Biochemistry I Lecture 21 Oct 19, 20051Lecture 21: Protein Purification and Quaternary and Tertiary StructureReading in Campbell: Chapter 5, 4.5Key Terms:• Chromatographic Steps• Molecular Weight Determination• SDS Gel ElectrophoresisTypeType of ResinPrincipal of SeparationHow to Elute the ProteinAnionExchangeSeparationby charge.• Beads with apositivechargeProtein sticks to resin becauseof:• Overall negative charge (anions)• Proteins have patches of negative charge• Increase salt concentration to weaken electrostatic interaction.• Change of pH to pH < pI(protein becomes positively charged)CationExchangeSeparationby charge.• Beads with anegativechargeProtein stick to resin because of:• Overall positive charge (cations)• Proteins have patches of positive charge• Increase salt concentration to weaken electrostatic interactions.• Change of pH to pH > pI(protein becomes negatively charged)Hydro-phobicChromato-graphySeparationby hydro-phobicity• Beads with ahydrophobicgroupProtein sticks to resin in high saltbecause of:• Hydrophobic patchesonsurface ofproteininteract withhydrophobicgroups onresin. (In high salt, less water isavailable to form a shell aroundthese hydrophobic patches).• Decrease saltconcentration to weakenhydrophobic interactions(hydrophobic patches are onceagain surrounded by a shell ofwater)Biochemistry I Lecture 21 Oct 19, 20052• Beads with aligand:Protein stick to resin because of:• Binding site for ligand• Excess ligand• Change in pH, salt, solvent to weaken protein- ligand interaction.AffinityChromato-graphySeparationby affinity.• or AntibodyProtein stick to resin because of:• Binding to antibody• Changes in solution conditions (pH, salt, solvent) to weaken protein-antibody interaction.GelFiltration:Separationby size• Proteins don't "stick".• Small proteins enter the interior of the beads, and therefore take longer to wash off of the column.• Simply washing thecolumn with buffer willeventually wash theproteins out of the column.• Smaller proteins elute last.Size & Quaternary Structure DeterminationNative Molecular Weight:Gel filtration (sizeexclusion).The matrix or beads in thegel filtration column containspores that allow smallermolecules to enter butexclude larger molecules.The larger the molecule theless time it spends in thebeads and the sooner it iswashed (eluted) from thecolumn.• Large proteins elute first.• Small proteins elute last.Biochemistry I Lecture 21 Oct 19, 20053The volume that it takes for a particular proteinto elute from a column is called the elutionvolume, Ve. For example, if a protein wascontained in the 69th mL of liquid that drippedfrom the column then its elution volume wouldbe 69 mL.The relationship between the molecular weightof the protein and its elution volume is given bythe following equation: † log(MW ) =aVE+bwhere a and b are constants obtained bymeasuring the elution volume ofproteins with known sizes. Gelfiltration columns must be calibratedwith known proteins.Note: Gel filtration is usually performed under conditions where the quaternary structure of theprotein is preserved, giving the native molecular weight.Denatured (subunit) Molecular weight: SDS polyacrylamide gel electrophoresis (SDS-PAGE):In SDS gel electrophoresis, proteins are denatured to form individual polypeptide chainsand then they are forced through a gel by an electric field. For proteins, acrylamide gels (achemically crosslinked polymer) are used. This material is very similar to Jello in its macroscopicproperties, i.e. a tightly knit web of polymer fibers. After the experiment the gels are stained witha protein specific stain to visualize the separated protein bands.These gels act as size exclusion media, impeding the motion of larger protein. As theproteins are forced through the gel, they separate according to size.• Smaller proteins have a much easier moving and thereforemigrate a larger distance.• Larger proteins have a more difficult time and therefore migratea smaller distance.Remarkably, the relationship between the distance migrated, d, and themolecular weight is:† log(MW ) =gd +e where g and e are constants determined bycalibration with known standards.Denaturation by SDS:The proteins are denatured by the use of a detergent called SDS(sodium dodecyl sulfate). This negatively charged detergent binds to theprotein, giving each protein a constant charge-to-mass ratio. It isestimated that each residue in the protein binds approximately 3 SDSmolecules.The formation of auniform charge to massratio is importantbecause the actual velocity of a charged particle in an electric field depends on the ratio of thecharge to the mass of the particle. † v µqM Since all proteins have the same charge to massBiochemistry I Lecture 21 Oct 19, 20054ratio after treatment with SDS, they will migrate in the same direction, toward the positiveelectrode, or anode. (Anions, or negatively charged particles, migrate towards the anode). In freesolution (e.g. without the gel) they would all migrate with the same velocity because their intrinsiccharge has been overwhelmed by the charge from the SDS. However, they separate accordingto molecular weight because of the sieving properties of the gel.Disulfide Bonds:If proteins are crosslinked by disulfide bonds (e.g. antibodies), and it is desirable to obtain thesizes of the individual subunits, then the S-S bonds have to be broken using b-mercaptoethanol(bME) before the electrophoretic separation. b-mercaptoethanol reduces a disulfide bond to freeCysteines:Example Determination of Quaternary Structure:An enzyme consists of four polypeptide chains. Two chainsare 20 kDa in size (a-chain) and two are 30 kDa in size (b-chain). There is a single disulfide bond between the a and bsubunits. The four chains associate as indicated in thediagram to form a hetero-tetramer, (ab)2.The following three experiments were performed:1. Gel filtration chromatography2. SDS-PAGE in the absence of b-mercaptoethanol3. SDS-PAGE in the presence of b-mercaptoethanolTwo proteins with known molecular weights, one witha molecular weight of 10 kDa and the other with a molecular weight of 160 kDa, were alsoincluded in these experiments. These two standards consist of a single polypeptide chainand therefore give a single