Download Analysis of Proteins and Peptides

Analysis of Proteins and Peptides
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Amino acid composition
Molecular weight
Isoelectric point
Subunit structure
Prosthetic groups
Solubility
Biological activity
Rule: Before a protein can be analyzed, it must first be
isolated in a pure state, i.e., purified
Rule: Protein purification means separating the protein of
interest from other proteins and components
Separation Strategies
Protein from H2O
Protein from salt and small ions
Protein from other proteins
Protein from H2O (Concentration)
Precipitation
Salting out (ammonium sulfate)
Organic solvents (ethanol, acetone)
Heavy metals or salts (trichloroacetic acid)
Force protein-protein interactions above protein solvent
Dehydration
Lyophilization (cold evaporation)
Osmosis
Protein from salt and small ions
Start
Size-dependent diffusion
Dialysis
Gel filtration
End
Dialysis
Gel Filtration
Principle of Gel Filtration
Penetrating the
beads slows the
migration rate
of the smaller
particles
Protein from Protein
Rule: Protein-Protein separations is based on differences
in physical properties of the proteins
Major:
Size or molecular weight
Charge at a given pH (isoelectric point)
Unique structural features
Minor:
Solubility in water, salt solutions, or organic solvents
Rule: All effective methods for separation of proteins
employ the techniques of CHROMATOGRAPHY
Chromatography:
Mobile phase
Stationary phase
(lit., separation by color)
Moving force
Opposing force
Types:
Column chromatography - gravity or pump driven
Electrophoresis - driven by electric current
Osmotic movement
Medium:
Porous gels
Charged resins
Gas-liquid
Affinity resins
Paper
(size exclusion)
(charge affinity)
(hydrophobic interactions)
(group recognition)
(no longer in use)
Properties of Proteins
Detecting Proteins
Ultraviolet Absorbance at 280 nm
Trichloroacetic acid precipitation
Quantifying Proteins
Nitrogen content (16%) Kjeldahl test
Colorimetric Tests
See Strategies
Bradford analysis
pp 61-67
Absorbance at 280 nm
E 1% = 10 (10 mg/ml)
STRUCTURE ANALYSIS
Composition
Acid Hydrolysis 6 N HCl, 105 oC, 18 hours
Breaks all peptide bonds
Sequence Analysis
Edman Degradation
Sequential removal from N-terminus
CHARACTERIZATION
MOLECULAR MASS
Kilodaltons
MOLECULAR WEIGHT No Units
TRUE Molecular Weight
Sedimentation Analysis
RELATIVE Molecular Weight (MR)
SDS (Sodium dodecylsulfate)
electrophoresis
Gel Filtration
TCA Precipitation
Protein Solution
White Precipitate
Signifies Protein
TCA will remove protein from a solution
CHARGE PROPERTIES
H+
OH
Chromatography based on Charge
Ion exchange (cation or anion)
+
+ +
+ +
DEAE
column
+
+
+
+
+
+
+
NaCl
eluent
Diethylaminoethyl
(DEAE) group attached
to the cellulose
H CH2-CH3
CH2-CH2-N
+ CH2-CH3
Salt gradient
Most negatively charged protein
comes off last
Electrophoresis
Movement of a charge particle in an electric field
pH-dependent (native protein)
Sodium dodecyl-sulfate (SDS)
Denatures protein
Uniform charge/mass ratio
Uses
Assess protein purity
Determine relative Mwt (SDS)
Relative Mwt dependent on chain length
Isoelectric Focusing (IEF)
Principal
A protein will move in an electric field only when
it has a net charge on its structure.
Application
Establish a stable pH gradient in the medium
Mixture of organic acids with different pKa’s (ampholytes)
Uses
Assess protein purity
Determine isoelectric point (pI) of a protein