Download Chapter 5 Separations: I) Based on Charge or pI A) Electrophoresis

Chapter 5
Separations:
I) Based on Charge or pI
A) Electrophoresis
An electric field is applied across a solid support (polymer gel, starch, paper). The solid support
is saturated with buffer/protein solution. Depending on the charge of the protein it will move towards
either the cathode (-) or the anode (+) or remain stationary (if pH=pI).
Example: Place four different proteins in a buffer solution at a particular pH (also use this pH for the
running buffer).
Protein A
+6
Protein B
0
Protein C
-12
Protein D
-5
B) Isoelectric focusing- electrophoresis technique
The solid support contains a pH gradient. The proteins placed onto the solid support will move
through the solid support until they reach the pH that is equal to their pI.
Protein A pI= 6.8
Protein B pI= 4.6
Protein C pI=9.2
C) Ion Exchange
Place either cation exchange resins (-) or anion exchange resins (+) in a column.
The degree to which proteins bind to the resins depends on the magnitude of charge on the protein at a
certain pH. For a cation exchange column, start with an elution buffer with a low pH (if the pI<pH the
protein will have a net positive charge and will stick to the resin) and increase the elution buffer pH
until all proteins are eluted from the column as pH>pI the protein becomes negative and elutes from the
column).
A pI= 6.8
B elutes first, then A, and finally C
B pI =4.6
Can use a UV-VIS detector to determine when the protein is eluting from the
C pI=9.2
column.
D) Capillary electrophoresis
Electrophoresis within a fused silica capillary. This technique has high separation efficiency,
utilizes very small sample amounts, and requires only minutes for a run.
More + charged molecules have the highest mobility through the column, while more – charged
molecules have the slowest mobility. Can use a UV-VIS detector to detect when proteins are eluting
from the column.
II) Based on Size or Molecular Weight
A) Ultracentrifugation
Proteins subjected to a centrifugal force move in the direction of the force at a velocity
dependent on mass. Measure the rate of sedimentation in Svedburg units (S=1x10-13 sec).
1-200 S for proteins
B) Size exclusion chromatography
Fine, porous beads (agarose, polyacrylamide) are packed into a chromatography column. The
pore size of these beads approximates the dimensions of macromolecules. As a solution of
macromolecules is passed through the column, the molecules distribute between the solution and the
pores, depending on their ability to enter the pores. The larger the molecule, the more quickly it passes
through the column, while smaller molecules spend more time in the pores and will elute from the
column later.
C) Polyacrylamide gel electrophoresis with detergent- SDS PAGE
An electrophoresis technique in which proteins migrate through the solid support based on
molecular weight. The SDS molecules (-2 charge) surround the protein, and the running buffer contains
SDS as well. The SDS molecules are attracted to the anode and sets up a flow through the PAG.
Smaller proteins can move more easily through the gel (which acts as a molecular sieve) and will travel
faster through the gel than larger proteins.
III) Other
A) HPLC-high-performance liquid chromatography
In reverse-phase HPLC, the molecules partition between a nonpolar stationary phase (C4, C8,
C18) and a polar liquid phase (MeCN). Solute molecules are eluted from the column in proportion to
their solubility in this more polar liquid. Here, the most polar solutes will come off the column first,
while the more nonpolar solutes elute last. Therefore, molecules are separated based on their
hydrophobicity.
B) Affinity Chromatography
Useful for separating a single desired molecule from a mixture of molecules. A ligand that
specifically binds the protein of interest is covalently attached to an inert matrix. Example polystyrene
bead-biotin is specific for the protein avidin.
An immunoaffinity column uses antibodies as the ligand. The antibody is attached to an inert
matrix and placed into a column. A protein mixture is then passed over the column. Only the antigen
(protein of interest) will bind to the antibody. Everything else is then washed out of the column. The
protein can be eluted from the column by an acidic solution (denatures antibody and releases antigen).
Determination of Amino Acid Sequence:
1→Must denature the protein first.
2→Then break the polypeptide chain into smaller fragments with:
proteolytic enzymes (endopeptidases)
Trypsin cleaves protein after R and K residues
Chymotrypsin cleaves after aromatics F, Y, W
Elastase cleaves after small hydrophobic residues
Chemical
cyanogens bromide cleaves after Met residues
3→Determine the amino acid sequence of each peptide fragment by:
1) Edman degradation (see figure 4.22)
In weakly basic solutions, phenylisothiocyanate will combine with the N-terminal amino acid in
a peptide and result in the cleavage of this amino acid from the chain as a phenylthiohydantoin (PTH)
derivative. The PTH derivative can then be identified by chromatographic techniques by its retention
time compared to standards.
Advantages: This can be fully automated.
Disadvantages: 1) Must have pure peptide (so will need to separate all tryptic fragment peptides
prior to analysis).
2) Need at least 10 pmol of peptide
3) Will not recognize derived amino acids
4) Can not do if N-terminus is blocked
2) Mass Spectrometry (see figure 4.25)
Tandem mass spectrometry can be used to determine the amino acid sequence of peptides.
Advantages: 1) Need only small amount of peptide (amol range 10-18)
2) Peptide does not need to be pure
3) Can do modified amino acids and peptides with blocked N-terminus
Disadvantages: Need an experienced person to run the instrument and interpret the spectra
4→ Repeat steps 2 and 3 using a different proteolytic enzyme to cleave the protein to produce
overlapping peptide fragments.
5→Reconstruct the overall amino acid sequence of the protein using the sequences in the overlapping
fragments.
* Exopeptidases cleave amino acids from the ends of a peptide or protein.
Carboxypeptidases will cleave amino acids sequentially from the C-terminus. There are
different types (A, B, C, and Y) that are effective for different amino acids.
Aminopeptidases will cleave amino acids sequentially from the N-terminus.
Determination of 3-D structure of proteins
X-ray diffraction (X-ray crystallography) – A technique that directly images molecules (see page 144).
Requires the formation of a protein crystal (hard for scarce proteins and membrane proteins). This
technique provides extensive knowledge of protein structure but it is only one static formation of a
dynamic protein.
Evaluating Protein Structure and Function
A) UV Spectroscopy- Can be used to study changes in a protein’s secondary or tertiary structure.
Examples:
1. Folded and unfolded proteins have absorbance maxima at different
wavelengths based upon the environment of the aromatic amino acids.
2. A disordered structure and an α-helix have absorbance maxima at different
wavelengths because the peptide bond in an α-helix interacts with electrons above
and below the bond.
3. Can evaluate substrate binding. The substrate may absorb at a certain
wavelength but does not absorb at that wavelength in the bound state.
B) NMR- Can use 2D NMR to obtain solution confirmation. Can study localized environments and
interactions using NOESy spectra (through space coupling).
C) Circular Dichroism (CD)- Can be used to determine the amount and type of secondary structure
in a protein. Example: can be used to study changes in protein folding by studying the
absorbance of α-helical structures in proteins.