lecture 2 biochemistry (week 1) Flashcards
(22 cards)
what are the factors affection resolution in gel filtration?
diffusion (particle size of column material, flow rate)
volume of the sample (~0.2% of bed volume)
viscosity of the sample (protein concentration < 30mg/ml)
relative amounts of the protein to be separated
where can gel filtration be used?
change of buffer, desalting
removal of recion products
removal of phenol red from culture medium prior to anion exchange chromatography
fractionation
determination of molecular mass (monomer, dimer, multimer, resolution of multimers by addition of 6M urea)
covalent chromatrography
low molecular thiols like glutathione have to be removed from the sample prior to chromatography (dialysis or gel filtration)
binding is monitored by measuring absorption at 343 nm in the flow through (thiopyridone)
reducing elution most often with 10-25mM DTT or 25-50 mM beta-mercaptoethanol –> 2-thipyridone which ends up in the eluate has to be removed by gel filtration
what are the types of ion exchangers?
anion exchangers and cation exchangers
what are anion exchangers?
basic ion exchanger with positive functional groups
what are cation exchangers?
acid ion exchanger with negative functional groups
what makes an ion exchanger strong or weak?
strong ion exchangers –> material ionised over a broad pH range –> capacity is not reduced by loss of charge at high or low PH
weak ion exchangers –> carry weak base or weak acid as functional group
what is the influence of pH on net protein charge?
proteins are amphoteric (carry positive and negative charges)
net charge is dependent on pH
isoelectric point is the pH where the net charge is zero
in acidic pH, amino groups of mainly lys, arg, and his, are protonated –> cationic behaviour
in basic pH, the chargers of aspartate, and glutamate take over –> anionic behaviour
IP> 8 basic protein
IP< 6 acidic protein
what is the buffer for ion exchange chromatography?
ionic strength has to be high enough to guarantee protein stability (10mM - 20mM)
high buffer capacity ( pKa of buffer not more than 0.5 pH units from pH of the sample)
buffer ions should have the same sigh as the charged groups of the ion exchangers otherwise they act as counter ions
if freeze drying is planned volatile buffers should be used
what does the elution by salt gradient using a non buffering salt lead to?
increases competition of free ions with the bound protein for binding sites on the matrix
describe pH-gradient elution as an alternative to the increase
of ionic strength (salt gradient)
alteration of the pH towards the isoelectric point of substance to be elutes
substances with weak interaction come off first.
Attention !!! many proteins have their lowest solubility close to their IP
aggregation by reduced repulsion
describe hydrophobic interaction chromatography
the interaction of hydrophobic regions on the surface of biomolecules with hydrophobic ligands (alkyl-aryl groups) on a gel matrix
protein binding is supported by increased concentration of anti chaotropic (neutral) salts.
elution by stepwise or continuous reduction in salt concentration in the adsorption buffer
chaotropic agents destabilize the hydrogen bonding and hydrophobic interactions
what are the important facts for hydrophobic interaction chromatography
well suited as next purification step following protein precipitation (ammonium sulphate)
most biological starting material already has a high ionic strength (high conductivity) HIC suitable as first chromatographic step
HIC suited after ion exchange chromatography as the sample is already in a –> buffer with high ionic strength
the longer the alkyl chains the higher the substitution –> stronger binding
high salt concentrations can result in the precipitation of proteins on the column –> and therefore have a negative influence on the selectivity
increase in pH reduces the strength of the hydrophobic interactions
lowering the temperature reduces the hydrophobic interactions
additives, which in low concentrations reduce the interactions: –> - H2O miscible alcohols (e.g. 30% isopropanol; 40% - 80% ethylene glycol) –> detergents –> chaotropic salt
what are the influences of salt in hydrophobic interaction chromatography?
Na2SO4, NaCl, and NH$2SO4 are the salts most often used in HIC
learn the hofmeister effect
describe the steps of the chromatogram of hydrophobic interaction chromatography
equilibration –> sample application –> gradient elution –> salt free wash –> re-equilibration
describe the principles of affinity chromatography
different biological affinities are used to bind to a solid phase
affinity for biologically functional partner
affinity for biometric ligand (eg, dye with similar structures as ATP; blue sepharose)
immunoadsorption –> antigen-antibody interaction (elution at pH = 2-3)
generation of adsorption matrix most often activated gel materials are used, which allow covalent coupling of the ligand (cyanogen bromide CNBr is most often used for activation) ligand concentration for small ligands 1 – 20 µmol/ml gel; for proteins 5 – 10 mg/ml gel
what are features of the ligand?
reversible complex formation with the protein to be isolated
high specificity
suitable dissociation constant to allow reversible complex formation without harming the protein –> KD = 1 – 10 µM
chemical features, which allow immobilisation on a matrix -
stable in the solvent used for coupling
at least one functional group for immobilisation (-NH2 – amino; -COOH – carboxyl; -CHO – aldehyde; -SH – thiol; -OH – hydroxyl)
functional group must not take part in the interaction involved in the purification covalent binding to the matrix
what are the classifications of ligands?
low molecular, monospecific: biotin-avidin
low molecular: group specific: biomimetic dyes
macromolecular, mono specific: protein-protein interactions __> transferrin receptor
macromolecular, group specific: lectins – glycoproteins, protein A or protein G – IgG
describe elution
changing pH –> neutralization immediately after elution
changing ionic strength –> mainly dye columns
changing in structure or changing polarity –> addition of chaotropic salts in the range of 1 – 3 M, addition of 4-6 M urea or guanidinium hydrochloride, decrease of polarity by addition of 20 – 40 % ethylene glycol (most gentle method !) or up to 10 % dioxane, addition of detergents close to the critical micelle concentration: Lubrol, Nonidet P-40, Octylglycoside
special elution by adition of free ligand (5-100mM) at neutral pH
what are the steps of affinity chromatography?
equilibration –> adsorption of sample and elution of unbound material –> wash away unbound material –> elute bound proteins –> gel regenaration
immobilised metal ion affinity chromatography
metal chelator (nitriloacetic acid NTA, iminodiacetic acid IDA) –> coupled to a matrix
NTA binds Ni2+ stronger than IDA higher protein yield, less –> liberation of metal ions
immobilised metal ion affinity chromatography
free coordination sites for binding of the protein to be purified
interfering ionic interactions can be avoided by increasing the ionic strength (0.15 – 1 M NaCl)
low pH results in elution
elution by competitive ligands, pH gradient, organic solvents, chelators
