? Differential centrifugation
? Chromatography
? Salt Fractionation
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Clinical applications
Protein Fractionation
Protein fractionation required to separate and characterize protein
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Protein differ in their molecular size and charge, can be separated on
basis of following properties:
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? Molecular size? Solubility
? Electric charge
? Binding affinity
? Adsorption properties
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? HydrophobicityCharacteristics of proteins utilized in various separation procedures
Biochemistry. 4th edition by Donald Voet and Judith G. Voet
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Differential CentrifugationFirst step in protein purification is to break and open these cells,
releasing their proteins into a solution called a crude extract.
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Differential centrifugation used to prepare subcellular fractions or to
isolate specific organelles.
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Once extract or organelle preparation is ready, various methods areavailable for purifying one or more of proteins.
Differential Centrifugation
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Fig1.8 a: Lehninger Principles of Biochemistry by David L Nelson
Chromatography
Sample to be examined called solute or analyte allowed to interact
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with two immiscible phases-mobile and stationary phase
These two phases could be solid and liquid, gas and liquid, liquid and
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liquidStationary phase may be solid or liquid supported on solid, does not
move.
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Mobile phase moves sample through stationary phase. Mobile phase
may be liquid or gas
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Cont--Chromatography techniques separate one protein from another
based upon difference in their:
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Based on shape of chromatographic bed:
? Planar (Thin layer and paper chromatography) and Column
Chromatography
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Based on physical state of mobile and stationary phase:
? Gas, Liquid, High-Pressure Liquid Chromatography
Cont--
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Based on mechanism of separation:? Size (size exclusion chromatography)
? Charge (ion-exchange chromatography)
? Ability to bind a specific ligand (affinity chromatography)
? Gel-Filtration/Molecular Exclusion Chromatography
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? Hydrophobicity (hydrophobic interaction chromatography)? Adsorption Chromatography
? Partition Chromatography
Planar Chromatography
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Stationary phase present as or on plane
Plane can be paper impregnated by substance acting as a stationary
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phase called paper chromatographyIf substance acting as stationary phase spread on glass, metal or
plastic plate called thin layer chromatography (TLC)
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In TLC, stationary phase is thin layer of silica gel or alumina on glass,
metal or plastic plate
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TLC detect aminoacidopathiesColumn Chromatography
Porous solid material with appropriate chemical properties (stationary
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phase) held in a column, and a buffered solution (mobile phase) passthrough it.
Protein-containing solution, layered on the top of column, pass
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through solid matrix as an ever-expanding band within larger mobile
phase.
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As mobile-phase liquid emerges from column, it is automaticallycollected in a series of small portions called fractions.
Column Chromatography
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Column chromatography: The standard elements of a chromatographic column include a solid, porous material supported inside a column, general y made of plastic or glass. The solid material (matrix)
makes up the stationary phase through which flows a solution, the mobile phase. The solution that passes out of the column at the bottom (the effluent) is constantly replaced by solution supplied from a
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reservoir at the top. The protein solution to be separated is layered on top of the column and al owed to percolate into the solid matrix. Additional solution is added on top. The protein solution forms a bandwithin the mobile phase that is initial y the depth of the protein solution applied to the column. As proteins migrate through the column, they are retarded to different degrees by their different interactions
with the matrix material. The overal protein band thus widens as it moves through the column. Individual types of proteins (such as A, B, and C, shown in blue, red, and green) gradual y separate from
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each other, forming bands within the broader protein band. Separation improves (resolution increases) as the length of the column increases. However, each individual protein band also broadens with
time due to diffusional spreading, a process that decreases resolution. In this example, protein A is wel separated from B and C, but diffusional spreading prevents complete separation of B and C under
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these conditions. Fig 3.17: Lehninger Principles of Biochemistry by David L NelsonHigh-Pressure Liquid Chromatography (HPLC)
It is liquid chromatography tech i.e; mobile phase is liquid, stationary phase
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may be solid or liquidInstead of solvent (mobile phase) being allowed to drip through a column
under gravity, it is forced through under high pressure
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Column materials are very finely divided and, as a consequence, more
interaction sites and greater resolving power.
