Chromatography

Chromatography● Chromos Greek color Chromatography: process of separation of a mixture of compounds by slow penetration of the mixture through a material that absorbs each compound differently. (Oxford Dictionary of English) Two phases – – ● ● Stationary phase Mobile phase A simple chromatographic experiment ● Filter paper, beaker, fluids, inkt, pens) Place a dot on a filter paper Immerse the paper in a liquid Wait Rf = dn/dtotal ● ● ● Separation of classed of biomolecules ● ● ● ● ● ● ● ● Cells Organelles Cell wall components Membranes and lipids DNA and RNA Proteins Metabolites Salts ● ● ● ● Filtration Centrifugation Precipitation Denaturation Separation of biomolecules Proteins Differences in physico-chemical properties of proteins lead to differences in interaction with a stationary phase. These differences are exploited to purify proteins. ● Overall charge Size Hydrophobicity Specific Interaction Interaction with other biomolecules (Solubility) ● ● ● ● ● Chromatography pump Solution in ABC Solutions Gradients Column pH meter Detector Solution out Conductivity . light scattering) Fractionation .Äkta High pressure system Liquid pumps Sample application Column Detection (UV/conductivity pH. .). Charged particle (matrix) bind reversibly to sample molecules (proteins.Physico-chemical property: charge Ion Exchange Chromatography Basic Principles The attractive forces between molecules carrying charged groups of opposite signs are used. etc. Desorption is then brought about by increasing the salt concentration or by altering the pH of the mobile phase. Iso electric point.0 overall charge – at pH 7.0 . pI The pI of a protein is the pH at which the net charge of the protein is zero Acidic proteins: pI < 7.0 overall charge + at pH 7.0 Basic proteins: pI > 7. pI .Iso electric point. size distribution chemical and mechanical stability chemical nature ● Capacity Separation Resolution Price ● ● .Column Material ● Type of exposed groups acidic basic The nature of the exposed groups weak acidic or basic groups strong acidic or basic groups The concentration of exposed groups Properties of the matrix size. CM Structure -O-CH2-COO- Abbrev.Acidic or Basic groups Cation exchangers Group carboxy methyl Anion exchangers Group aminoethyl Abbrev. AE Structure -O -(CH2) 2NH3+ phosphate P -O-PO3- Diethyl aminoethyl DEAE -O -(CH2)2-N (C2H5) 2H+ sulfoethyl SE -O-(CH2) 2.SO3- Triethyl aminoethyl TEAE -O -(CH2)2-N (C2H 5) 3+ sulfopropyl SP -O-(CH2) 3.SO3- Trimethyl amino Q N+(CH 3)3 . 5 . Strong exchangers DEAE anion exchanger.Weak vs. bead NH+(C2H5)2 Q anion exchanger bead N+(CH3)3 Completely positively charged from pH 3-10 pKa = 9. Matrix material Agarose (Sepharose) ● ● Chemical stability Mechanical stability Exposed “ non-functional” groups Exclusion limits Size 10-200 µm Size distribution ● ● Dextran (Sephadex) ● ● ● Cellulose (DEAE Whatman 52) ● ● ● Acrylamide ● Polystyren (Dowex 8) ● 45-165 µm 15 µm 10 µm ● ● . Capacity The matrix material and number of exposed groups determine the capacity Two definitions: Capacity of the material for protons of other monovalent ions. This equals the number of functional groups per ml or gram material. Determined by titration. . Capacity of binding a certain weight of protein to a ml or gram column material. Often x mg BSA/ml. Resolution Resolution describes how well peaks can be resolved Experiments checking broadening of a standard compound AU/cond FWHH Resolution depends on bead size. distribution and column packing Resolution depends also on experimental conditions . Elution: Gradients Solution in Salt Column Salt Salt Solution out Salt Elution volume . 05% DDM Wash of unbound sample Elute proteins with gradient Wash Buffer B:20% glycerol 10 mM Tris HCl pH 8.5 15 12.5 45 42.5 5 2. 0.5 5 7.5 35 47.5 35 32.5 Absorption in mAU ml Equilibration Sample application Re-equilibration Buffer A: 20% glycerol 10 mM Tris HCl pH 8.5 10 12.5 10 7.0 1M NaCl.5 40 37.5 25 22.5 0 37.5 30 32.5 15 17.5 30 27. mS/cm 220 200 180 Chromatography Protein of interest SecYEG complex 160 140 120 100 80 60 40 20 0 -20 0 2.5 20 17. 0.0 0 M NaCl.A280 A410 cond.5 25 27.5 20 22.05% DDM . 