Transcript Analysis of amino acids and peptide primary structure determination

Amino Acids:
Structure, Analysis,
and Sequence (in peptides)
Structures of the Amino Acids
O
C
H2N C
OH
O
H
H2N C
C
OH
O
H
HO C
C
general structure
of an amino acid
L-serine
O
C
H2N
H
C
HO
L-glyceraldehyde
H
H
CH2OH
CH2OH
CH2OH
R
O
H
Fischer projection
of L-glyceraldehyde
OH
H
Fischer projection
of an L-amino acid
R
Examples of the 20 common amino acids:
Neutral amino acids: -R = -H, -CH3, -CH(CH3)2, -CH2SH, -CH2OH, -CH2C6H5, -CH2C6H4OH
Acidic amino acids: -R = -CH2CO2H, -CH2CH2CO2H
Basic amino acids:
-R = -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH2)2
Abbreviations of Amino Acids
• Amino acids have 1-letter and 3-letter abbreviations; the
1-letter abbreviations are used almost exclusively today,
but you should also be aware of the older 3-letter
abbreviations.
• Some examples:
–
–
–
–
–
–
–
–
glycine (R = H)
alanine (R = CH3)
phenylalanine (R = CH2C6H5)
tyrosine (R = CH2C6H4OH)
serine (R = CH2OH)
cysteine (R = CH2SH)
methionine (R = CH2CH2SCH3)
leucine (R = CH2CH(CH3)2)
Gly
Ala
Phe
Tyr
Ser
Cys
Met
Leu
G
A
F
Y
S
C
M
L
Isoelectric Point
• Each amino acid has an isoelectric point, (pI) numerically equal to
the pH at which the zwitterion concentration is at a maximum.
• The amino acid has no NET charge at its pI; it has one positive and
one negative charge.
• At a pH less than the value of the isoelectric point, the amino acid is
protonated and has a POSITIVE charge; at a pH greater than the pI
the amino acid is deprotonated and has a NEGATIVE charge.
O
C
H3N
O
OH
C
H
H
R
H3N
O
O
C
OH
H
R
O
H2N
H
R
@ pH < pI
@ pH = pI
@ pH > pI
Cation
Neutral
Anion
(zwitterion form)
Separation and Analysis using pI values
• Differences in isoelectric points (and therefore charges) are used to
separate mixtures of amino acids by two common methods:
– Ion exchange chromatography
– Polyacrylamide gel electrophoresis (PAGE)
These methods will be illustrated with a simple mixture
of three amino acids having very different isoelectric points:
H O
H3N C CO
Mixture of:
buffered at pH 6.0
CH2CO2
H O
H O
+
D (pI=2.8)
aspartic acid
H3N C CO
+
CH3
A (pI=6.0)
alanine
H3 N C CO
CH2CH2CH2CH2NH3
K (pI= 9.7)
lysine
Ion Exchange Chromatography
H O
H3N C CO
Mixture of:
buffered at pH 6.0
CH2CO2
+
H3N C CO
D (pI=2.8)
SO3 K
sulfonated
polystyrene
H O
H O
SO3
+
H3 N C CO
CH2CH2CH2CH2NH3
CH3
A (pI=6.0)
K (pI= 9.7)
(strongly retained)
A (slightly retained,
&
D (unretained)
SO3
D- elutes first, followed by A; K+ elutes last, and only
after pH of buffer is increased and K+ is deprotonated.
Ion Exchange Chromatography
• Recall that in our simple mixture D- elutes first, followed
by A; K+ elutes last, and only after the pH of buffer is
increased and K+ is deprotonated.
• But there is a problem in detecting amino acids; they are
colorless, and most of them have very little absorption in
the UV region (they have no conjugation, except in the
four aromatic amino acids)
• To overcome this difficulty, amino acids are converted
(after separation by ion exchange chromatography) to a
derivative using ninhydrin.
Derivatization with Ninhydrin
O
O
H O
OH
2
+
OH
O
N
H3N C CO
O
R(any)
O O
Ninhydrin (2 mol) reacts with one mol of ANY amino acid to give
the SAME blue colored product. This reaction is performed post-column,
after Ion Exchange Chromatography separation of a mixture of amino
acids. The area of each peak in the chromatogram is proportional
to the relative molar amount of the amino acid of that retention time.
