proteins - lecture4

Proteins need to be purified in order to study 

Biological methods 

  1. Must have an assay 

  2. Means to quantify total protein 

  3. Homogenise the material 

Method one 

Column Chromatography

1. Selection of Medium/Resin

  • Choose resin based on desired separation properties (size, charge, hydrophobicity).

  • Resin contains porous beads with fixed diameters.

2. Column Preparation

  • Pack column with chosen resin, ensuring a settled and evenly packed bed.

3. Equilibration

  • Use a buffer matching separation conditions.

  • Helps prepare resin and stabilize column for sample loading.

4. Sample Loading

  • Apply sample to the top of the column.

  • Proteins interact with resin based on affinity.

5. Elution

  • Elute bound proteins using a gradient (salt concentration, pH, hydrophobicity).

  • Eluted proteins exit the column at different times.

Additional Details:

  • Color of resin: Typically white.

  • Texture: Slurry feels slippery due to pores in beads.

  • Pores: Have fixed diameters, proteins interact and diffuse in/out.

  • Time for diffusion varies based on substance affinity.

  • Reservoir contains solvent aiding elution.

  • Not all proteins are colored.

  • Fraction collector at the bottom collects eluted proteins.

  • Test collected proteins using acid (for example) for further analysis.


Ion Exchange Chromatography

Process Overview:

  1. Principle:

    • Separation based on charge differences of proteins or molecules.

  2. Medium/Resin:

    • Contains charged functional groups (anion/cation exchange) on the beads.

  3. Column Preparation:

    • Pack the column with the ion exchange resin.

  4. Equilibration:

    • Use a buffer to establish an appropriate ionic environment.

  5. Sample Loading:

    • Apply the sample containing mixed charged molecules.

  6. Binding:

    • Molecules bind based on charge interactions with the resin.

  7. Washing:

    • Remove unbound substances with buffer.

  8. Elution:

    • Alter ionic strength or pH to release bound molecules.

  9. Eluted Fractions:

    • Collected in tubes or fractions based on their elution profile.

Key Details:

  • Anion/Cation Exchange: Determines resin choice based on molecule charge.

  • Ion Interaction: Molecules bind to resin based on opposite charges.

  • Selectivity: Separates molecules based on charge differences.

  • Elution: Change in conditions (pH/ionic strength) for molecule release.

  • Fractions Collection: Different molecules elute at varying conditions.

Applications:

  • Purification of proteins, nucleic acids, and other charged biomolecules.

  • Separation of molecules with varying charges in a sample.

Size Exclusion Chromatography

Process Overview:

  1. Principle:

    • Separation based on differences in molecular size and shape.

  2. Medium/Resin:

    • Porous beads with specific pore sizes.

  3. Column Preparation:

    • Pack the column with the size exclusion resin.

  4. Equilibration:

    • Use a buffer matching separation conditions.

  5. Sample Loading:

    • Apply the sample containing molecules of various sizes.

  6. Separation:

    • Larger molecules pass through the column faster as they don't enter the pores, while smaller ones enter pores and take longer.

  7. Elution:

    • Collect molecules in order of their size as they elute out.

Key Details:

  • Pore Size Selection: Determines resin choice based on the range of molecule sizes to be separated.

  • Exclusion Effect: Large molecules elute first as they don't enter pores, smaller ones are delayed.

  • Separation Mechanism: Size-based separation without binding to the resin.

  • Elution Profile: Collection of molecules based on their size distribution.

  • Applications:

    • Purification of proteins, polysaccharides, nucleic acids based on size differences.

    • Analyzing molecular size and oligomeric state of proteins. 

Affinity Chromatography

Process Overview:

  1. Principle:

    • Separation based on specific interactions between a molecule of interest and a ligand immobilized on the resin.

  2. Medium/Resin:

    • Resin contains a ligand that binds selectively to the target molecule.

  3. Column Preparation:

    • Pack the column with the affinity resin containing the immobilized ligand.

  4. Equilibration:

    • Use a buffer matching the conditions favorable for binding.

  5. Sample Loading:

    • Apply the sample containing the mixture of molecules, including the target molecule.

  6. Binding:

    • Target molecule selectively binds to the immobilized ligand while non-specific molecules pass through.

  7. Washing:

    • Remove unbound molecules to ensure specificity.

  8. Elution:

    • Disrupt the binding between the target molecule and ligand, releasing the target molecule.

  9. Eluted Fractions:

    • Collected with the specifically bound target molecule.

Key Details:

  • Specific Interaction: Ligand on the resin interacts selectively with the target molecule.