5 45 42.5 25 22.5 20 17. salt.5 35 47.5 25 27.5 35 32.5 5 7.5 30 32.5 15 17.A280 A410 cond.5 15 12.5 30 27.5 5 2.5 20 22. mS/cm 220 200 180 Chromatography Protein of interest SecYEG complex 160 140 120 100 80 60 40 20 0 -20 0 2.5 0 37.5 10 7.5 40 37.5 10 12. gradient Resolution: Choice of column .5 Absorption in mAU ml Optimisation: pH. mS/cm 900 800 Overloading 50 45 40 35 30 25 Absorption in mAU 700 600 500 400 300 200 100 0 0 5 10 15 20 25 30 35 40 20 15 10 5 0 45 ml Equilibration Sample application Wash of unbound sample Elute proteins with gradient Wash Re-equilibration Sample overloading.A280 A410 cond. exceeding the column binding capacity . Na Beads equilibrated with NaCl and buffer. bead SO3Protein+ Proteins with positive patches bind to column and may be released again by a higher concentration of NaCl .Cation exchange chromatography S cation exchanger bead SO3carboxy-groups bead CH2CO2- Beads with exposed acidic groups that are negative at pH > pKa bead + SO3. inhibitors) ●Lipids ●Air bubbles in column ●Aggregation in columns ● Net charge. Charge distribution Negative or positive patches .Expect the unexpected? Incorrect equilibration ●Overloading ●History of the column ●Small molecules (ATP. Hydroxyapatite Hydroxyapatite material is crystaline Calcium phosphate Negative charges on the protein interact with Ca2+ Positive charges interact with PO43.buffers Basic protein eluted by increased ionic strength (NaCl) .groups Some ion exchange character Acid/neutral proteins elute with increasing PO43. low detergent concentration . high salt.bead Phenyl Hydrophobic interaction Hydrophobic interaction matrix interacts with exposed hydrophobic surfaces on the protein Group butyl octyl phenyl Formula -O -(CH2) 3CH3 Hydrophobicity + ++ ++ -O -(CH2) 7CH3 -O -(C6H5) Binding: polar solvent. optimize elution conditions Bead size and column issues similar Example: Halorhodopsin Binding to Source-Phenyl material in cholate detergent Impurities do not bind Elution in a gradient from 0% β-OG to 0.Hydrophobic interaction Detergents may interact with the exposed hydrophobic area's on protein and column material Low recovery. Chem. M. Biol.5% β-OG detergent (Duschl et al. J. 1988. Andersson master thesis 2003) .. All rights reserved . Elution with stronger binder or with binding analogue © Amersham Biosciences Limited .Affinity Chromatography Specific binding of a protein with a substrate (analog) or binding partner is used for affinity purification. Matrix Affinity Chromatography Little unspecific absorption Good flow proterties Good stability Many activatable groups High porosity Proper coupling reaction Support activation and ligand immobilization O O -O.CH2CHOH-CH2-NH-Ligand .CH2CH-CH2 -OH + Cl-CH2CH-CH2 epichlorohydrin + H2N-Ligand -O. dependent enzymes.All rights reserved . aldehyde and formate dehydrogenases. polysaccharides containing N-acetyl-D-glucosaminyl residues Serine proteases.Examples Affinity Chromatography Groups/material Protein A Streptavidin Purification of: Antibody Biotinilated proteins Strep-tag AWAHPQPGG NWSHPQPEK Glycoproteins. ATP-dependent enzymes. trypsin and trypsin-like proteases NADP+. Maltose binding protein (MBP) and MBP constructs GlutathionS-transferase (GST) and GST constructs Proteins with exposed histidines Agarose Wheat Germ Lectin Benzamidine 5' AMP amylose glutathion immobilized metal ions CNBr activated material Epoxy activated material © Amersham Biosciences Limited . 2 M KCl ATP binding sites bacterium phosphate .Dye Affinity Chromatography Dyes: aromatic ring systems with specific groups Poteins often bind to dyes reversible or irreversible Coupling of dyes to chromatographic matrix material Cibracron-blue Hexokinase Ribonuclease Red-agarose ABC ATPase NAD/NADP adeninenucleotide binding sites Rat brain elution 10 mM NADP Rabbit muscle elution 1. iron. tyrosine Strong binding of engineered 6-10 histidine tags . cobalt. cysteines. zink.Immobilized metal affinity chromatography (IMAC) Column material binds divalent metal ions: Copper. nickle. calcium Metal ions specifically interact with exposed residues on the protein: Histidines. tryptophans. 