Biochemistry Chapter on Purification Techniques

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Questions and Answers

What is typically done before the extraction of an enzyme during purification?

Conduct cell fractionation (correct)

Introduce nuclease enzymes

Heat the extraction medium

Add divalent metal ions

What is a potential issue caused by extracted nucleic acids during enzyme purification?

They increase the viscosity of the solution (correct)

They reduce the temperature of the medium

They enhance enzyme activity

They precipitate proteins immediately

Which method is commonly used to precipitate proteins during enzyme purification?

Use sonication to shear nucleic acids

Raise the pH of the solution

Increase the salt concentration (correct)

Centrifuge the solution at high speed

What is the main advantage of using ammonium sulphate for precipitation?

It is highly soluble in water Signup and view all the answers

What occurs at an enzyme's isoelectric point (pI)?

The enzyme precipitates from the solution Signup and view all the answers

Which of the following can be used to digest nucleic acids during enzyme purification?

Nuclease enzymes Signup and view all the answers

What is a disadvantage of isoelectric precipitation?

It is incompatible with enzyme activity recovery Signup and view all the answers

What is the purpose of adding divalent metal ions during purification?

To precipitate nucleic acids Signup and view all the answers

What is the primary aim of increasing salt concentration in protein purification?

To selectively precipitate proteins that are not needed. Signup and view all the answers

Which method is commonly used to remove residual ammonium sulfate after enzyme purification?

Dialysis. Signup and view all the answers

In enzyme purification, what is one reason adding the substrate may increase enzyme stability?

Enzymes are often more stable in the presence of their substrates. Signup and view all the answers

What is the advantage of using chromatography for enzyme purification?

It exploits the differences in protein structure for separation. Signup and view all the answers

Which of the following is NOT a characteristic that can be utilized for enzyme purification?

Color absorption. Signup and view all the answers

When purifying enzymes, why might the temperature of the medium be raised?

To denature and precipitate unwanted proteins. Signup and view all the answers

What is one potential method for purification at an industrial scale?

Ultrafiltration. Signup and view all the answers

Which purification method involves using resins with functional groups attached?

Ion exchange chromatography. Signup and view all the answers

What role do lyotropic salts, such as ammonium sulfate, play in hydrophobic interaction chromatography (HIC)?

They increase the ionic strength, enhancing hydrophobic interactions. Signup and view all the answers

Which of the following statements about reversed-phase HPLC is true?

It relies on the interaction of hydrophobic areas of proteins with aliphatic ligands. Signup and view all the answers

In hydrophobic charge induction chromatography (HCIC), what effect does decreasing the pH have?

It triggers the elution of proteins from the resin. Signup and view all the answers

What is a characteristic of mixed mode chromatography (MMC)?

It may utilize various interactions for protein binding. Signup and view all the answers

What is the primary ligand used in hydrophobic charge induction chromatography (HCIC)?

4-mercapto-ethylpyridine (4-MEP) Signup and view all the answers

What happens to the ionic strength during the elution phase of hydrophobic interaction chromatography (HIC)?

It is decreased to promote elution of bound proteins. Signup and view all the answers

Why is the recovery of enzyme activity from HIC usually high?

The enzymes are less likely to denature in HIC. Signup and view all the answers

Which of the following is NOT a feature of reversed-phase HPLC?

Competes effectively for binding with immunoglobulins. Signup and view all the answers

What primarily determines the interactions between proteins and HA in affinity chromatography?

The amino acid content of the proteins Signup and view all the answers

Why is affinity chromatography rarely used in isolation for protein purification?

It is limited in application to specific enzymes Signup and view all the answers

What is the advantage of using an immobilized monoclonal antibody in immunoaffinity chromatography?

It increases the specificity of the purification Signup and view all the answers

In covalent chromatography, how are thiol-containing proteins and peptides isolated?

By forming a covalent mixed disulphide with a resin Signup and view all the answers

What type of enzymes can be bound using immobilized triazine-based dyes?

NAD- and NADP-dependent dehydrogenases Signup and view all the answers

What is the key difference between covalent bonding and coordinate (dative) bonding?

In coordinate bonding, both electrons come from one atom. Signup and view all the answers

Which of the following amino acids contains a functional group that is electron-rich and can participate in dative bonding?

Cysteine Signup and view all the answers

At what pH level can amino acids with carboxylic acid residues typically form coordinate bonds with trivalent metal ions?

Acidic conditions (around 3.0) Signup and view all the answers

Which of the following resins is typically used in Immobilized Metal Ion Affinity Chromatography (IMAC)?

Sepharose Signup and view all the answers

How is the binding strength of a protein to a metal ion in IMAC primarily determined?

By the number and local environment of histidine residues. Signup and view all the answers

What role does the 6His tag play in the purification of recombinant proteins during IMAC?

It facilitates strong binding to the metal-charged resin. Signup and view all the answers

What is the primary chemical composition of hydroxyapatite (HA)?

Calcium phosphate and hydroxyl groups Signup and view all the answers

How can the elution of bound enzymes from resin in IMAC be accomplished?

