CHEMICALS AND DETERGENTS
UREA + THIOUREA
Both are chaotropic agents that are commonly used in 2D-GE. Urea
(7M) is the best chaotropic agent – it breaks efficiently
hydrogen bonds, but it is less efficient in breaking hydrophobic
interactions. It is important to remember to not increase the
temperature of urea solution above 37 degrees as urea can form
ammonium cyanate that can react with amino groups and change the
charge of the proteins.
Thiourea (2M) is more efficient in breaking the hydrophobic
interactions and it is good agent used for membrane proteins.
CHAPS
It is a detergent that belongs to the class of zwitterionic
detergents. Detergents contain a hydrophobic and hydrophilic
parts. They can be classified depending on the electrical charge.
Zwitterionic detergent possess a net zero charge arising from the
presence of equal numbers of +1 and -1 charged groups.
Beta-mercaptoethanol
This is a detergent used to break disulphide bonds. High
concentrations are needed (700mM). It ionizes at high pH = this
will have consequences with IEF (isoelectric focusing), because
of ionization at basic pH it will enter the basic part of IEF and
ruin the pH gradient.
DTT
This is an alternative to beta-mercaptoethanol. Much lower
concentrations are needed (50mM). It also ionizes at high pH, but
because of its lower concentration, this is acceptable in the use
with IEF.
, AMINO ACID MODIFICATIONS
Cysteine Residue
Problem:
Cysteine residues in proteins are reactive and readily form
disulphide bonds with other cysteine resides (due to oxidation by
the oxygen presence in the air). Unmodified cysteine is destroyed
in N-terminal protein sequencing (Edman degradation) and in amino
acid analysis.
Solution:
Derivation of the thiol group. By deriving the cysteine residues,
they are prevented from reacting and forming disulphide linkages.
How:
Modification of the -SH group using different chemical reagents.
Titration of -SH groups
In proteins with multiple cysteine residues the reactivity of an
individual cysteine can be very dependent on both its local
environment and the hydrophobicity of the reactive dye.
Depending on the function and the structure of the protein the
cysteines can be in a reduced or oxidized states. It is important
to know for functional and structural analyses how many thiols
still are susceptible and accessible for reaction with a reagent.
Lysine
Problem:
, Trypsin cleaves the protein at the C-terminal end of a Lysine or
Arginine residue. Sometimes this gives very short fragments.
Solution:
Modify Lysine so that trypsin will not be able to cleave the
peptide bond at the C-terminal end of the residue
How:
Modification of the -NH2 group using different chemical reagents
Pre- and Post-column derivatization
Derivatization = it is a technique which converts a chemical
compound into a product of similar structure (a derivative).
Both of these techniques are used in detection of proteins and
polypeptides.
Pre-Column Derivatization:
It is considered appropriate to use when intention is to analyze
a somewhat limited variety of samples with high sensitivity.
Commonly used reagents are OPA and PITC (also used in Edman
degradation). Pre-column derivatization allows modification of
the sample in such way that it can be used in reversed-phase
chromatography. Normally, reverse phase chromatography Is used to
separate reaction products, and it is not suited for separation
of highly hydrophilic substances. The amount of protein sample
required is about 0.5-1ug.
In this case, the amino acids are modified before injection, and
then the reaction products are separated and detected. This
provides number of advantages:
- Reagent consumption rates can be minimized by specifying a
small reaction system
UREA + THIOUREA
Both are chaotropic agents that are commonly used in 2D-GE. Urea
(7M) is the best chaotropic agent – it breaks efficiently
hydrogen bonds, but it is less efficient in breaking hydrophobic
interactions. It is important to remember to not increase the
temperature of urea solution above 37 degrees as urea can form
ammonium cyanate that can react with amino groups and change the
charge of the proteins.
Thiourea (2M) is more efficient in breaking the hydrophobic
interactions and it is good agent used for membrane proteins.
CHAPS
It is a detergent that belongs to the class of zwitterionic
detergents. Detergents contain a hydrophobic and hydrophilic
parts. They can be classified depending on the electrical charge.
Zwitterionic detergent possess a net zero charge arising from the
presence of equal numbers of +1 and -1 charged groups.
Beta-mercaptoethanol
This is a detergent used to break disulphide bonds. High
concentrations are needed (700mM). It ionizes at high pH = this
will have consequences with IEF (isoelectric focusing), because
of ionization at basic pH it will enter the basic part of IEF and
ruin the pH gradient.
DTT
This is an alternative to beta-mercaptoethanol. Much lower
concentrations are needed (50mM). It also ionizes at high pH, but
because of its lower concentration, this is acceptable in the use
with IEF.
, AMINO ACID MODIFICATIONS
Cysteine Residue
Problem:
Cysteine residues in proteins are reactive and readily form
disulphide bonds with other cysteine resides (due to oxidation by
the oxygen presence in the air). Unmodified cysteine is destroyed
in N-terminal protein sequencing (Edman degradation) and in amino
acid analysis.
Solution:
Derivation of the thiol group. By deriving the cysteine residues,
they are prevented from reacting and forming disulphide linkages.
How:
Modification of the -SH group using different chemical reagents.
Titration of -SH groups
In proteins with multiple cysteine residues the reactivity of an
individual cysteine can be very dependent on both its local
environment and the hydrophobicity of the reactive dye.
Depending on the function and the structure of the protein the
cysteines can be in a reduced or oxidized states. It is important
to know for functional and structural analyses how many thiols
still are susceptible and accessible for reaction with a reagent.
Lysine
Problem:
, Trypsin cleaves the protein at the C-terminal end of a Lysine or
Arginine residue. Sometimes this gives very short fragments.
Solution:
Modify Lysine so that trypsin will not be able to cleave the
peptide bond at the C-terminal end of the residue
How:
Modification of the -NH2 group using different chemical reagents
Pre- and Post-column derivatization
Derivatization = it is a technique which converts a chemical
compound into a product of similar structure (a derivative).
Both of these techniques are used in detection of proteins and
polypeptides.
Pre-Column Derivatization:
It is considered appropriate to use when intention is to analyze
a somewhat limited variety of samples with high sensitivity.
Commonly used reagents are OPA and PITC (also used in Edman
degradation). Pre-column derivatization allows modification of
the sample in such way that it can be used in reversed-phase
chromatography. Normally, reverse phase chromatography Is used to
separate reaction products, and it is not suited for separation
of highly hydrophilic substances. The amount of protein sample
required is about 0.5-1ug.
In this case, the amino acids are modified before injection, and
then the reaction products are separated and detected. This
provides number of advantages:
- Reagent consumption rates can be minimized by specifying a
small reaction system
