specific organelles.
? Purification methods are based on properties of proteins.
--- Content provided by FirstRanker.com ---
? Methods of Proteins Purification includes chromatography techniques.
? Protein denaturation and renaturation.
Introduction
--- Content provided by FirstRanker.com ---
? First step in protein purification is to break and open these cells, releasing their
proteins into a solution called a crude extract.
--- Content provided by FirstRanker.com ---
? Differential centrifugation can be used to prepare subcellular fractions or toisolate specific organelles.
? Once the extract or organel e preparation is ready, various methods are available
--- Content provided by FirstRanker.com ---
for purifying one or more of the proteins.
Differential Centrifugation
--- Content provided by FirstRanker.com ---
Fig1.8 a: Lehninger Principles of Biochemistry by David L NelsonMethods of Proteins Purification
All purification techniques are based on:
? Stability
--- Content provided by FirstRanker.com ---
? Solubility? Size
? Charge
? Binding affinity
? Hydrophobicity
--- Content provided by FirstRanker.com ---
Chromatographic techniques separate one protein from another based upondifference in their:
? Size (size exclusion chromatography),
--- Content provided by FirstRanker.com ---
? Charge (ion-exchange chromatography),? Hydrophobicity (hydrophobic interaction chromatography),
? Ability to bind a specific ligand (affinity chromatography).
Dialysis
--- Content provided by FirstRanker.com ---
? Proteins separated from small molecules such as salt by dialysis through a
semipermeable membrane with pores.
--- Content provided by FirstRanker.com ---
? Protein mixture is placed inside the dialysis bag, submerged in a buffer solutionthat devoid of small molecules to be separated away.
? Molecules dimensions significantly greater than the pore diameter are retained
--- Content provided by FirstRanker.com ---
inside the dialysis bag.
Cont--
? Smaller molecules and ions
--- Content provided by FirstRanker.com ---
capable of passing through thepores of the membrane diffuse
down
--- Content provided by FirstRanker.com ---
their
concentration
--- Content provided by FirstRanker.com ---
gradients and emerge in thesolution outside the bag.
? Useful for removing a salt or other
--- Content provided by FirstRanker.com ---
small molecule from a cell
fractionate, but it will not
--- Content provided by FirstRanker.com ---
distinguish between proteinseffectively.
Fig. 3.2. Protein molecules (red) are retained within the dialysis bag, whereas smal molecules
--- Content provided by FirstRanker.com ---
(blue) diffuse into the surrounding medium. Biochemistry 7th edition by Jeremy M. Berg, John
L. Tymoczko and Lubert Stryer.
--- Content provided by FirstRanker.com ---
Hydrophobic Interaction Chromatography? It separates proteins based on their tendency to associate with a stationary phase
matrix coated with hydrophobic groups (eg, phenyl Sepharose, octyl Sephadex).
--- Content provided by FirstRanker.com ---
? Proteins with exposed hydrophobic surfaces adhere to the matrix via hydrophobic
interactions enhanced by employing a mobile phase of high ionic strength.
Cont--
--- Content provided by FirstRanker.com ---
? After non-adherent proteins are washed away, polarity of the mobile phase is
decreased by gradually lowering its salt concentration.
--- Content provided by FirstRanker.com ---
? If the interaction between protein and stationary phase is strong, ethanol orglycerol added to the mobile phase to decrease its polarity and weaken
hydrophobic interactions.
--- Content provided by FirstRanker.com ---
Short hydrophobic-interaction chromatography video attached with email
Column Chromatography
--- Content provided by FirstRanker.com ---
? Porous solid material with appropriate chemical properties (the stationary phase)held in a column, and a buffered solution (the mobile phase) pass through it.
? Protein-containing solution, layered on the top of the column, pass through the
--- Content provided by FirstRanker.com ---
solid matrix as an ever-expanding band within the larger mobile phase.
