LDH: Function, Extraction, and Methods

Overview of LDH and Its Role

• Lactate dehydrogenase (LDH) is crucial in lactate-pyruvate conversion, essential for homolactic fermentation.
• During homolactic fermentation, muscle tissues convert pyruvate to lactate.
• Gluconeogenesis also requires this conversion, where the liver converts lactate back to pyruvate for glucose synthesis.
• These pathways are key for energy production.

Extraction of LDH from Beef Heart

• LDH will be extracted from beef heart tissue.
• Releasing the enzyme from its cellular environment is the initial step in purification.

Methods Used for Cell Disruption

• Gentle methods will break open the cells, since beef heart cells lack cell walls.
• Homogenization involves grinding with a mortar and pestle.
• Osmotic lysis involves creating osmotic pressure differences.
• Ultrasonic vibration uses sound waves to disrupt cell membranes.
• Homogenization breaks connective and fatty tissue barriers gently, maintaining enzyme integrity.

Maintaining LDH Stability During Extraction

• Enzyme degradation occurs once released from cell protection.
• Oxidation of proteins is more likely outside the cellular environment.
• Proteolytic degradation happens when proteases break down proteins upon cell lysis.
• The extraction is carried out cold (0–4 °C) to prevent enzyme deactivation by slowing down reactions.
• Control of ionic strength, polarity, and pH balance are also key.

Buffer Composition for LDH Extraction

• The extraction buffer protects LDH from degradation.
• 50 mM Tris-HCl at pH 7.5 maintains pH stability.
• Ethylenediaminetetraacetic acid (EDTA) chelates calcium ions, which reduces protease activity.
• 2-Mercaptoethanol prevents oxidation of cysteine residues by acting as a reducing agent.
• These chemicals help stabilize LDH by reducing unwanted interactions outside the cellular environment.

Key Extraction Considerations

• Gentle cell disruption is required to extract LDH while maintaining its activity.
• Cold temperatures (0–4°C) is used to prevent oxidation and proteolytic degradation.
• Protective buffers containing EDTA and 2-Mercaptoethanol are used to ensure LDH remains functional.
• This process forms the basis for LDH purification.

Affinity Chromatography: A Targeted Protein Purification Approach

• Affinity chromatography isolates proteins through specific binding interactions.
• A ligand immobilized on a solid matrix is used to selectively capture the protein of interest.

Key Steps in Affinity Chromatography

• Binding Phase: The protein mixture is applied to the affinity column, and target proteins bind to the ligand while others pass through.
• Wash Step: Removes unbound proteins with a buffer.
• Elution Step: Releases the bound protein from the ligand using a specific elution buffer, like a high concentration of substrate or competitor molecules.
• This technique purifies LDH effectively because LDH has a natural affinity for specific substrates involved in its reaction.

Reaction Catalyzed by Lactate Dehydrogenase (LDH)

• LDH is vital in energy metabolism, specifically anaerobic glycolysis and gluconeogenesis.
• The enzyme facilitates the interconversion of lactate and pyruvate

LDH-Catalyzed Reaction

• Pyruvate + NADH + H⁺ converts to Lactate + NAD⁺
• LDH reduces pyruvate to lactate, regenerating NAD⁺, which is vital for glycolysis, to continue producing ATP under low oxygen conditions.

How Affinity Chromatography Helps Purify LDH

• Affinity chromatography columns can be functionalized with NADH and pyruvate due to their interaction with LDH.
• Specific LDH capture allows controlled elution using a competing ligand.

Key Chromatography Considerations

• Affinity chromatography selectively purifies LDH using specific ligand interactions.
• LDH catalyzes pyruvate-to-lactate conversion, essential in cellular respiration.
• NADH-dependent affinity columns enhance LDH purification efficiency.

Detection of LDH Activity: Measuring Enzyme Function

• Focus is confirming Lactate Dehydrogenase (LDH) is active by monitoring its enzyme-catalyzed reaction.