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Column is made of finer material, high-pressure applied to column to obtainadequate flow rates.
Net result is both high resolution and rapid separation.
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Cont--
In a typical HPLC setup, a detector that monitors absorbance of
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eluate at a particular wavelength is placed after column.In sample HPLC elution profile, proteins are detected by setting
detector to 220 nm (characteristic absorbance wavelength of peptide
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bond).
In a short span of 10 minutes, a number of sharp peaks representing
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individual proteins can be readily identified.Cont--
Gel filtration by HPLC clearly defines individual
proteins because of its greater resolving power:
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1. Thyroglobulin (669 kd): Large protein
2. Catalase (232 kd),
3. Bovine serum albumin (67 kd),
4. Ovalbumin (43 kd), and
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5. Ribonuclease (13.4 kd).Fig. 4.6. High-Pressure Liquid Chromatography (HPLC): Biochemistry 7th
edition by Jeremy M. Berg, John L. Tymoczko and Lubert Stryer.
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Ion-Exchange Chromatography
In mobile phase, proteins with a net positive charge migrate through
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matrix more slowly than those with a net negative charge, becausemigration of former is retarded more by interaction with stationary
phase.
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Expansion of protein band in mobile phase caused both by separation
of proteins with different properties and by diffusional spreading.
Cont--
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As length of column increases, resolution of two types of protein with
different net charges generally improves.
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Rate at which protein solution can flow through column decreaseswith column.
Ion-Exchange Chromatography
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Ion-exchange chromatography exploits differences in the sign and magnitude of the net electric charges of proteins at a given pH. The column matrix is a synthetic polymer containing bound charged groups;
those with bound anionic groups are cal ed cation exchangers, and those with bound cationic groups are cal ed anion exchangers. Ion-exchange chromatography on a cation exchanger is shown here. The
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affinity of each protein for the charged groups on the column is affected by the pH (which determines the ionization state of the molecule) and the concentration of competing free salt ions in the surroundingsolution. Separation can be optimized by gradual y changing the pH and/or salt concentration of the mobile phase so as to create a pH or salt gradient. Fig 3.18 a: Lehninger Principles of Biochemistry by David L Nelson.
Affinity Chromatography
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Based on binding affinity of a proteinBeads in column covalently attached to chemical group.
Protein with affinity for this particular chemical group bind to beads in
column, and its migration retarded.
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Enabling purification of a biomolecules w.r.t. biological function
Substance to be purified is specifically and reversibly adsorbed to a
ligand, immobilized by a covalent bond to a chromatography bed material
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Substances of interest bound to ligand while unbound substance washed
away
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Affinity ChromatographyAffinity chromatography separates proteins by their binding specificities. The proteins retained on the column are those that bind specifical y to a ligand cross-linked to the beads. (In biochemistry, the term
"ligand" is used to refer to a group or molecule that binds to a macromolecule such as a protein.) After proteins that do not bind to the ligand are washed through the column, the bound protein of particular
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interest is eluted (washed out of the column) by a solution containing free ligand. Fig 3.18 c: Lehninger Principles of Biochemistry by David L Nelson.
Gel-Filtration/Molecular Exclusion Chromatography
More-discriminating separations on basis of size and shape
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Sample is applied to top of a column consisting of porous beads made
of an insoluble but highly hydrated polymer such as dextran or agarose
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or polyacrylamide (act as stationary phase)Small molecules can enter these beads, but large ones cannot
Gel filtration uses aqueous mobile solvent to separate and characterize
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molecules
Cont--
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Small molecules distributed in aqueous solution both inside beadsand between them, whereas large molecules located only in solution
between beads.
