Rapidly degraded in water; stock solutions are made in anhydrous ethanol, isopropanol, or DMSO.

  • Effective concentration range: 0.1 - 1 mM for proteolytic inhibition.

• Mechanism of Action

  • PMSF binds covalently to the active site serine residue in serine proteases.

  • This binding prevents the protease from catalyzing reactions.

• Chemical Reaction

  • $ext{Enzyme(active)Ser-O-H} + ext{F-SO}<em>2 ext{CH}</em>2 ext{C}<em>6 ext{H}</em>5 <br>ightarrow ext{EnzymeSer-O-SO}<em>2 ext{CH}</em>2 ext{C}<em>6 ext{H}</em>5 + ext{HF}$

• Comparison with EDTA

  • EDTA inactivates proteases that require metal cofactors by chelating metal ions.

Ammonium Sulfate Precipitation Mechanism

• Role of Ammonium Sulfate

  • Acts as anti-chaotropic or kosmotropic salt.

  • Affects water structure by removing structured waters around hydrophobic protein patches.

  • Promotes hydrophobic interactions leading to protein aggregation and precipitation.

  • Stabilizes protein structure, minimizing denaturation.

  • Reduces protein solubility through shielding charged groups, allowing proteins to aggregate.

Effects of Ammonium Sulfate on Protein Solubility

• Salting-In Effect

  • At low concentrations, increased salt concentration leads to enhanced protein solubility.

• Salting-Out Effect

  • At higher concentrations, solubility significantly decreases with increasing salt concentration.

  • This results in protein precipitation.

• Purification Method

  • Ammonium sulfate precipitation is a method for crude purification and concentration of protein extracts.

Applications of G-25 Sephadex

• Use of G-25 Sephadex

  • Fast removal of salt from protein solutions post-ammonium sulfate precipitation.

  • Structure: Small cross-linked dextran beads used in gel filtration and ion exchange chromatography.

  • Cross-linking degree alters pore size, affecting molecule separation based on size.

• Separation Mechanism

  • Larger molecules are excluded from pores and exit first.

  • Intermediate molecules enter some pores and exit in the middle.

  • Smaller molecules aggregate and must navigate through all pores, exiting last.

Size Exclusion Chromatography (SEC)

• Column Chromatography Process

  • Proteins in solution applied to the top of a column filled with a permeable matrix immersed in solvent.

  • A solvent is pumped through the column: different proteins are separated based on interactions with the matrix.

Column Parameters for SEC

• Parameters

  • $V_o = ext{Void Volume}$

  • $V_t = ext{Total Volume}$

  • $V<em>e = V</em>o + K<em>{av}(V</em>t - V_o)$

  • $K_{av} = ext{Proportion of pores available to the molecule}$

Fractionation Range of G-25 Sephadex

• Fractionation Ranges

  • G-10: ≤700 Da

  • G-15: ≤1500 Da

  • G-25: 1000–5000 Da

  • G-50: 1500–30,000 Da

  • G-75: 3000–80,000 Da

  • G-100: 4000–150,000 Da

  • G-150: 5000–300,000 Da

  • G-200: 5000–600,000 Da

Purification of Chicken LDH by Affinity Chromatography

• Desalting Step

  • G-25 column to remove ammonium sulfate.

• Affinity Chromatography Steps

  • Run the desalted protein solution over Cibacron Blue column (LDH binds).

  • Wash with NAD+ (weakly binding proteins removed).

  • Elution with NADH to release LDH.

Cibacron Blue Agarose in Protein Purification

• Mimicking Nucleotide Structure

  • Cibacron Blue moiety mimics NAD+ and ATP structures, binding to nucleotide-binding sites on enzymes like LDH.

• Procedure for Cibacron-Blue Chromatography

  • Collect 5 mL fractions, label them.

  • Steps include loading, rinsing, washing with NAD+, and elution with NADH, all while reading A280 to monitor protein concentration.

Lecture Objectives / Questions

• 1. Define LDH and illustrate the reaction it catalyzes. Explain differences between muscle and liver LDH functions.

• 2. State purposes of Tris-HCl, β-mercaptoethanol, PMSF, and EDTA during protein purification.

• 3. Describe β-mercaptoethanol's chemical structure and disulfide bond reduction.

• 4. Explain the need for fast cell disruption in protein purification.

• 5. Define anti-chaotropic salt and its laboratory applications.

• 6. Compare salting-in and salting-out effects.

• 7. Describe ammonium sulfate precipitation mechanisms: salting-in and salting-out effects. Address rationale for 40% and 60% precipitations.

• 8. Discuss the principles of gel filtration chromatography and its applications in protein separation.

• 9. Elaborate on the fractionation range concept for Sephadex resins, specifically G-25, and its use in desalting.

10. Explain the rationale for using a Cibacron-Blue Agarose column in protein purification.

11. Summarize each step in the Cibacron-Blue chromatography process, including buffer components.

12. Clarify the selective elution of LDH by NADH versus NAD+.