Key Reaction Monitored

• LDH catalyzes the oxidation of NADH to NAD⁺, a process that's crucial for measuring its activity.

Why NADH Absorbance is Useful

• NADH absorbs strongly at 340 nm, whereas NAD⁺ does not.
• This facilitates the monitoring of NADH to NAD⁺ conversion spectrophotometrically.
• A decrease in absorbance at 340 nm (A₃₄₀) is indicative of LDH activity.

Graph Interpretation: A₃₄₀ vs. Time

• A graph illustrating absorbance (A₃₄₀) decreasing over time indicates LDH catalysis of NADH oxidation.

How the Graph Works

• X-axis shows time.
• Y-axis displays A₃₄₀ (absorbance at 340 nm).
• The negative slope of the curve indicates the enzyme’s initial velocity, indicating how fast LDH converts NADH to NAD⁺.

Assay Conditions

• Substrate concentrations (pyruvate and NADH) are kept at non-limiting levels.
• This ensures the rate of NADH oxidation is proportional to enzyme concentration.

Enzyme Units: Expressing LDH Activity

• Standard Enzyme Unit (U): 1 unit (U) converts 1 µmol of NADH per minute under standard conditions.
• Katal (SI Unit): 1 katal converts 1 mole of substrate per second.
• Biochemists commonly use enzyme units (U), despite katal being the recommended SI unit.

Key Activity Considerations

• LDH activity is spotted by monitoring NADH oxidation at A₃₄₀.
• The absorbance graph's negative slope measures the reaction velocity.
• Enzyme activity is measured in units (U) or katals.

Protein Purity and Specific Activity

• The degree of purity of a protein is expressed in specific activity, measuring enzyme activity per mg of protein.
• Specific activity increases with each purification step, reaching a max when the protein is completely pure.
• Units of specific activity include U/mg (enzyme units per mg of protein) and µmol min⁻¹ mg⁻¹ (micromoles of substrate converted per minute per mg of protein).

Bradford Protein Assay

• Determining protein concentrations is done through the Bradford assay method.
• The Bradford assay is simple, quick, and relatively unaffected by contaminants, except detergents.

How It Works

• Coomassie Brilliant Blue G-250 dye is added to the protein solution.
• Interacts with proteins, causing a color shift from reddish to blue.
• Absorbance is measured at 595 nm, constructing a standard curve using bovine serum albumin (BSA).
• Determining the protein concentration of unknown samples happens by comparing to the A595 values to the standard curve.
• The Bradford assay gives a linear plot of absorbance vs. concentration within a limited range (typically 2–120 μg/mL of protein), with proper sample dilution needed for results.

Elution Profile and Enzyme Purification

• Included is an elution profile a β-N-acetyl-D-glucosaminidase purification from green crab viscera (Zhang et al., 2006).

Key Observations from the Profile

• Curve 1 (Total Protein) shows protein present in virtually every collected fraction.
• Curve 2 (Enzyme Activity) indicates enzyme activity eluted in a sharp peak over ~five fractions, partly purifying the target enzyme.
• Further purification steps (like DEAE-cellulose chromatography) were needed, since proteins with similar shape and size co-elute, achieving a high-purity enzyme preparation.

Key Protein Considerations

• Protein purity increases with purification steps, measured by specific activity.
• The Bradford Assay provides as a method of measuring protein concentration.
• Distint peaks will be present if a target enzyme is eluded, which becomes apparent with elution profiles, ideally separated from other proteins.

Affinity Chromatography for LDH Purification

• Affinity chromatography relies on specific ligand-protein interactions to selectively retain LDH while performing purification.
• Cibacron Blue 3GA is used because it's an analogue of NADH, which LDH binds naturally.
• Proteins that don't bind NADH will pass through the column without being retained.

