Summary Enzymology practical
course
Chapter 1
Purification: the isolation of groups of biomolecules from specific sources. Purification can be
chemical or physical. Reasons for purification can be: the unravelling of cellular processes,
determining the cause of diseases, rational design (= design of new enzymes or development of novel
drugs) and to obtain valuable biomolecules for, for example, the food industry.
Recombinant DNA technology can be used to increase purification yield (overexpress):
The gene expressing the protein of interest can be cloned into a suitable expression plasmid
By adding a signal sequence (for secretion) to the gene of interest, the gene of interest can
be exported from the cell
By adding an affinity tag, the tagged protein can be purified using highly selective purification
methods.
Overexpressed proteins are not always identical to the proteins isolated from the native source, due
to posttranslational modifications. Overexpression can sometimes lead to improper protein folding.
Group-separation is often very easy, since the physical properties of biomolecules are very different.
For example compare lipids with nucleic acids. Separation of biomolecules within a group is often
more challenging. Several analytical tools are used to follow the purification progress of a protein
after each purification step. The activity of a protein can be measured during the process if it is an
enzyme that catalyses a specific reaction.
In general, proteins are purified from an extract, in which the protein is present in a soluble form.
Cells can be removed from an extract by centrifugation or by flocculation and filtration. Cells are
removed when the protein of interest is excreted by cells.
If the protein is not excreted, but accumulates within the cell, the cell has to be broken.
Physical methods applied during purification should be used within the boundaries of the physical
stability of the protein. The most important parameters are the pH and the ionic strength of the
buffer in which the protein is purified.
Methods for purification of the desired protein from cell-free extract can be divided into four
categories:
1. Selective precipitation methods
2. Separation methods based on affinity
3. Separation methods based on hydrophobicity or charge’
4. Separation methods based on size.
The purity of the resulting protein preparation can be tested with SDS-page, with which you can
determine if also other proteins are still present in the sample. UV-VIS absorption profiles, activity
measurements and mass spectrometry can also provide insights in the purity of the product.
Chapter 2
Removal of proteins for the cellular environment can result in loss of activity or in alternation of the
structure. Causes of loss of proteins are:
, Denaturation of the protein due to pH, ionic strength or temperature conditions
Aggregation due to pH, ionic strength or temperature conditions
Proteolytic degradation
Tight binding of a protein to a column (requiring extreme
elution conditions)
Removal of a component required for its activity
Inactivation of the enzyme by inhibitors or protein modifying
components
Non covalent interactions and covalent cross-links are the major forces
that keep a polypeptide chain in a stable conformation. High
concentrations of specific ions or denaturants may competitively disrupt the interactions and thus
cause unfolding. Hydrophobic substances in the buffer solution can attach to hydrophobic patches on
the protein, thereby reorganizing the water layer. In most folded proteins, hydrophobic patches are
present in the interior. The exterior can interact with water molecules. The solubility of a protein can
be influenced by:
A rise in temperature
A decrease of pH or an increase of pH (protonation and deprotonation)
Low ionic strengths (results in protein-protein interactions,
aggregation). Proteins can be salted in to a solution by adding salt
High ionic strengths (mineral ions decrease availability of water
molecules, which decreases protein hydration and result in salting out)
Proteolytic activity is generally
the greatest in the initial crude
extract, because purification
usually removes proteases from the sample. A small
contamination with a protease in the final sample will
rapidly degrade the purified protein. Common
protease inhibitors are PMSF, pefablock, benzamidine
and EDTA. The first three irreversibly block the active
sites of serine proteases, EDTA inhibits
metalloproteases. Microbial or fungal growth can also
cause proteolysis, so sodium azide can be added as an
inhibitor of the respiratory chain.
The buffer pH is a huge influence during the
purification. When the pH > pI (pI =isoelectric point),
the protein will have a negative net charge. When pH
< pI, the protein will have a positive net charge. At high protein concentrations, a neutral net charge
can result in protein aggregation due to reduced electrostatic repulsive forces. The protein will have
its minimum solubility. The pH of solutions is sensitive to addition of H or OH ions, and therefore,
protein purifications are performed in pH-controlled buffer solutions. The pH of a solution of a weak
acid/base can be calculated with Henderson-Hasselbalch: pH = pKa + log[(basic species)/(acidic
species)]. When choosing a buffer, you should consider interactions of the buffer with the enzyme
and the pH-optimum of the enzyme. For the determination of the pH optimum of an enzyme, it is
useful to test a series of buffers that span a wide pH-range. Many buffers are temperature sensitive.