Ion Exchange Chromatography
Recall that in our simple mixture D- elutes first,
followed by A; K elutes last, and only after the
pH of buffer is increased and K+ is deprotonated.
D
injection
A
Increase pH of buffer
Retention time
K
Polyacrylamide Gel Electrophoresis (PAGE)
H O
H3N C CO
Mixture of:
buffered at pH 6.0
CH2CO2
H O
H O
+
H3N C CO
H3 N C CO
+
CH2CH2CH2CH2NH3
CH3
D (pI=2.8)
A (pI=6.0)
Before current is turned on:
K
A
D
After current is turned on:
K
A
D
K (pI= 9.7)
The Strecker amino acid synthesis
O
CH3CH
NH
NH3
KCN, H2O
NH2
CH3CH
CN
CH3CH
H3O
heat
+
NH2
CH3CH
CO2H
(racemic alanine)
Resolution of racemic amino acids
CO2H
D-
H
NH2
H
O O
R
CH3COCCH3
+
NHCCH3
H
NHCCH3
R
R
Carboxypeptidase
+
CO2H
L- H2N
CO2H O
CO2H O
H
R
Racemic amino acid
O
+
CO2H
CH3CHN
H
R
Racemic N-acetyl amino acid
CO2H
H2N
H
R
L-amino acid +
D-N-acetylamino acid
Carboxypeptidase hydrolyzes the amide bond ONLY of the L-aa,
leaving the unnatural D-N-acetylamino acid unreacted; separation is simple
Covalent bonding in peptides
• Amino acids are covalently bonded to one another by amide
linkages (bonds) between the carboxylic acid group of one amino
acid and the amino group of the next amino acid.
• Amide bonds are strong and are resistant to hydrolysis, but there
are enzymes that catalyze their hydrolysis (to the amino acids).
H O
H2N C C OH
R1
H O
+
H2N C C OH
R2
peptidase
enzyme
H O
H2N C C
R1
H H O
N C C OH
R2
• In addition to amide bonds, a second kind of covalent bond exists
in some peptides in which two cysteine residues (amino acid units)
are connected through a disulfide bond formed by oxidation
(dehydrogenation) of the sulfhydryl (SH) groups (next slide).
Disulfide bonding in peptides
H H O H H O H
H O H
H O H
H O H
H O
N C C N C C N
C C N
C C N
C C N
C C
CH3
CH(CH3)2 H
CH3
CH2
CH2CH2SCH3
SH
SH
CH2
C
H
CH(CH3)2
CH3
C
N
C C
N C C
N C
O H
H
O H
H O H
H O H
C
CH2OH
N C
CH3
C N C C N
H O H H O H H
[O]
H H O H H O H
H O H
H O H
H O H
H O
N C C N C C N
C C N
C C N
C
C
CH3
CH(CH3)2 H
CH3
CH2
C N
C
CH2CH2SCH3
S
S
CH2
C
H
CH(CH3)2
CH3
C
C
C
N
C C
N C
O H
H
O H
H O H
N C
H O H
CH2OH
N C
CH3
C N C C N
H O H H O H H
Total Hydrolysis: conversion of a peptide
into a mixture of its component amino acids
H O H H O H
H3N C C N C
CH3
C N
CH2OH
A
H O H
H O H
H O H
H O
C
C
C
C
C N
CH2
C N
CH(CH3)2 H
F
S
C N
CH2CH2SCH3
M
G
V
C O
H3O, heat
(total hydrolysis)
H O
H3N C CO
CH3
A
H O
+
H3N C CO
CH2OH
S
H O
H O
+
H3N C CO
CH2
F
+
H3N C CO
H O
H O
+
H3 N C CO
CH(CH3)2
+
H3N C CO
CH2CH2SCH3
H
V
G
M
(equimolar mixture of
A, S, F, V, G, and M )
S
Ion Exchange Chromatogram:
G
A
V
M
F
2. Amino Acid Sequence: Primary
Structure Determination of Peptides
• Total hydrolysis followed by ninhydrin derivatization and
ion exchange chromatography tells us the identity and
relative amount of each amino acid present in the
peptide
• It gives NO INFORMATION about the sequence, or order
of attachment of the amino acids, however.
• For this, we need to perform selective hydrolysis of the
peptide.
