Amino Acids: Structure, Analysis, and Sequence in peptides

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Amino Acids Structure, Analysis, and Sequence
(in peptides)
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Structures of the Amino Acids
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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) Gly G
• alanine (R CH3) Ala A
• phenylalanine (R CH2C6H5) Phe F
• tyrosine (R CH2C6H4OH) Tyr Y
• serine (R CH2OH) Ser S
• cysteine (R CH2SH) Cys C
• methionine (R CH2CH2SCH3) Met M
• leucine (R CH2CH(CH3)2) Leu L

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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.

Cation
Neutral
Anion
(zwitterion form)
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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
aspartic acid alanine lysine
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Ion Exchange Chromatography
D- elutes first, followed by A K elutes last,
and only after pH of buffer is increased and K
is deprotonated.
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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.

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Derivatization with Ninhydrin
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.
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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.
injection
Increase pH of buffer
Retention time
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Polyacrylamide Gel Electrophoresis (PAGE)
Before current is turned on
After current is turned on
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The Strecker amino acid synthesis
(racemic alanine)
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Resolution of racemic amino acids
D-
L-
L-amino acid D-N-acetylamino acid
Racemic amino acid
Racemic N-acetyl amino acid
Carboxypeptidase hydrolyzes the amide bond ONLY
of the L-aa, leaving the unnatural
D-N-acetylamino acid unreacted separation is
simple
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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).
• In addition to amide bonds, a second kind of
  covalent bond exists in some peptides in which
  two cysteine residues (amino acids) are connected
  through a disulfide bond formed by oxidation
  (dehydrogenation) of the sulfhydryl (SH) groups
  (next slide).

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Disulfide bonding in peptides
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Total Hydrolysis conversion of a peptide
into a mixture of its component amino
acids
Ion Exchange Chromatogram
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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.
• Well learn three methods
• Sangers reagent followed by total hydrolysis
• Carboxypeptidase
• Leucine aminopeptidase

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Sangers Reagent N-terminal Amino Acid Analysis
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Sangers Reagent, contd
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Carboxypeptidase C-terminal AA Analysis
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Ion Exchange Chromatograms following
Carboxypeptidase
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Leucine aminopeptidase N-terminal AA Analysis
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Ion Exchange Chromatograms following Leucine
Aminopeptidase
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Partial Hydrolysis
Peptide represented schematically
(some molecules)
(other molecules)
(some other molecules)
(different molecules of the peptide can fragment
differently, leading to a mixture)
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Putting it all together!

• Suppose an unknown hexapeptide gave tagged A
  (alanine) upon treatment with Sangers 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?