Last Lecture: Amino Acids, Peptide Bonds

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Last Lecture Amino Acids, Peptide Bonds Today
Protein 1 Structure,
Purification/Characterization
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Problems to Consider 1. Draw a chemical
mechanism for the reduction of a
disulfide (RS-SR) by (i) b-mercaptoethanol
(BME) (ii) DTT 2. DTT is
a stronger reducing agent than BME. Why? 3.
Draw a chemical mechanism for the formation of an
amide from the reaction of an ester RC(O)OR
with an amine RNH2 (i) in acidic aqueous
media (ie H3O/H2O) (ii) in
basic aqueous media (ie HO-/H2O)
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Proteins - Isolation and Characterization
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Isoelectric Point of Polypeptides
pI pH at which charge is 0
Consider Asp-Ala-Lys
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Ion-Exchange Chromatography
-Separation based on charge Column polystyrene
or cellulose beads with attached functional
groups Anion exchange resin (DEAE) Cation
exchange resin (CM)
DEAE
CM
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Ion-Exchange Chromatography
-Separation based on charge Column polystyrene
or cellulose beads with attached functional
groups Anion exchange resin (DEAE) Cation
exchange resin (CM)
DEAE
CM
(increase salt in buffer)
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Size-Exclusion Chromatography
-Separation based on size/shape Column
crosslinked sephadex beads
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Hydrophobic-Interaction Chromatography
A /- B neutral, hydrophobic
-phenyl (butyl) sepharose
Elution salt (NaCl, KCl) high
to low
A elutes first, B elutes second
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Affinity Chromatography
Antibody
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Affinity Chromatography
Tagged Proteins GST-Tag
Glutathione S-Transferase
Glutatione (GSH)
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Affinity Chromatography
Tagged Proteins His6-Tag Ni2-chelation
Imidazole
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Electrophoresis
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SDS-PAGE
PAGE - polyacrylamide gel electrophoresis
SDS - sodium dodecyl sulfate
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SDS-PAGE
molecular weight markers
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Coomassie blue stain
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SDS-PAGE
-
200 kD
14 kD

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LYMPHOMA SWISS-2DPAGE map
Source http//www.expasy.ch/ch2d/
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Protein Structure
Primary (1) amino acid sequence, disulfide
bridges
Secondary (2) regular structure
- alpha helix (a)
-beta sheet (b)
Tertiary (3) fold of structure
Quaternary (4) interaction of sub-units
-higher order structures
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Protein Folding
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Folding/Unfolding
Folding small energetic advantage
-chaperones
Unfolding temperature, denaturants (SDS, urea,
guanidine) reducing agents
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Peptide Mapping/Sequencing Reagents for Specific
Cleavages
1. Enzymes Trypsin (Lys, Arg) Chymotrypsin
(Phe, Tyr, Trp) Elastase (Ala, Ser, Val,
Gly) Lys-C (Lys),
Trypsin
WDGK TL
ie. WDGKTL
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Peptide Mapping/Sequencing Reagents for Specific
Cleavages
2. Cyanogen Bromide Br-CN, cleavage after Met
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Amino Acid Analysis
- total hydrolysis 6 N HCl 24 hrs - release
of amino acids
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Amino Acid Analysis
sulfonated polystyrene
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Ninhydrin Reaction
purple-blue (lmax 570 nm)
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Peptide Sequencing
http//www.ncbi.nlm.nih.gov/BLAST/
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Peptide Sequencing -Chemistry
Edman Degradation
Phenylisothiocyanate (PTC)
PTC
PTC derivative
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