AMINO ACID, PEPTIDE AND

PROTEIN
General Structure of Amino acid
H
Side chain group
+
amino group H3N C R
(+ ion or cation)
protonated
C Carboxyl group
- (- ion or anion)
O O
Deprotonated

Zwitter ions, Natural pH

2 Negative charge on O delocalized

 Naturally occurring polypeptide

20 amino acid 11 can be synthesize by the adult body

Essential 9 Non essential 4 Conditional 7

Histidine, Alanine, Arginine

Isoleucine, Asparagine, Cysteine,
Leucine, Lysine, Aspartic Acid, Glutamine,
Methionine, Glutamic Acid Glycine, Proline,
Phenylalanine, Serine, and
Threonine, Tyrosine.
Tryptophan, and
Valine.
 NH3+ O
20 amino acid
NH3+ O H C C O-

H C C O- CH3

H
Alanine (Ala) NE

Glycine (gly) C
NH3+ O NH3+ O

H C C O- H C C O-

CH2
Leucine (Leu) E CH
CH H3C CH3
H3C CH3

Valine (Val) E
 NH3+ O
20 amino acid H O
+
NH3 O H C C O-
+
NH2 C C O-
-
H C C O
CH2
H C CH3
CH2
H2C
C SH
CH2 H2

CH3 NH3+ O Cysteine (Cys) C

Proline (Pro) C NH3+ O
-
Isoleucine(Ile) E H C C O

H C C O-
CH2

CH2
CH2

S
Methionine (Met) E
Phenylalanine
CH3 E
 NH3+ O
NH3+ O NH3+ O
H C C O-
H C C O-
H C C O- E
CH2 H C OH
H C OH Serine (Ser) Thereonine (Thr)
CH3
C NH3+ O
H
N
NH3+ O H C C O-
H NH3+ O

Tryptophan (Trp) E H C C O- CH2

H C C O-
CH2 CH2
CH2
C
C NE O NH2
O NH2

C C
10 Non polar Asparagine (Asp)
Glutamine (Glm)
OH Tyrosine (Tyr)
5 polar
2 acidic

NH3+ O
NH3+ O
H C C O-
H C C O-
CH2
CH2
CH2

C
C
O O- O O-

Aspartate (Asp)
Glutamate (Glu)
Aspartic Acid
Glutamic Acid

NE NE
 NH3+ O
NH3+ O
NH3+ O
H C C O-
H C C O-
H C C O-
CH2
CH2

CH2 CH2
CH2

CH2 CH2 C CH

CH2 C NH2+ NH
N
NH3+ NH2
C
H

Arginine (Arg) Histidine (His)

Lysine (Lys)
E
E C

3 Basic
 Amino acid Classes
Amino acids are classified according to their capacity to interact with water.

Nonpolar hydrophobic effects They cannot H-bond with water

Polar H-bond with water and other amino acid

Acidic Contain a carboxylic acid functional group with a negative

charge at neutral pH. Can H-bond with water, can form ionic
interactions, and can also serve as nucleophiles
Nitrogen containing with a net positive charge at neutral pH. Can
Basic serve as proton donors in chemical reactions, and form ionic
interactions.
10
Two compounds with the exact
same connectivity, that are mirror
images of each other but that are
not identical to each other are
called enantiomers.

two enantiomers have the same

physical properties. they have the
same melting point, the same
solubility,
13
Tyrosine (Tyr)
Cysteine (Cys)
 NH3+ O

H C C O-
red-shift or a bathochromic shift.
CH2

NH3+ O

H C C O-

CH2

OH
  Titration
 Add strong base
 Lose proton
 Higher pKa value
 Lower the acid strength

The pH at which this occurs is called

the isoelectric point (pI)

pK1  pK 2
pI 
2
Amino acid with ionizable side
chains have more complex
titration curves.
Eg Glutamic acid
Net charge +1
Base added – alpha carboxyl group lose proton no net charge 0, more base added second carboxyl
group proton and net charge -1, adding additional base ammonium ion lose proton net charge -2

The pI value for glutamate is the pH halfway between the pKa values for the two carboxyl group.

2.19  4.25
pI 
2
Key Concept
Titration is useful in determining the relative ionization potential of acidic and
basic groups in an amino acid or peptide.
The pH at which an amino acid has no net charge is called its isoelectric point.
Amino acid reactions
H
Amino group
Peptide bond
+
H3N C R
Carboxyl group
Disulfide bridge formation
C
Functional group -
O O Schiff base formation
Peptide bond = amide linkage  unshared electron pair of the a amino nitrogen
atom + a carboxyl of another  nucleophilic acyl substitution

A peptide bond forms when the a carboxyl group of one amino acid reacts with the
amino group of another.
A water molecule is formed in the reaction. Amino acid residue

peptide bond formation is a

dehydration reaction
two amino acid molecules  linked  a dipeptide.
glycine + serine  glycylserine or serylglycine.
As amino acids are added and the chain lengthens, the prefix reflects the number of
residues:
a tripeptide contains three amino acid residues, a tetrapeptide four, and so on.
By convention, the amino acid residue with the free amino group is called the N-terminal
residue and is written to the left. The free carboxyl group on the C-terminal residue
appears on the right.
Peptides are named by using their amino acid sequences, beginning from their N-
terminal residue.

is a tetrapeptide named tyrosylalanylcysteinylglycine.

