protein purification Flashcards
how were proteins originally defined
Defined originally according to their solubility
Globulins are usually large structures which are
insoluble in water but soluble in salt solutions
e.g. immunoglobulins
B) Albumins are smaller structures with a compact shape and they are soluble in water.
e.g. human serum albumin (HSA) or bovine serum albumin (BSA).
What properties of proteins can be used to purify them from a complex mixture?
A) charge (positive and negative charge from lysine and arginine or aspartate and glutamate respectively). Nearly all proteins in the cytoplasm will have both negative and positive charge at pH 7.0
B) hydrophobicity (polarity)
C) size (relative molecular mass Mr)
D) biospecificity (including posttranslational modifications such as glycosylation or phosphorylation)
Proteins embedded in the plasma membrane will almost certainly be glycosylated on the part of the protein exposed to the extracellular matrix (ECM). Those exported from the cell Proteins exported from the cell will almost always have a disulphide bond for strength.
It has been estimated that at any time up to 40% of the cellular proteins will be phosphorylated on serine, threonine or tyrosine (100:10:1).
Some will be glycosylated (N-acetylglucosamine).
why do proteins have to be pure
- humanised monoclonal antibodies are in a large amount of drugs and for people to have these they must be pure as if not pure they can be unsafe.
- diagnostics- monoclonal and polyclonal antibodies used to confirm presence of many compounds e.g. in pregnancy tests
- pure proteins are also required to form crystals for x ray diffraction studies.
What bioanalytical techniques can be used to purify proteins?
A) charge……ion exchange chromatography (IEXC) and isoelectric focussing
B) hydrophobicity (polarity)……. hydrophobic interaction chromatography (HIC) and reversed phase chromatography (RPC)
C) size… size exclusion chromatography (SEC) and denaturing (use a detergent) polyacrylamide gel electrophoresis (SDS-PAGE
D) biospecificity including posttranslational modifications (glycosylation and phosphorylation)….. affinity chromatography, immunoaffinity chromatography and metal ion affinity chromatography (IMAC)
The charge on a protein
This involves a number of different components which require evaluation and revision.
The properties of water…….forming a solvation shell around the protein.
pH……..different pH values will dramatically alter the charge on a protein due to amino acid side chains in the proteins structure.
Buffers…..required to maintain the protein in the correct shape for structure/function and participating in catalytic events
most important property of the Structure of water: H2O
Water has a dipole, in that one pole of the molecule is more electronegative than the other pole. The dipole in water allows water molecules to interact. Positive ends are attracted to negative ends. This is referred to as Hydrogen bonding which contributes to the many properties of water. A hydrogen bond is longer and weaker than a covalent bond. proteins contain many hydrogen bonds.The hydrogen bonds in water can form over distances. The surface tension in water and the movement of water by capillary action is due to hydrogen bonding
The ionisation of water
The dipole moment in the structure of water allows adjacent water molecules to form H-bonds.
In the dipole the relatively large oxygen atom tends to hog the electrons making the hydrogen protons loosely held in the structure allowing protons to be exchanged between water molecules (also other hydrogen containing molecules).
This exchange of protons does not alter the charge on the molecule but at a relatively low level (2 molecules in every billion) water molecules can ionise into a negatively charged hydroxyl group (OH-) and a positively charged hydronium ion (H3O+). This represents another very important property of water.
H2O ⇄ H+ + OH- (1)
This is what you see in many books but it would leave a proton (H+) without a home. In reality the proton engages with another water molecule to become a hydronium ion (H3O+ )
H2O + H+ ⇄ H3O+(2)
Summary: 2 H2O ⇄ H3O+ + OH-
how to calculate pH
pH = - log10 [H+]
pH at room temp
At 25oC the pH of a neutral solution is pH 7.0
Due to carbon dioxide dissolving in water to form carbonic acid (H2CO3) water that we come across is usually pH 5.5.
what did Bronsted propose
Brønsted proposed that acid-base reactions involve the transfer of an H+ ion (proton).
A Brønsted acid is a proton donor and a Brønsted base is a proton acceptor.
Every Brønsted acid has a conjugate base and every Brønsted base has a conjugate acid.
HCl + H2O « H3O+ + Cl- acid base acid base
what is a strong acid
Acids are often referred to as being “strong” or “weak” and a measure of the strength of an acid is the acid-dissociation equilibrium/dissociation constant (Ka) for that acid.
HA + H2O « H3O+ + A-
Acid base acid base
Ka= [H3O+][A-]
[HA]
A strong acid (proton donor) readily dissociates at all pH values and has a high Ka value (HCl: Ka = 1x 103)
A weak acid has a small Ka value (CH3COOH Ka = 1.8 x10-5) and its dissociation is pH dependent.
e.g. (a) HCl « H+ + Cl-
Strong acid dissociation
(dissociates completely from pH 0-14)
pKa
pKa: The negative log10 of the acid dissociation constant(Ka) of a solution.
pKa = -log10KaThe lower the pKavalue, the acid has a greater tendency to dissociate.
Weak acids
A weak acid has a small Ka value (CH3COOH Ka = 1.8 x10-5) and its dissociation is pH dependent.
CH3COOH « H+ + CH3COO-
Weak acid dissociation of acetic acid
Below pH 3.0 the carboxyl group is undissociated.
Dissociation of the carboxyl hydrogen begins above pH 3.0 and is fully dissociated above pH 7.0.
see graph
A titration curve for acetic acid: the inflection point
in the plot is the point on the pH scale where there are
equal molar concentrations of the ionised and unionised forms.
buffers
Proteins have pH optima for their activity and for their ideal storage conditions. Outside these pH values a protein may become denatured and inactivated.
In biology maintaining the pH of a solution is vitally important.
A buffer can maintain the pH of a solution by soaking up or releasing protons upon the addition of an acid or an alkali.