1-0.5 Tris HCl.5 Tris HCl.1-0.5 M NaCl glycerol 5-20 mM Imidazole Elution buffer: pH > 7.5 M NaCl glycerol EDTA OR low pH OR .5 Tris HCl 0.IMAC-His6 Chromatography Loading buffer: pH > 7.5 M NaCl glycerol Wash buffer: pH > 7. 0.1-0. 0. IMAC Chromatography Purification of a 6His tag protein on Ni-agarose column material elution with imidazole . GSH/GSSG ● . overnight binding ● use a 10-histidine construct ● use urea/denaturing conditions ●Proteolytic degradation ● add protease inhibitors ● protease site? ●Interaction with gel material ● increased salt concentration ● glycerol ● detergents ●Reducing conditions ● metals may get reduced in presence of DTT.IMAC Chromatography Poor binding of His 6 constructs ● detergents/lipids prevent binding. pre-clean in another step ● slow kinetics of binding. Choice of Chromatography Steps What needs to be seperated ●How much impurities do I expect ●What is the volume of the starting material ●Can affinity-tags be engineered ●What is the pI ● Initially low resolution Fast Flow with cheap material ● Ion exchange step ● IMAC step ●Better separation with higher resolution ion exchange step ●Polishing with gel filtration step ● . Small scale experiments Normal chromatography Small scale experiments pasteur pipet or spin columns 100-500 ul column screen pH. salts Advantages good control high yield Advantages less buffer required faster . Batch experiments Normal chromatography Batch Column material is mixed with solution and separated by centrifugation Advantages good control high yield Advantages less control faster larger particles . smaller molecules penetrate into the porous column material and elute slower .Gelfiltration or Size exclusion chromatorgraphy Size differences are exploited to separate complexes or molecules. what fraction of the stationary phase is available for the molecule Kd = (Ve – Vvoid)/(Vtotal.Vvoid) KAV = 0 Vvoid high Mw Vintern Vtotal low Mw Vvoid Vvoid-Vtotal Vintern Vtotal KAV = 1 .Vvoid – Vgel) Vtotal = h x 2π r2 Vvoid determined from Ve of large molecules KAV = (Ve – Vvoid)/(Vtotal.Gelfiltration or Size exclusion chromatography Vtotal=Vvoid + Vintern + Vgel Elution volume Ve = Vvoid + KdVintern (absence of specific interactions) Partitioning coefficient. Gelfiltration:KAV log Mw 5.2 0.9 1 KAV Lineair relation between KAV (or Ve) and log Mw.7 0.3 0.5 0.25 0 0. with a standard gelfiltration columns can be calibrated .6 0.75 2.4 0.1 0.75 3.25 3 2.5 2.75 4.8 0.25 5 4.25 Log Mw 4 3.5 3.5 4. Gelfiltration:sample size . Gelfiltration:sample size . Gelfiltration:column size . Gelfiltration Gelfiltration analysis technique Aggregation state: monomers. Estimation of Mw for globular molecules Gelfiltration separation Separation on size Removal of large aggregates Desalting/removal of small molecules Issues Slow flow. trimers etc. small-medium scale Detergent micelles add to size of membrane proteins Weight differences are “ smaller” . dimers. Strategy Purification without chromatographic steps Affinity tags? His-tag Anion exchange (FF) Affinity columns High resolution anion exchange Gelfiltration . Proteins migrate in a pH gradient under the influence of a electric field. until their net charge is zero when the environment pH equals the pI of the protein. overall charge pH Anode + pI Cathode - .Iso Electric Focusing Proteins are seperated on the basis of their pI. Iso Electric Focusing Iso electric focusing can be performed in acryl-amide gels. in large gel-filtration gel beds. polyamino polycarboxy-buffers . multi-charged organic buffer mixtures with spaced pI. specialized devices Analytical to determine pI and purity Preparative for purification and separation + anode + lowers pH + + increase pH cathode - - pH gradients are developed using ampholine solutions. 32.39. 4. 4. 4.42.36. 4.20 .24.Iso Electric Focusing IEF separation of LHC complexes with pI 4.27. 4. 4. Iso Electric Focusing Excellent separation method Proteins need to be soluble at pI Preparative IEF requires some additional equipment . .Gelfiltration ● Globular protein: M = ¾ pi r3 ρprot N log M = 3 log r + log b non-globular proteins – – ● ● D = kT/f f = 6 π η Rs diffusion coeff. η viscosity R=Rs Rs R1<Rs<R2 . Rs stokes radius. f is friction coeff.