25
Large polypeptides  three-dimensional structures.
® amino acid sequence  single bonds,
® each polypeptide  conformational changes caused by rotation around the single
bonds.
® most polypeptides spontaneously fold into a single biologically active form.
® Using X-ray diffraction studies, they characterized the peptide bond (1.33 Å) as rigid
and planar (flat). Having discovered that the C—N bonds joining each two amino
acids are shorter than other types of C—N bonds (1.45 Å), Pauling deduced that
peptide bonds have a partial double-bond character.
27
CYSTEINE OXIDATION = reversible oxidation that forms a disulfide.
disulfide bridge  intermolecular bridge
help stabilize many polypeptides and proteins
O O

C O- C O-

+
+
H3N C H H3N C H

H C H H C H
2H+ , 2e
SH S

SH S
2H+ , 2e
H C H H C H

H C NH3+ H C NH3+

-
O C -
O C

O O

28
SCHIFF BASE FORMATION
primary amine groups react with carbonyl gp  imine product  Schiff bases
nucleophilic addition reaction,
attacks the electrophilic carbon of a carbonyl group to form an alkoxide product. The transfer of a
proton from the amine group to the oxygen to form a carbinolamine, followed by the transfer of
another proton from an acid catalyst, converts the oxygen into a good leaving group (OH 2). The
subsequent elimination of a water molecule followed by loss of a proton from the nitrogen yields the
imine product. The most important examples of Schiff base formation in biochemistry occur in
amino acid metabolism.
30
Key Concept
 Polypeptides are polymers composed of amino acids linked by peptide bonds. The
order of the amino acids in a polypeptide is called the amino acid sequence.
 Disulfide bridges, formed by the oxidation of cysteine residues, are an important
structural element in polypeptides and proteins.
 Schiff bases are imines that form when amine groups react reversibly with carbonyl
groups.

31
 Isolation (extraction)

Biochemistry in Lab cell disruption and homogenization

isolating,
differential centrifugation
purifying,
(density gradient centrifugation) component of
Characterizing proteins exploit
an organelle
differences in charge,
molecular weight,
and binding affinities.
Purification
In salting out, high concentrations of salts such as ammonium sulfate [(NH4)2SO4] are
used to precipitate proteins.
When proteins are tightly bound to membrane, organic solvents or detergents often aid
in their extraction.
Dialysis
to remove low-molecular-weight impurities such as salts, solvents, and detergents.

more pure,
more sophisticated methods
chromatography and electrophoresis.
Chromatography

In all chromatographic methods the protein mixture

is dissolved in a liquid known as the mobile phase.
As the protein molecules pass across the
stationary phase (a solid matrix), they separate
from each other because they are differently
distributed between the two phases. The relative
movement of each molecule results from its
capacity to remain associated with the stationary
phase while the mobile phase continues to flow.

35
Three chromatographic methods
gel-filtration chromatography, ion-exchange chromatography, and affinity chromatography. Gel-
filtration chromatography is a form of size-exclusion chromatography
in which particles in an aqueous solution flow through a column (a hollow tube) filled with gel and
are separated according to size. Molecules that are larger than the gel pores are excluded and
therefore move through the column quickly. Molecules that are smaller than the gel pores diffuse in
and out of the pores, so their movement through the column is retarded. Differences in the rates of
particle movement separate the protein mixture into bands, which are then collected separately.

36
Ion-exchange chromatography separates proteins on the basis of their charge. Anion-exchange
resins, which consist of positively charged materials, bind reversibly with a protein’s negatively
charged groups. Similarly, cation-exchange resins bind positively charged groups. After proteins
that do not bind to the resin have been removed, the protein of interest is recovered by an
appropriate change in the solvent pH and/or salt concentration.

Affinity chromatography takes advantage of the unique biological properties of proteins. That is,
it uses a special noncovalent binding affinity between the protein and a special molecule (the
ligand). The ligand is covalently bound to an insoluble matrix, which is placed in a column. After
nonbinding protein molecules have passed through the column, the protein of interest is removed
by altering the conditions that affect binding (i.e., pH or salt concentration).