Elution strategy

• Crude LDH Extract is loaded onto the Cibacron Blue 3GA column.
• Non-NADH binding proteins elute during the Wash Step.
• Remaining contaminants are followed by a buffer containing free NADH, during the Elution Step.
• LDH releases Cibacron Blue to elute from the column using the free NADH.
• LDH is highly enriched by this method, compared to standard ion-exchange techniques.

Elution Profile for Enzyme Purification

• Included is a graph of elution profiles: β-N-acetyl-D-glucosaminidase purification using Sephadex G-100 gel filtration chromatography.

What the Graph Shows

• Curve 1 (Total Protein Concentration) represents all protein fractions collected.
• Displays enzyme activity peaks which corresponds to where the target enzyme eluted (Curve 2)

Recovery and Purification Factors

• Total enzyme recovered (compared to initial amounts in crude extract) and purification fold (increase in enzyme purity across steps) are two parameters determined by biochemists, in addition to elution profiles.
• Beyond basic chromatography steps, purification efficiency will get assessed.

Key Profile Considerations

• Cibacron Blue 3GA affinity chromatography selectively purifies LDH by targeting NADH-binding proteins.
• When the enzyme separates from contaminants the elution profiles visually track purification progress.
• Recovery and purification calculations is a method by which to quantify purification efficiency beyond chromatography data.

Experiment 3, Week 1: Extraction and Purification of Lactate Dehydrogenase (LDH)

• There will be an affinity chromatography extracting and purifying LDH using beef heart.
• LDH plays a role in metabolism because it it converts lactate to pyruvate.

Objective

• The goal is to extract LDH from beef heart tissue efficiently by maintaining enzyme activity through careful conditions.
• The purification will be performed using Cibacron Blue 3GA affinity chromatography, since LDH naturally binds to NADH.

Chemicals & Reagents

• For extraction a precise buffer compositions is necessary to stabilize LDH and prevent degradation:
• Key reagents include Tris-HCl buffer (0.05 M, pH 7.5), which maintains enzyme stability.
• Ethylenediaminetetraacetic acid (EDTA, 1 mM) chelates metal ions, which will reduce unwanted protein interactions.
• 2-Mercaptoethanol (1 mM) acts as a reducing agent, preventing oxidation.
• Identical to the Tris-HCl buffer Elution Buffer, but has supplementation with 2 mM NADH, to support LDH recovery.
• Thimerosal (0.02%) helps preserves enzyme integrity.
• Phosphate Buffer (0.052 M, pH 7.5) is used in the enzyme activity assay with the help of 0.103 mM NADH and 2.07 mM pyruvate to measure LDH-catalyzed conversion.

Equipment Used

• To extract and analyze LDH efficiently, the following tools are required: Cold Mortar & Pestle + Acid-Washed Sand for gentle tissue homogenization.
• Separating cellular debris from soluble LDH Beckman Coulter Allegra 64R Centrifuge (F1010 Rotor).
• Cibacron Blue 3GA Affinity Column (2.5 mL Bed Volume) for binding LDH for purification.
• Jenway 6320D Spectrophotometer can measure enzyme activity by tracking NADH oxidation at 340 nm.
• Sample handing requires the use of Plastic Cuvettes & Pasteur Pipettes.
• Controlled handling and temperature maintenance demands Micropipettes & Ice.

Key Experiment Considerations

• LDH purification depends on specific ligand interactions with Cibacron Blue 3GA.
• The buffer prevents enzyme oxidation and proteolysis.
• Affinity chromatography isolates LDH with high specificity.
• Accurate extraction calculations need precise tissue weight measurement ("0.822g").

Method: LDH Extraction via Tissue Homogenization

• The step-by-step protocol, for isolating LDH from beef or chicken heart tissue has just been prescribed.

Key Considerations Before Extraction

• All preparations should be kept cold.
• For enzyme integrity and to preserve LDH activity the mortar and pestle were pre-chilled.
• Buffers are to be retained on ice throughout the procedure.

Step-by-Step Extraction

• Selecting & Preparing Tissue has two steps:
• Weigh 0.9 g of beef/chicken heart tissue documenting exact weight.
• Avoid connective or fatty tissue to avoid homogenization and little LDH.