? As the mobile-phase liquid emerges from the column, it is automatically collected
--- Content provided by FirstRanker.com ---
in a series of small portions called fractions.Column Chromatography
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
--- Content provided by FirstRanker.com ---
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 reservoir at the top. Theprotein 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 band within 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
--- Content provided by FirstRanker.com ---
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 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
--- Content provided by FirstRanker.com ---
example, protein A is wel separated from B and C, but diffusional spreading prevents complete separation of B and C under these conditions. Fig 3.17: Lehninger Principles of Biochemistry by David L NelsonShort column chromatography video attached with email
High-Pressure Liquid Chromatography (HPLC)
--- Content provided by FirstRanker.com ---
? Column materials are very finely divided and, as a consequence, more interaction
sites and greater resolving power.
--- Content provided by FirstRanker.com ---
? Column is made of finer material, high-pressure applied to the column to obtainadequate flow rates.
? Net result is both high resolution and rapid separation.
--- Content provided by FirstRanker.com ---
Cont--? In a typical HPLC setup, a detector that monitors the absorbance of the eluate at
a particular wavelength is placed after the column.
--- Content provided by FirstRanker.com ---
? In the sample HPLC elution profile, proteins are detected by setting the detector
to 220 nm (the characteristic absorbance wavelength of the peptide bond).
--- Content provided by FirstRanker.com ---
? In a short span of 10 minutes, a number of sharp peaks representing individualproteins can be readily identified.
Cont--
--- Content provided by FirstRanker.com ---
Gel filtration by HPLC clearly defines
the individual proteins because of its
--- Content provided by FirstRanker.com ---
greater resolving power:1. Thyroglobulin (669 kd),
2. Catalase (232 kd),
3. Bovine serum albumin (67 kd),
--- Content provided by FirstRanker.com ---
4. Ovalbumin (43 kd), and5. Ribonuclease (13.4 kd).
Fig. 4.6. High-Pressure Liquid Chromatography (HPLC): Biochemistry 7th edition by Jeremy M.
--- Content provided by FirstRanker.com ---
Berg, John L. Tymoczko and Lubert Stryer.Short HPLC video attached with email
Ion-Exchange Chromatography
--- Content provided by FirstRanker.com ---
? In mobile phase, proteins with a net positive charge migrate through the matrixmore slowly than those with a net negative charge, because the migration of the
former is retarded more by interaction with the stationary phase.
--- Content provided by FirstRanker.com ---
? Expansion of the protein band in mobile phase caused both by separation of
proteins with different properties and by diffusional spreading.
--- Content provided by FirstRanker.com ---
Cont--? As the length of the column increases, the resolution of two types of protein with
different net charges generally improves.
--- Content provided by FirstRanker.com ---
? Rate at which the protein solution can flow through the column decreases with
column.
Ion-Exchange Chromatography
--- Content provided by FirstRanker.com ---
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 affinity of each protein for the
--- Content provided by FirstRanker.com ---
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 surrounding solution. Separation can be optimized bygradual 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. Short Ion-exchange video attached with email
Affinity Chromatography
--- Content provided by FirstRanker.com ---
? Based on binding affinity of a protein.
? Beads in column covalently attached to chemical group.
? Protein with affinity for this particular chemical group bind to the beads in the
--- Content provided by FirstRanker.com ---
column, and its migration retarded.Affinity Chromatography
Affinity 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
--- Content provided by FirstRanker.com ---
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 interest is eluted (washed out of thecolumn) by a solution containing free ligand. Fig 3.18 c: Lehninger Principles of Biochemistry by David L Nelson.
Short affinity-chromatography video attached with email
--- Content provided by FirstRanker.com ---
Gel-Filtration/Molecular Exclusion Chromatography
? More-discriminating separations on the basis of size.
--- Content provided by FirstRanker.com ---
? Sample is applied to the top of a column consisting of porous beads made of aninsoluble but highly hydrated polymer such as dextran or agarose (which are
carbohydrates) or polyacrylamide.
--- Content provided by FirstRanker.com ---
? Sephadex, Sepharose, and Biogel are commonly used for preparations of these
beads (0.1 mm).
--- Content provided by FirstRanker.com ---
? Small molecules can enter these beads, but large ones cannot.Cont--
? Small molecules distributed in the aqueous solution both inside the beads and
--- Content provided by FirstRanker.com ---
between them, whereas large molecules located only in solution between thebeads.
? Large molecules flow more rapidly through this column and emerge first because
--- Content provided by FirstRanker.com ---
a smaller volume is accessible to them.
