Amino Acids,

Peptides and Proteins

Introduction
• Asam amino adalah senyawa yang mempunyai gugus karboksil (satu at lebih)
dan gugus amino (satu at lebih).
• Proteins : biopolymers of α-amino acids joined by amide linkages called peptide
bonds
• Biological functions:
- Enzymes and hormones : proteins catalyze and regulate the reactions that occur
in the body
- Muscles and tendons : provide the body with the means for movement
- Skin and hair : give it an outer covering
- Hemoglobin molecules : transfer all-important oxygen to its most remote
corners
- Antibodies : provide protection against disease
Introduction
Structure and Stereochemistry of the
α-Amino Acids
• Except for glycine, the α-amino acids are all chiral
• In every case (except glycine), the chirality center is the asymmetric α carbon
atom
 The Standard Amino Acids of Proteins
• There are 20 a-amino acids, called the standard amino acids, that are
found in nearly all proteins
The Standard Amino Acids of Proteins
The Standard Amino Acids of Proteins
 Amino Acids as Dipolar Ions
• Amino acids contain both a basic group (-NH2) and an acidic group (-CO2H).
• In the dry solid state, amino acids exist as dipolar ions, a form in which the
carboxyl group is present as a carboxylate ion, -CO2-, and the amino group is
present as an aminium ion, -NH3+ (Dipolar ions are also called zwitterions)
• In aqueous solution, an equilibrium exists between the dipolar ion and the
anionic and cationic forms of an amino acid.
Struktur dipolar ini membrikan sifat yang khas, yaitu :
1. Pada umumnya asam amino berupa kristal dan terdekomposisi pada suhu
tinggi dibandingkan dengan amina dan asam karboksilat yang besesuaian
2. Tidak larut dalam pelarut nonpolar, tetapi larut dalam air.
3. Mempunyai momen dipol yang tinggi dibandingkan senyawa asam atau basa
pada umumnya.
4. Mempunyai sifat asam dan basa.
5. Mempunyai struktur ion dipolar.
 Amino Acids as Dipolar Ions

• The predominant form of the amino acid present in a solution depends on

the pH of the solution and on the nature of the amino acid.
• In strongly acidic solutions all amino acids are present primarily as cations
• in strongly basic solutions they are present as anions
• The isoelectric point (pI) is the pH at which the concentration of the dipolar
ion is at its maximum and the concentrations of the anions and cations are
equal
Assignment
Explain the Acid-Base Properties of Amino Acids and how to
calculate Isoelectric Point
 Polypeptides and Proteins
• Enzymes polymerize amino acids by forming amide linkages
• Peptide : A molecule formed by joining amino acids together
• Peptide bonds or peptide linkages : the amide linkages in peptide
• Each amino acid in the peptide is called an amino acid residue
• Peptides that contain 2, 3, a few (3–10), or many amino acids are called
dipeptides, tripeptides, oligopeptides, and polypeptides

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 Polypeptides and Proteins
• N-terminal residue: One end of a polypeptide chain terminates in an amino acid
residue that has a free -NH3+ group
• C-terminal residues : One end of a polypeptide chain terminates in an amino acid
residue that has a free -CO2- group

• Peptide and protein structures written by the N-terminal amino acid residue on the left
and the C-terminal residue on the right

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 Hydrolysis of Protein
• A polypeptide can be hydrolyzed by refluxing with 6M hydrochloric acid for 24h
• The individual amino acids can be separated from each other using a cation-exchange
resin
• An acidic solution of the amino acids is passed through the cation-exchange column
 the strength of adsorption varies with the basicity of each amino acid (the most
basic are held most strongly)
• Washing the column with a sequence of buffered solutions causes the amino acids to
move through it at different rates

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Hydrolysis of Protein
• Ninhydrin : the hydrate of indane-1,2,3-trione.
• the α-amino acids in proteins react with ninhydrin to give the same
intensely colored purple anion (λmax 570 nm)

• the most common method now: High-performance liquid

chromatography (HPLC)

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Primary Structure of Polypeptides and Proteins

• Primary structure : The sequence of amino acid residues in a polypeptide

or protein
• Terminal residue analysis techniques used to identify the N- and C-
terminal amino acids:
- Edman Degradation
- Sanger N-Terminal Analysis
- C-Terminal Analysis
- Peptide Sequencing Using Mass Spectrometry and Sequence Databases

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 Edman Degradation
• Edman degradation method can be used to sequence peptides up to about 60 residues in
length
• Principle: labeling reaction between the N-terminal amino group and phenyl isothiocyanate
to form a phenylthiocarbamyl derivative

• Phenylthiocarbamyl derivative then cleaved from the peptide chain by acid  an unstable
anilinothioazolinone (ATZ), which rearranges to a stable phenylthiohydantoin (PTH) derivative of
the amino acid.