  • High Selectivity: Enables highly specific isolation of the desired molecule.

  • Elution Condition: Changes (pH, ionic strength) disrupt binding for target molecule release.

  • Applications:

    • Purification of proteins, antibodies, enzymes, and other biomolecules.

    • Isolation of specific targets from complex mixtures with high purity.

gel electrophoresis is a widely used technique for separating biomolecules based on their size and charge. Sodium dodecyl sulfate (SDS) is often used in SDS-PAGE (polyacrylamide gel electrophoresis) to denature proteins and provide a uniform charge density.

SDS: Sodium Dodecyl Sulfate

  • Structure: Sodium dodecyl sulfate is a type of anionic detergent. Its structure includes a hydrophilic sulfate head and a long hydrophobic carbon chain (dodecyl group).

  • Hydrophilic Property: The sulfate group in SDS is highly hydrophilic, allowing it to interact strongly with water molecules.

  • Function: In SDS-PAGE, SDS binds to proteins, unfolding them and providing a negative charge proportional to their length.

Gel Electrophoresis:

  • Gel Composition: Gel is a polymerized matrix, forming a mesh-like structure.

  • Principle: Biomolecules are separated based on their size and charge as they move through the gel in response to an electric field.

  • Percentage of Gel: Determines the pore size, affecting the migration rate. Higher percentage gels have smaller pores, slowing larger molecules' movement.

  • Separation: Smaller molecules migrate more quickly through the gel than larger ones.

  • Enrichment: Sequential steps in the process aim to enrich the desired molecules and purify them from contaminants.

Activity and Yield:

  • Activity Increase: As you move through the steps, the concentration or activity of the desired biomolecule may increase.

  • Yield Limitation: Despite the enrichment, maintaining all the activity or concentration of the desired molecule might be challenging, leading to some loss during the purification process.

Gel electrophoresis is a powerful tool for separating biomolecules and enriching specific targets, but maintaining high yields throughout the purification process can be a challenge due to various factors involved in the procedure.

Isoelectric focusing 

 i) When pI = pH, the  electrophoretic mobility is  zero.  

ii) A stable pH gradient is  established beforehand ampholytes . 

 iii) Sample is added, then an  electric field is applied.  The  porosity of the matrix is  very large to allow max.  mobility.

Characterising purified proteins 

The edman degradation 

Sample Preparation:

The protein of interest is first purified and isolated from other components in the sample to obtain a relatively pure protein sample.

Derivatization of Amino Terminus:

The N-terminal amino acid of the protein is selectively reacted with a phenylisothiocyanate (PITC) reagent, specifically reacting with the amino group to form a phenylthiocarbamoyl (PTC) derivative.

Cleavage of Amino Acid:

The PTC-derivatized N-terminal amino acid is then cleaved off from the protein under mild anhydrous acidic conditions (usually 6 N hydrochloric acid) without affecting other amino acids.

Purification and Analysis:

The cleaved amino acid is then purified and analyzed using chromatographic techniques, such as high-performance liquid chromatography (HPLC), to identify the amino acid and determine its quantity.

Cycle Repeats:

The process is repeated for each cycle to determine the amino acid sequence step by step, starting from the N-terminus and proceeding towards the C-terminus.

By repeating these steps in a cyclic manner, researchers can determine the complete amino acid sequence of the protein. The Edman degradation method is accurate and efficient for sequencing peptides and small proteins.

However, there are limitations to Edman degradation:

The method is most effective for peptides and small proteins (up to about 50-70 amino acids in length).

Over extended cycles, accumulation of errors can occur due to side reactions or incomplete reactions, making it less accurate for longer proteins.

Complex proteins or proteins with post-translational modifications can present challenges for this technique.

In modern proteomics, mass spectrometry-based techniques have largely replaced Edman degradation for large-scale and high-throughput protein sequencing due to their speed, sensitivity, and capability to handle more complex protein samples.

Fragmenting a peptide using tandem mass spectrometry 

  • Fragmented by bombarding with inert gas ions 

How does one sequence proteins 

Ie insulin - connected by two inter chain disuphide bonds 

Cyctine residue must be cleaved various procedures 

The two chains separated 

If still too long chemical / enzymatic fragmentation 

Order the fragments by identifying overlaps 

Another method of characterising protein - the sanger method

This sequences the dna instead of the protein itself 

Once protein is sequenced 

Compare sequence with known proteins to see inference of structure and function 

Compare the protein to other species to see genetic relationship 

Slide on 3d structure