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Large molecules flow more rapidly through this column and emerge
first because a smaller volume is accessible to them.
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Molecules enter a bead wil flow from column at an intermediateposition, and small molecules, which take a longer path, exit last.
Gel-Filtration Chromatography
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Fig. 4.3. Biochemistry 7th edition by Jeremy M. Berg, John L. Tymoczko and Lubert Stryer.Adsorption Chromatography
Separation of components present in mixture based on relative
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differences in adsorption of components to stationary phase present in
chromatography column
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Components of mixture travel with different rates due to differences intheir non-covalent interactions with stationary phase
Adsorption involves weak non-covalent interactions bet components of
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mixture and stationary phase
Partition Chromatography
Separation based on solute partitioning between two liquid phases
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Paper chromatography: End of paper dipped into solvent mixture
consists of aqueous and organic components.
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? Solvent soaks into paper by capil ary action because of fibrous natureof paper
? Aqueous component of solvent binds to cellulose of paper and form
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stationary phase with it.
? Organic component of solvent continuous migrating forming mobile
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phaseHydrophobic Interaction Chromatography
It separates proteins based on their tendency to associate with a
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stationary phase matrix coated with hydrophobic groups (eg, phenyl
Sepharose, octyl Sephadex).
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Proteins with exposed hydrophobic surfaces adhere to matrix viahydrophobic interactions enhanced by employing a mobile phase of
high ionic strength.
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Cont--Clinical applications:
? Screening and diagnosis of inherited disorders of aa metabolism e.g.
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phenylketonuria and Tyrosinemia? Peptide fingerprinting for diagnosis of disease like sickle cell anemia
Cont--
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After non-adherent proteins are washed away, polarity of mobile
phase is decreased by gradually lowering its salt concentration.
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If interaction between protein and stationary phase is strong, ethanolor glycerol added to mobile phase to decrease its polarity and
weaken hydrophobic interactions.
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Salt FractionationProtein show variation in solubility depends on conc of salts in
solution.
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Used to separate serum proteins into albumins and globulins,
albumins are soluble and globulins are not soluble in water
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Salting in: Globulins are soluble in weak salt solutions, going intosolution at salt conc 0.1mol/L
? Due to electrostatic attraction between salt ion and charged group on
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protein, which decrease intermolecular electrostatic attraction of
proteins and increases interaction of proteins molecules with water,
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polar solvent making them solubleCont--
Salting Out: Solubility of proteins lowered at high salt concentrations,
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an effect called "salting out."
? As the salt concentration increased, salt ions compete for water
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molecules of hydration of hydrated groups of proteins, resulting indecreased solubility and precipitation of protein out of solution
? Ammonium sulfate ((NH4)2SO4) used for salting out proteins,
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albumin precipitated on fully saturating solution
? Useful to isolate a protein of interest from a mixture of different
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proteins.Summary
Proteins are separated and purified based on their properties.
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Proteins precipitated by addition of certain salts.Chromatographic procedures makes use of differences in size,
binding affinities, charge, and other properties.
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These include ion-exchange, size-exclusion, affinity, and high-
performance liquid chromatography.
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Cont--Polyacrylamide gels (PAG) provide a porous matrix for separating
proteins on basis of their mobility in an applied direct current
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electrical field.
Constant ratio at which anionic detergent SDS binds proteins enables
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SDS-PAGE to separate polypeptides on basis of relative size.Reference Books
33
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1) Harper's Illustrated Biochemistry-30th Ed2) Biochemistry 7th edition by Jeremy M. Berg, John L. Tymoczko and Lubert
Stryer.
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3) Lehninger Principles of Biochemistry by David L Nelson, 6th Ed4) Fundamentals and techniques of biophysics and molecular biology 2nd Ed.
by Pranav Kumar
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5) Principles and Techniques of Biochemistry and Molecular Biology 7th Ed.By Keith Wilson and John Walker
Thank you
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