Download Classification of Amino Acids

Chapter 3
Amino Acids, Peptides,
and Proteins
Amino Acids
Amino Acids
 20 Residues
 Numbering of carbons
 1,2,3... from C of COO a , b, g… from C bonded
to NH3+ and COO-
 Absolute configuration of
amino acids
 L amino acids in
biological system
1
2
a
Classification of Amino Acids
Nonpolar
UV absorption at 280 nm
Classification of Amino Acids
• Nonpolar
• Structural role
Classification of Amino Acids
Uncommon Amino Acids
 Collagen
 Myosin
 Cell wall
 Collagen
 Prothrombin
 Ca2+ binding proteins
 Elastin
 A few proteins
 Incorporation
during
translation
Amino Acids as Acids and Bases
 Zwitterion
 Acts as either an acid or a base
 Ampholytes
 Amphoteric substances
 Substances with dual nature
 Amino acid is a diprotic acid
Titration of Amino Acids
 Two pKa and two
buffering regions
 pI: ioselectric point or
isoelectric pH
 The point with zero
electric charge
 Above pI : negative
charge
 Below pI : positive
charge
 pI = (pK1 + pK2)/2 = 5.92
Effect of Chemical Environment on
pKa
Amino Acids with Ionizable R Group
 pI = (pK1 + pKR)/2 = 3.22
 pI = (pKR+ pK2)/2 = 7.59
Peptides and Proteins
Peptides and Proteins
 Peptide bond
 Dehydration reaction
 Polypeptide vs. protein
 Polypeptide: Mr<10,000
 Amino-terminal (N-terminal)
 Carboxyl-terminal (C-terminal)
Ionization of Peptide
 Ionization of peptide
 One free a-amino group
 One free a-carboxyl
group
 Inonizable R groups
 pKa of R groups in
peptide
 Different from pKa of free
amino acid
Biologically Active Peptides and
Polypeptides
 Size
 Small peptide
 Vertebrate hormones

 Oxytocin (9), thyrotropin-releasing factor (3), insulin (30 + 21)
Antibiotics

Large proteins

Most of the proteins
 Titin: vertebrate muscle protein (27,000 a.a.)
 < 2,000 a.a.
 Oligomeric status
 Single polypeptide chain
 Multisubunit proteins
 Oligomeric : at least two subunits are identical
 Protomers : identical units
 Calculation of the number of amino acid residues
 Mr / 110
 Average Mr of 20 a.a. : 138
 Average Mr of protein a.a : 128
 Removal of water during peptide bond formation : 128 -18 =110
Conjugated Proteins
 Conjugated proteins
 Proteins with permanently associated chemical
components
 Prosthetic group
 Non-amino acid part of a conjugated protein
Working with Proteins
Protein Purification
 Cell lysis
 Crude extract
 Fractionation
 Use differences in protein solubility
 Depending on pH, temperature,

salt concentration etc.
Salting out
 Addition of ammonium sulfate
((NH4)2SO4) for differential
precipitation of proteins
 Dialysis
 Exchange of salts and buffer using
semipermeable membrane
 Column chromatography
 Separation of proteins based on
charge, size, binding affinity etc.
 Stationary phase and mobile
phase containing protein
Ion-exchange chromatography
 Cation-exchange
chromatography
 Solid matrix :
negatively charged
group
 Positive charged
proteins migrate
slowly
 Anion-exchange
chromatography
 Solid matrix :
positively charged
group
Size-Exclusion Chromatography
 Solid matrix :
Beads with
engineered pores
of cavities
 Small proteins
enter the pores
 Slow migration
Affinity Chromatography
 Beads with covalently attached chemical group
 Binding of proteins with affinity for the chemical
group
Protein Purification
 HPLC (high-performance liquid chromatography)
 Use high pressure pump that speed the movement
of the protein molecules
 Limited diffusion  High resolution
 Determining the methods for protein purification
 Mostly empirical
Separation of Protein by
Electrophoresis
 Electrophoresis
 Separation of charged proteins in an electric
field
 Electrophoretic mobility of proteins
 Depending on size and shape of proteins
 SDS gel electrophoresis (SDS PAGE)
 SDS (sodium dodecyl sulfate) binds to
proteins roughly proportional to the
molecular weight of the protein
 Binding of 1 SDS/ 2 amino acids


 Similar chare to mass ratio for all the proteins
 Similar shape for all the proteins
Separation of proteins depending on the
mass
 Useful to determine molecular weight
Visualization of the bands by staining (e.g.
Goomassie blue)
Determining Molecular Weight of a
Protein
SDS PAGE
(polyacrylamide gel electrophoresis)
Isoelectric Focusing
 Procedure to determine
the pI of a protein
 Establishment of pH
gradient
 Gel containing a mixture
of low molecular weight
organic acids and bases
(ampholytes)
 Application of electric
field
 Each protein migrates
until it reaches the pH
corresponding to its pI
Two-Dimensional Electrophoresis
 1o : Isoelectric focusing
 2o : SDS-PAGE
Quantification of Proteins During the
Purification
 Enzyme assay
 1 unit of enzyme activity
 The amount of enzymeo causing transformation of 1.0 mmol
of substrate/min at 25 C under optimal conditions
 Activity
 Total units of enzyme in solution
 Decrease during purification
 Specific activity
 The number of enzyme units/ mg of total proteins
 Measure of enzyme purity
 Increase during purification
 Other assays
 Biological activities
 DNA binding, alteration of cell growth
 Detection of the presence of the target protein
 Western blotting