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 Edman Degradation
• PTH derivative  HPLC  comparison of its retention time with known
amino acid PTH derivatives

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 Sanger N-Terminal Analysis
• The N-terminal end of the polypeptide is labeled with 2,4-dinitrofluorobenzene
in mildly basic solution and the polypeptide is hydrolyzed
• The labeled N-terminal amino acid is separated from the mixture and identified

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 C-Terminal Analysis
• C-Terminal residues can be identified through the use of digestive enzymes
called carboxypeptidases.
• Carboxypeptidases specifically catalyze the hydrolysis of the amide bond of the
amino acid residue containing a free -CO2H group, liberating it as a free amino
acid.
• A carboxypeptidase will continue to attack the polypeptide chain that remains
 it is necessary to follow the amino acids released as a function of time

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Peptide Sequencing Using Mass Spectrometry and
Sequence Databases
Ladder Sequencing
• Mass analysis of proteins with very high precision
• Enzymatic digest is prepared that yields a mixture of peptide fragments
that each differ in length by one amino acid residue
• Mass spectrometric analysis of this mixture yields a family of peaks
corresponding to the molecular weight of each peptide
• Each peak in the spectrum differs from the next by the molecular
weight of the amino acid that is the difference in their structures
Struktur Protein
• Protein yang tersusun dari rantai asam amino akan memiliki berbagai macam
struktur yang khas pada masing-masing protein. Karena protein disusun oleh
asam amino yang berbeda secara kimiawinya, maka suatu protein akan
terangkai melalui ikatan peptida dan bahkan terkadang dihubungkan oleh
ikatan sulfida. Selanjutnya protein bisa mengalami pelipatan-pelipatan
membentuk struktur yang bermacam-macam. Adapun struktur protein meliputi
struktur primer, struktur sekunder, struktur tersier, dan struktur kuartener.
Examples of Polypeptide and Protein Primary Structure

Oxytocin and Vasopressin

• Their amino acid sequences similar but different physiological effects.
• Oxytocin occurs only in the female of a species and stimulates uterine
contractions during childbirth.
• Vasopressin occurs in males and females; it causes contraction of
peripheral blood vessels and an increase in blood pressure.
Its major function is as an antidiuretic; physiologists often refer to
vasopressin as an antidiuretic hormone.

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Struktur primer
• Struktur primer merupakan struktur yang sederhana dengan urutan-urutan
asam amino yang tersusun secara linear yang mirip seperti tatanan huruf
dalam sebuah kata dan tidak terjadi percabangan rantai
Struktur Sekunder
• Struktur sekunder protein terbentuk akibat adanya ikatan hidrogen dari atom
C, O, H dan N yang melekat pada asam amino penyusunnya. Secara umum,
terdapat dua jenis struktur sekunder protein, yakni alpha-helix dan beta-sheet.
Struktur alpha helix menyerupai lilitan tali dan dapat terlihat dari dua arah
berbeda yakni arah putar kanan (right-handed) ataupun putar kiri (left-
handed). Sementara itu, struktur beta-sheet menyerupai pelat datar yang saling
bertindihan dengan rantai polipeptida yang berikatan sejajar juga melalui
ikatan hidrogen. Hampir serupa dengan alpha helix, beta-sheet memiliki dapat
berorientasi paralel atau antiparalel.
Struktur Tertier
• Struktur tersier protein mencapai bentuk tiga dimensi dari interaksi asam
amino, muatan ion, ataupun gugus yang terdapat pada struktur sekunder.
Interaksi ini memungkinkan terjadinya pelipatan protein (protein folding) dari
struktur sekunder. Protein folding dapat terjadi melalui ikatan hidrogen, atau
ikatan disulfida yang terdapat pada struktur alpha-helix ataupun beta-sheet.
Struktur Quarterner
• Struktur kuarterner adalah gambaran dari pengaturan sub-unit atau
promoter protein dalam ruang. Struktur ini memiliki dua atau lebih dari sub-
unit protein dengan struktur tersier yang akan membentuk protein kompleks
yang fungsional.
Examples of Polypeptide and Protein Primary Structure
Insulin
• a hormone secreted by the pancreas, regulates glucose metabolism
• Insulin deficiency in humans is the major problem in diabetes mellitus
• Bovine insulin has a total of 51 amino acid residues in two polypeptide chains, called
the A and B chains, joined by two disulfide linkages
• The A chain contains an additional disulfide linkage between cysteine residues at
positions 6 and 11
• Human insulin differs from bovine insulin at only three amino acid residues:
Threonine replaces alanine once in the A chain (residue 8) and once in the B chain
(residue 30), and isoleucine replaces valine once in the A chain (residue 10).

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Sintesis Asam Amino

1. Aminasi asam α-halogen

2. Sintesis Gabriel Ftalamida
Salah satu metode sintesis asam amino yangpaling baik adalah
kombinasi antara sintesis amina dari gabriel dan sintesis ester
malonat dari asam karboksilat. Sintesis ester malonat dari Gabriel
dimulai dengan ester N-ftalimido malonat, kemudian direaksikan
dengan etil kloroasetat. Setelahdihidrolisis dengan HCl panas,
-
dilanjutkan dengan penambahan basa kuat (OH ) sehinga diperoleh
produk berupa asam amino aspartat.
3. Sintesis Strecker
Reaksi ini terdiri dari 2 tahap, mula-mula aseltaldehid ditambahkan ke dalam
larutan amonia dan asam sianida, menghasilkan α-aminopropionitril, kemudian
dihidrolisis sehingga diperoleh 60% campuran (D,L)-alanina
Sintesis Peptida
• Gugus karboksilat & amino dapat bereaksi  dilindungi
• Benzil klorokarbonat, t-butil oksikarbonilazida, benzil kloroformat, benzil
oksikarbonil , dll  harus mudah dilepaskan diakhir reaksi
TERIMA KASIH