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高等生化學
Advanced Biochemistry
Amino Acids, Peptides and
Proteins
陳威戎
2011. 03. 14
Amino Acids, Peptides and Proteins
1. Amino Acids
2. Peptides and Proteins
3. Working with Proteins
4. Covalent Structure of Proteins
Amino Acids
Proteins are polymers of amino acids, with each amino acid
residues joined to its neighbor by a specific covalent bond.
Twenty different amino acids are commonly found in
proteins.
First: asparagine (1806) ; last: threonine (1938).
Names derived from the sources:
Asparagine – asparagus
Glutamate – wheat gluten
Tyrosine – cheese
Glycine – sweet taste
1. Amino acids share common structural features.
General structure of an amino acid
Two conventions used to identify the carbons in an
amino acid
The a-carbon atom is a chiral center.
The absolute
configuration of
substituents of
the asymmetric
carbon atom
Stereoisomerism in
a-amino acids
2. The amino acids residues in proteins are
L stereoisomers
Steric relationship of the stereoisomers of alanine
to the absolute configuration of glyceraldehyde
3. Amino acids can be classified by R group.
3. Amino acids can be classified by R group.
Absorption of ultraviolet light by aromatic amino acids
Reversible formation of the disulfide bond
4. Uncommon amino acids also have important
functions
4. Uncommon amino acids also have important
functions
5. Amino acids can act as acids and bases
Beer-Lambert Law
Absorbance (Optical density) = log I0/I = ecl
6. Amino acids have characteristic titration curves
Effect of the chemical environment on pKa
7. Titration curves predict the electric charge
of amino acids
Isoelectric point,
Isoelectric pH, pI
■
環
境
影
響
分
子
的
帶
電
性
質
：
Buffer pH
10
9
8
7
Isoelectric point,
pI
+
6
5
4
3
0
Net Charge of a Protein
-
8. Amino acids differ in their acid-base properties
8. Amino acids differ in their acid-base properties
Peptides and Proteins
1. Peptides are chains of amino acids
Formation of a peptide bond by condensation
1. Peptides are chains of amino acids
The pentapeptide serylglycyltyrosylalanylleucine, or
Ser-Gly-Tyr-Ala-Leu
2. Peptides can be distinguished by their
ionization behavior
3. Biologically active peptides and polypeptides
occur in a vast range of sizes
Many small peptides exert effects at very low concentrations:
Aspartame (2 a.a.): artificial sweetener
Oxytocin (9 a.a.): secreted by posterior pituitary
stimulate uterine contractions
Bradykinin (9 a.a.): inhibits inflammation of tissues
Thyrotropin-releasing factor (3 a.a.): formed in hypothalamus
stimulates the release of thyrotropin from the anterior pituitary
Amanitin: toxic mushroom poison
Antibiotics
3. Biologically active peptides and polypeptides
occur in a vast range of sizes
4. Peptides have characteristic
amino acid compositions
5. Some proteins contain chemical groups other
than amino acids
6. There are several levels of protein structure
Working with Proteins
1. Proteins can be
separated and
purified
Crude extract
Fractionation
Ammonium sulfate
(salting out)
Dialysis
Column chromatography
Column
chromatography
Ion-exchange
chromatography
Column
chromatography
Size-exclusion
(Gel filtration)
chromatography
Column
chromatography
Affinity
chromatography
Purification Table
3. Unseparated proteins can be quantified
Activity versus specific activity
2. Proteins can be separated and characterized by
electrophoresis
SDS 在蛋白質表面均勻附上一層負電荷：
SDS
原態蛋白質
boiling
變性蛋白質成一線狀分子
■ 單元體分子量的測定： SDS-PAGE
Mol mass
kD
330
220
kD
300
200
kD
94
67
60
100
80
60
50
40
30
67
43
36
30
20
20.1
10
0.5
Migration (Rf)
1.0
18.5
14.4
Pharmacia: Molecular Markers for electrophoresis
Isoelectirc focusing
Two-dimensional electrophoresis
Covalent Structures of Proteins
1. The amino acid sequences of
millions of proteins have been
determined.
Frederick Sanger
2. Short polypeptides are sequenced using
automated procedures.
3. Large proteins must be sequenced in
smaller segments.
1. Breaking disulfide bonds
2. Cleaving the polypeptide chain: proteases
3. Sequencing of peptides
4. Ordering peptide fragments
5. Locating disulfide bonds
Breaking disulfide bonds in proteins
Cleaving the polypeptide
chain: proteases
Cleaving proteins and sequencing and
ordering the peptide fragments
4. Amino acid sequences can also be
deduced by other methods
1. New methods based on mass spectrometry permit
sequencing of short polypeptides (20-30 a.a.) in just a few
minutes.
2. Development of rapid DNA sequencing methods.