• We’ll learn three methods:
– Sanger’s reagent followed by total hydrolysis
– Carboxypeptidase
– Leucine aminopeptidase
Sanger’s Reagent: N-terminal Amino Acid Analysis
H O H H O H H O H H O H H O H
H3N C C N C C N
CH3
A
CH2OH
S
H O
C C N
C C N C C N
C C O
CH2
CH(CH3)2 H
CH2CH2SCH3
F
V
M
G
Sanger's Reagent
(2,4-dinitrofluorobenzene)
O2N
H H O H H O H H O H H O H H O H
H O
N C C N C C N
CH3
NO2
CH2OH
C C N
C C N C C N
C C OH
CH2
CH(CH3)2 H
CH2CH2SCH3
Sanger’s Reagent, cont’d
O2N
H H O H H O H
H O H
H O H
H O H
H O
N C C N C
C C N
C
C C N
C
CH2
CH(CH3)2 H
CH3
C N
CH2OH
C N
C OH
CH2CH2SCH3
NO2
H3O, heat
(total hydrolysis)
H H O
O2N
N C C OH
+
CH3
"tagged" A
H O
H3N C CO
CH2OH
S
H3N C CO
CH2
F
+
H3N C CO
H O
H O
H O
H O
+
NO2
+
H3N C CO
+
H3N C CO
CH(CH3)2
H
CH2CH2SCH3
V
G
M
("tagged" A plus an equimolar mixture of S, F, V, G, and M )
Carboxypeptidase: C-terminal AA Analysis
H O H H O H
H3N C C N C C N
CH3
A
CH2OH
H O H
H O H
H O H
H O
C
C
C C N
C C O
C N
CH2
CH(CH3)2 H
F
S
C N
CH2CH2SCH3
M
G
V
Carboxypeptidase
H O H H O H
H O H
H O H
H O
H3N C C N C C N
C C N
C C N
C CO
CH2
CH(CH3)2 H
CH3
CH2OH
H O
H3N C CO
+
CH2CH2SCH3
M
Carboxypeptidase
H O H H O H
H O H
H O
H3N C C N C C N
C C N
C
CH2
CH(CH3)2
CH3
CH2OH
CO
H O
+
H3 N C CO
H
G
Ion Exchange Chromatograms
following Carboxypeptidase
10 min
S
G
A
V
M
F
S
G
A
V
M
F
20 min
30 min
40 min
Leucine aminopeptidase: N-terminal AA Analysis
H O H H O H
H O H
H O H
H O H
H O
H3N C C N C C N
C C N
C C N
C C N
C C O
CH2
CH(CH3)2 H
CH3
CH2OH
A
F
S
CH2CH2SCH3
M
G
V
Leucineaminopeptidase, 10 min
H O H
H O H
H O H
H O H
H O
H3 N C C N
C C N
C C N
C C N
C C O
CH2
CH(CH3)2 H
H O
H3N C CO
+
CH2OH
CH3
A (first aa released)
F
S
CH2CH2SCH3
G
V
M
Leucineaminopeptidase, 10 more min
H O
H3 N C
CO
CH2OH
S (2nd aa released)
+
H3N
H O H
H O H
H O H
H O
C C N
C
C C N
C
CH2
CH(CH3)2 H
F
C N
V
C O
CH2CH2SCH3
G
M
Ion Exchange Chromatograms
following Leucine Aminopeptidase
10 min
S
G
A
V
M
F
S
G
A
V
M
F
20 min
30 min
40 min
Partial Hydrolysis
H O H H O H
H3N C C N C
CH3
C N
CH2OH
A
H O H
H O H
H O H
H O
C
C
C
C
C N
CH2
C N
CH(CH3)2 H
F
S
C N
V
C O
CH2CH2SCH3
G
M
Peptide represented schematically:
A S
F V G M
+
dil H3O
A S
A
A
S
F
F V
+
+
V G M
+
S
F
V
+
+
G M
G M
(some molecules)
(other molecules)
(some other molecules)
(different molecules of the peptide can fragment differently, leading to a mixture)
Putting it all together!
• Suppose an unknown hexapeptide gave “tagged” A
(alanine) upon treatment with Sanger’s reagent, and
upon treatment with carboxypeptidase, the first amino
acid released was M (methionine) followed by G
(glycine)
• Partial hydrolysis gave the following identifiable
tripeptides: V-G-M, A-S-F, and S-F-V. What is the 1º
structure of the hexapeptide?
A S F V G M
A S F V G M
V G M
A S F V G M