38
Electrophoresis
Because proteins are electrically charged, they move in an electric field. In this process, called
electrophoresis, molecules separate from each other because of differences in their net charge. For
example, molecules with a positive net charge migrate toward the negatively charged electrode
(cathode). Molecules with a net negative charge will move toward the positively charged electrode
(anode). Molecules with no net charge will not move at all. Electrophoresis, one of the most widely
used techniques in biochemistry, is nearly always carried out by using gels such as polyacrylamide
or agarose. The gel, functioning much as it does in gel-filtration chromatography, also acts to
separate proteins on the basis of their molecular weight and shape. Consequently, gel
electrophoresis is highly effective at separating complex mixtures of proteins or other molecules.

39
SDS–polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used variation of electrophoresis
that can be used to determine molecular weight (Figure 5H). SDS, a negatively charged detergent,
binds to the hydrophobic regions of protein molecules, causing the proteins to denature and
assume rodlike shapes. Because most molecules bind SDS in a ratio roughly proportional to their
molecular weights, during electrophoresis SDS-treated proteins migrate toward the anode (+ pole)
only in relation to their molecular weight.

40
41
44
 Mass Spectrometry

Mass spectrometry (MS) is a powerful and sensitive technique for separating, identifying, and determining the
mass of molecules. It exploits differences in their mass-to-charge (m/z) ratios. In a mass spectrometer, ionized
molecules flow through a magnetic field. The magnetic field force deflects the ions depending on their m/z ratios
with lighter ions being more deflected from a straight-line path than heavier ions. A detector measures the
deflection of each ion. In addition to protein identity and mass determinations, MS is also used to detect bound
cofactors and protein modifications. Because MS analysis involves the ionization and vaporization of the
substances to be investigated, its use in the analysis of thermally unstable macromolecules such as proteins and
nucleic acids did not become feasible until methods such as electrospray ionization and matrix-assisted laser
desorption ionization (MALDI) had been developed. In electrospray ionization a solution containing the protein of
interest is sprayed in the presence of a strong electrical field into a port in the spectrometer. As the protein
droplets exit the injection device, typically an ultrafine glass tube, the protein molecules becomes charged. In
MALDI, a laser pulse vaporizes the protein, which is embedded in a solid matrix. Once the sample has been
ionized, its molecules, now in the gas phase, are separated according to their individual m/z ratios. A detector
within the mass spectrometer produces a peak for each ion. In a computer-assisted process, information
concerning each ion’s mass is compared against data for ions of known structure and used to determine the
sample’s molecular identity.

45
Protein sequencing analysis makes use of tandem MS (two mass spectrometers linked in series, MS/MS). A
protein of interest often extracted from a band in a gel, is then digested by a proteolytic enzyme. Subsequently,
the enzyme digest is injected into the first mass spectrometer, which separates the oligopeptides according to
their m/z ratios. One by one, each oligopeptide ion is directed into a collision chamber, where it is fragmented by
collisions with hot inert gas molecules. Product ions, peptides that differ from each other in size by one amino
acid residue, are then sequentially directed into the second mass spectrometer. A computer identifies each peak
and automatically determines the amino acid sequence of the peptides. The process is then repeated with
oligopeptides derived from digestion with another enzyme. The computer uses the sequence information derived
from both digests to determine the amino acid sequence of the original polypeptide.

46
47
 Classification of amino acid

Base on structure
Aliphatic amino acids
A. Mono amino mono carboxylic acids:
Simple amino acids: Glycine, Alanine
Branched chain amino acids:
Valine, Leucine, Isoleucine
Hydroxy amino acids:
Serine, Threonine
Sulphur containing amino acids:
Cysteine, Methionine
Amino acids with amide group:
Asparagine,
48 Glutamine
Aliphatic amino acids
B. Mono amino dicarboxylic acids:
Aspartic acid, Glutamic acid
C. Di basic mono carboxylic acids:
Lysine, Arginine
D. Aromatic amino acids:
Phenylalanine, Tyrosine
E. Heterocyclic amino acids:
Tryptophan, Histidine
F. Imino acid:
Proline

49
 Base on Side chain
Amino acids having nonpolar side chains:
These include Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine and Tryptophan.
These groups are hydrophobic (water repellant) and lipophilic. Therefore, the parts of proteins madeup of
these amino acids will be hydrophobic in nature.

Memory Aid for Essential Amino Acids

"Any Help In Learning These Little Molecules Proves Truely Valuable"

This stands for
Arginine, Histidine, Isoleucine, Leucine, Threonine, Lysine, Methionine, Phenylalanine, Tryptophan
and Valine in that order.
Arginine and Histidine are semi-essential amino acids; while others are essential

50
PROPERTIES OF AMINO ACIDS

Glycine, alanine, valine, serine, tryptophan, histidine and proline are sweet in taste;
leucine is tasteless;
while isoleucine and arginine are bitter.
Sodium glutamate is a flavoring agent.
Aspartame, an artificial sweetener contains aspartic acid and phenyl alanine.
All amino acids have high melting points (more than 200°C).
All amino acids are soluble in water and alcohol (polar solvents); but
insoluble in nonpolar solvents (benzene).

51
 THANKS!
Any questions?

52