? Molecules enter a bead will flow from the column at an intermediate position,
--- Content provided by FirstRanker.com ---
and small molecules, which take a longer path, exit last.Gel-Filtration Chromatography
Fig. 4.3. Biochemistry 7th edition by Jeremy M. Berg, John L. Tymoczko and Lubert Stryer.
--- Content provided by FirstRanker.com ---
Short gel-filtration chromatography video attached with email
Denaturation at the protein levels
At primary structure: Sequence of aa held together by covalent peptide bonds, is
--- Content provided by FirstRanker.com ---
not disrupted by denaturation.
At secondary structure: Proteins lose all regular repeating patterns such as -
--- Content provided by FirstRanker.com ---
helices and -pleated sheets and adopt a random coil shape.Cont--
At tertiary structure: Disruption of covalent interactions between aa side chains
--- Content provided by FirstRanker.com ---
(such as disulfide-bridges bet cysteine groups), non-covalent interactions between
polar aa side-chains, van-der waals interactions between non-polar aa side chains.
--- Content provided by FirstRanker.com ---
At quaternary structure: Protein sub-units are dissociated and/or spatialarrangement of protein subunits is disrupted.
Cont--
--- Content provided by FirstRanker.com ---
? Native Protein denaturate in presence of reducing agent, alter pH, temp, ionic strength,and solubility.
? Denature protein causes loss of structure and function.
--- Content provided by FirstRanker.com ---
Various denaturants are:
1. Heat disrupts hydrogen bonds and hydrophobic interactions bet non-polar residues. Ex.
Albumin in egg denature and coagulate during cooking.
--- Content provided by FirstRanker.com ---
1. Strong acids and bases disrupts the salt bridges formed in a protein structure. Ex. In
digestive system, acidic gastric juices causes coagulation of milk by proteolytic enzyme
--- Content provided by FirstRanker.com ---
renin.Cont--
2. Urea disrupts the stabilizing hydrophobic interactions, thus freeing entire
--- Content provided by FirstRanker.com ---
polypeptide from its folded conformation.
3. Reducing agents like guanidine hydrochloride (GdnHCL) or -mercaptoethanol
--- Content provided by FirstRanker.com ---
reduces the disulphide bonds to sulfhydryl group and breaks intra and interchaindisulphide bonds.
4. Detergents (SDS) disrupts hydrophobic interactions.
--- Content provided by FirstRanker.com ---
Renaturation of Unfolded Proteins? The Nobel prize in chemistry 1972 was divided to Christian B Anfinsen provided
the first evidence that:
--- Content provided by FirstRanker.com ---
Aa sequence of a polypeptide chain contains all information required to fold the
chain into its native, 3-D structure.
--- Content provided by FirstRanker.com ---
? Certain globular proteins denatured by heat, extremes of pH, or denaturingreagents will regain their native structure and their biological activity if returned
to conditions in which the native conformation is stable. This process is called
--- Content provided by FirstRanker.com ---
renaturation.
Renaturation of Unfolded, Denatured Ribonuclease
--- Content provided by FirstRanker.com ---
Lehninger Principles of Biochemistry by David L NelsonSummary
? Proteins are separated and purified based on their properties.
--- Content provided by FirstRanker.com ---
? Proteins precipitated by the addition of certain salts.? Chromatographic procedures makes use of differences in size, binding affinities,
charge, and other properties.
--- Content provided by FirstRanker.com ---
? These include ion-exchange, size-exclusion, affinity, and high-performance liquid
chromatography.
--- Content provided by FirstRanker.com ---
Cont--? The 3-D structure and the function of proteins destroyed by denaturation, which
demonstrate the relationship between structure and function.
--- Content provided by FirstRanker.com ---
? Some denatured proteins can renature spontaneously to form biologically active
protein.
Reference Books
--- Content provided by FirstRanker.com ---
1) Harper 's Il ustrated Biochemistry-30th edition.
2) Biochemistry 7th edition by Jeremy M. Berg, John L. Tymoczko and Lubert Stryer.
3) Lehninger Principles of Biochemistry by David L Nelson.
4) Short you-tube video clips on types of chromatography.
--- Content provided by FirstRanker.com ---
33
Thank you
--- Content provided by FirstRanker.com ---