Homogenization & Centrifugation

• Bring 7 mL of cold Tris-HCl buffer to the workstation.
• Grind the tissue in a cold mortar with buffer including acid-washed sand if needed.
• Add remaining buffer with the transfer of homogenate to a centrifuge tube rinsing.
• Balance centrifuge against other the other or another water-filled tube followed up Centrifugation (20,000 rpm, 4°C, 5min).
• Lastly, the Supernatant is decanted into a cylinder to record the volume.

Purification of LDH via Affinity Chromatography

• Affinity chromatography isolates LDH using Cibacron Blue 3GA, which mimics NADH, a natural LDH ligand.

Step-by-Step Purification

• Column equilibrate with 5 volumes of Tris-HCl.
• Exceeding will require extract to be removed before loading
• First, pipette 2.5mL into the column, collecting 1.5ml in seperate microfuge tubes, remembering not to dip below the resin bed with 14ml of HCL buffer.
• It must not run dry through the course of the wash & elution protocol, or it will skew purity.

Key chromatography Takeaways

• Maintaing enzyme activity will require a precise execution during the preparation.
• Achieving the correct LDH isolation happens when you isolate selectively.
• Don't let the column run dry at any point, and keep it consistant throughout the process.

Main Steps

• Increase liquid flow with air pressure through the blue bulb.
• Ensure that 1.5mL is collected properly as buffer flows in the correct mirco tubes and labeled according.
• For elution purposes 15mil is added as a elution buffer contaning 2mM NADH.
• Add 1.5 mirco liters and label, while the others are being stored for future purposes.
• Finally 2.5mL thimerosal solution as a biochemical preservative.

Spectrophotometer Setup

• Use Cuvettes of 3mL for activity solution with the proper temperature, with the appropriate blank reference of wash buffer to calibrate.

Reaction Readings

• By placing a curvente, add solution to calibrate, with 340mm readings every 10 seconds in less than a minute.

Fraction label and enzyme stability

• Proper recording is the best practice for success.
• By rerunning the crude later and adjusting the reaction, may help to extract more reaction as an attempt to maintain reaction.
• Label, and add any notes to prevent delays when preparing the report.
• And prepare dilutions!

After Purification

• The fractions you get, store them in microfuge in appropriate tubes for further activity for the week after.

Analysis Takeaways

• Remember to dilute, if rerun required after first few steps.
• If the extracts are too high, be promptful to adjust.
• The correct enzyme equation is required to identify, or find further.

Objective Analysis

• When determining fractions, the main goal is to quanitfy.
• Remember to accurately quanitfy to assess when measuring fractions.

Quantification Bradford Assay

• Bradford quantification will stain reagent, protein molecules.

Bradford Assay Requirements

• The BSA is the proper measurement with the standard serum required.
• In order to prevent contamination, BSA solution will be needed to inhibit bacterial action.

Preparation Tips

• A 21-fold of extraction will give a reliable range.
• In the range of accurate samples, 11-fold should be applied.

Procedure Tips

• The solution mix should be a BSA 30 mirco, or 1mL Bradoford.
• The final step involves a meter to determine results.

Key procedure Takeaways

• A dilution is accurate for analysis, and samples require a specter read result.
• Follow linear and best practive.

Fraction tips for purity

• If the activity is present in samples, use the other sample for electrophoresis.
• Store this in your freezer for the next test.

Quantification Process

• Correct all factor and wash tests.
• Using your results to find protein concentrations.

Factors to keep in mind.

• When plotting fractions be aware of where to measure concentrations.
• This helps when LDH are present, as purity can affect outcome.

Tips and Reminders

• Purity balance on which best suits with enzymes.
• Equations are to quantify enrichment after all testing.

The final report

• Will include activity assays, recovery factors, all findings and conclusions.
• Most important graphing results to quantify and visualize the results.