Strong dilution of stock buffer solution or addition of salts can also have an effect on the pH of the
buffer.
course
Chapter 1
Purification: the isolation of groups of biomolecules from specific sources. Purification can be
chemical or physical. Reasons for purification can be: the unravelling of cellular processes,
determining the cause of diseases, rational design (= design of new enzymes or development of novel
drugs) and to obtain valuable biomolecules for, for example, the food industry.
Recombinant DNA technology can be used to increase purification yield (overexpress):
The gene expressing the protein of interest can be cloned into a suitable expression plasmid
By adding a signal sequence (for secretion) to the gene of interest, the gene of interest can
be exported from the cell
By adding an affinity tag, the tagged protein can be purified using highly selective purification
methods.
Overexpressed proteins are not always identical to the proteins isolated from the native source, due
to posttranslational modifications. Overexpression can sometimes lead to improper protein folding.
Group-separation is often very easy, since the physical properties of biomolecules are very different.
For example compare lipids with nucleic acids. Separation of biomolecules within a group is often
more challenging. Several analytical tools are used to follow the purification progress of a protein
after each purification step. The activity of a protein can be measured during the process if it is an
enzyme that catalyses a specific reaction.
In general, proteins are purified from an extract, in which the protein is present in a soluble form.
Cells can be removed from an extract by centrifugation or by flocculation and filtration. Cells are
removed when the protein of interest is excreted by cells.
If the protein is not excreted, but accumulates within the cell, the cell has to be broken.
Physical methods applied during purification should be used within the boundaries of the physical
stability of the protein. The most important parameters are the pH and the ionic strength of the
buffer in which the protein is purified.
Methods for purification of the desired protein from cell-free extract can be divided into four
categories:
1. Selective precipitation methods
2. Separation methods based on affinity
3. Separation methods based on hydrophobicity or charge’
4. Separation methods based on size.
The purity of the resulting protein preparation can be tested with SDS-page, with which you can
determine if also other proteins are still present in the sample. UV-VIS absorption profiles, activity
measurements and mass spectrometry can also provide insights in the purity of the product.
Chapter 2
Removal of proteins for the cellular environment can result in loss of activity or in alternation of the
structure. Causes of loss of proteins are:
, Denaturation of the protein due to pH, ionic strength or temperature conditions
Aggregation due to pH, ionic strength or temperature conditions
Proteolytic degradation
Tight binding of a protein to a column (requiring extreme
elution conditions)
Removal of a component required for its activity
Inactivation of the enzyme by inhibitors or protein modifying
components
Non covalent interactions and covalent cross-links are the major forces
that keep a polypeptide chain in a stable conformation. High
concentrations of specific ions or denaturants may competitively disrupt the interactions and thus
cause unfolding. Hydrophobic substances in the buffer solution can attach to hydrophobic patches on
the protein, thereby reorganizing the water layer. In most folded proteins, hydrophobic patches are
present in the interior. The exterior can interact with water molecules. The solubility of a protein can
be influenced by:
A rise in temperature
A decrease of pH or an increase of pH (protonation and deprotonation)
Low ionic strengths (results in protein-protein interactions,
aggregation). Proteins can be salted in to a solution by adding salt
High ionic strengths (mineral ions decrease availability of water
molecules, which decreases protein hydration and result in salting out)
Proteolytic activity is generally
the greatest in the initial crude
extract, because purification
usually removes proteases from the sample. A small
contamination with a protease in the final sample will
rapidly degrade the purified protein. Common
protease inhibitors are PMSF, pefablock, benzamidine
and EDTA. The first three irreversibly block the active
sites of serine proteases, EDTA inhibits
metalloproteases. Microbial or fungal growth can also
cause proteolysis, so sodium azide can be added as an
inhibitor of the respiratory chain.
The buffer pH is a huge influence during the
purification. When the pH > pI (pI =isoelectric point),
the protein will have a negative net charge. When pH
< pI, the protein will have a positive net charge. At high protein concentrations, a neutral net charge
can result in protein aggregation due to reduced electrostatic repulsive forces. The protein will have
its minimum solubility. The pH of solutions is sensitive to addition of H or OH ions, and therefore,
protein purifications are performed in pH-controlled buffer solutions. The pH of a solution of a weak
acid/base can be calculated with Henderson-Hasselbalch: pH = pKa + log[(basic species)/(acidic
species)]. When choosing a buffer, you should consider interactions of the buffer with the enzyme
and the pH-optimum of the enzyme. For the determination of the pH optimum of an enzyme, it is
useful to test a series of buffers that span a wide pH-range. Many buffers are temperature sensitive.
Strong dilution of stock buffer solution or addition of salts can also have an effect on the pH of the
buffer.