By lowering the pH to below 5.0 or using imidazole at pH 7.2. Signup and view all the answers

Study Notes

Preliminary Purification

Cell Fractionation is often used in initial purification steps to isolate the target enzyme.
The extraction medium is chosen to ensure the targeted enzyme remains soluble while others are not.
Nucleic acids can be removed by:
- Nuclease enzymes
- Precipitation with basic substances (streptomycin, protamine, polyethyleneimine)
- Precipitation with divalent metal ions (MnCl2 or MgCl2)
- Sonication to shear long nucleic acid fragments
Centrifugation removes cell debris, precipitates, and other unwanted materials.
Precipitation concentrates the enzyme and separates it from other molecules like mono- and oligosaccharides, nucleotides, and free amino acids.

Protein Precipitation

Isoelectric precipitation is used to precipitate proteins at their isoelectric point, but can be difficult to recover biological activity.
Increase in salt concentration is a common method for precipitation using ammonium sulfate.
Ammonium sulfate precipitation is carried out in stages to precipitate unwanted proteins before the targeted enzyme.
Dialysis or size-exclusion chromatography is used to remove residual ammonium sulfate.

Laboratory-Scale Purification Techniques

Ultrafiltration, polymer precipitation (PEG), and chromatographic resins (ion exchange, hydrophobic interaction) are effective for laboratory-scale purification.

Industrial-Scale Purification Techniques

Ultrafiltration, stirred cells, and expanded beds are considered for industrial-scale purification.

Additional Purification Steps

Increasing the temperature of the medium can denature and precipitate proteins while keeping the targeted enzyme stable.
The presence of the substrate can enhance enzyme stability and improve the effectiveness of temperature-based purification.

Chromatography

Chromatography separates materials based on their distribution between a stationary and a mobile phase.
Resins with specific functional groups are used to exploit differences in protein structure.

Types of Chromatography

Hydrophobic Interaction Chromatography (HIC):
- Exploits hydrophobic interactions between non-polar amino acids and hydrophobic column packings (phenyl-Sepharose, octyl-Sepharose).
- Elution is achieved by decreasing ionic strength or introducing an organic solvent.
- Protein binding to the ligand is determined by the lyotropic salt concentration (e.g. ammonium sulfate).
Reversed-phase HPLC:
- Utilizes hydrophobic interactions between the protein and aliphatic ligands (C18, C12, C8, C4) attached to silica beads.
- Strong interactions due to a higher density of ligands compared to HIC.
- Elution conditions often result in the loss of biological activity.
Hydrophobic Charge Induction Chromatography (HCIC):
- pH controls protein binding to the hydrophobic ligand (4-mercapto-ethylpyridine).
- Primarily used for immunoglobulin purification, relying on interactions with the Fc region and hydrophobic interactions.
- Elution at pH 5.0 facilitates enrichment of immunoglobulins.
Mixed Mode Chromatography (MMC):
- Utilizes resins like mercapto-benzimidazole sulphonic acid (MBI), hexylethylamino (HEA), phenylpropylamino (PPA), and Capto™ MMC.
- Protein interactions are complex and combine different interaction types.
- Offers potential for purifying enzymes that are difficult to resolve with other methods.
Immobilized Metal Ion Affinity Chromatography (IMAC):
- Utilizes resins with iminodiacetate (IDA) or nitrilotriacetate (NTA) bound to Sepharose or agarose.
- Di- or trivalent metal ions bind to the resin, leaving open spaces for interactions with amino acid functional groups (histidine, cysteine, tryptophan).
- Elution can be achieved by lowering pH to below 5.0 or by displacement with increasing imidazole concentrations.
- The strength of interaction depends on the number and environment of histidine residues.
- Recombinant proteins with a hexahistidine tag (6His) are often purified by binding to IMAC resins.
Hydroxyapatite Chromatography (HA):
- Crystalline form of calcium phosphate used in resins.
- Protein interactions are complex, involving calcium ions, phosphate groups, and hydroxyl groups.
- Interactions depend on the protein's amino acid content, pH, and ionic strength.
Affinity Chromatography:
- Exploits the bio-specificity of enzymes.
- Provides high purification but is rarely used in isolation besides recombinant protein purification.
Immunoaffinity Chromatography:
- Uses immobilized antibodies specific to the targeted enzyme.
- Utilizes monoclonal antibodies for even higher specificity.
Reactive Triazine Based Dyes:
- Dyes like Cibacron Blue F3G-A and Procion series are used as ligands.
- Dyes bind to a wide range of enzymes like NAD- and NADP-dependent dehydrogenases.
Covalent Chromatography:
- Targets thiol-containing proteins and peptides.
- Thiol residues on the resin form covalent bonds with sulphydryl groups on the protein.
- Elution with reducing agents like L-cysteine, 2-ME, or dithiothreitol breaks the covalent bonds.

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Description

This quiz focuses on enzyme purification methods such as cell fractionation and protein precipitation. It covers techniques for removing nucleic acids and other unwanted materials, highlighting the importance of choosing the right extraction medium. Test your knowledge on the various methods used in the preliminary purification of enzymes.