REVIEW PROPER 1: AMINO ACIDS

PART I: INTRODUCTION AND CHARACTERISTICS
In the structural sense, amino acids are amino-group
containing carboxylic acids.
carboxyl groups. Thus, they are chiral. Based on
absolute stereochemistry:
L isomer - more commonly found in nature, with D
isomers existing but less common.
Glycine only achiral amino acid of the 20.

An amino acid.

However, we do not consider all amino acids as

important for discussion. In biochemistry, we only
use 20: only those which are essential for the
creation of proteins. They are specifically called alpha
amino acids, because the amino and carboxyl groups The chirality of the tetrahedral alpha amino acid.
lie on the same (alpha) carbon.

The general structure of all 20 amino acids.

Glycine is the only achiral alpha amino acid.
.
Generally, an (alpha) amino acid is attached to
hydrogen and a R group in addition to the amino and

Zwitterionic form - +1 and -1 charge lying on the amino and carboxyl groups, respectively at neutral pH, giving a
net charge of 0. Amino acids always exist with at least one formal charge in the body.

Even with a positive and negative charge, the zwitterionic form of

an amino acid is electrically neutral.

PART II: THE 20 AMINO ACIDS

An amino acid may be distinguished from all others by their R groups.
They can be grouped together according to what charge their R group possesses in the neutral pH (neutral, basic,
acidic). Sometimes they are grouped based on the structural similarities among their R groups (aromatic, aliphatic,
amidic, etc.)

NEUTRAL AMINO ACIDS
Further classified in to non-polar and polar. The R
groups of polar amino acids only have a tendency to
be charged in the neutral pH.
Non-polar amino acids are listed in the sequence
aliphatic (G to M), aromatic (F, W) and finally the only
secondary amino acid (P); polar amino acids are listed
in the sequence amides (N, Q), alcohols (S, T), thiol
(C), and phenol (Y).

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 NON-POLAR AMINO ACIDS Asparagine (N, asn)
Amino Acid Structure R: Carbamoyl (amide) +
Glycine (G, gly) methyl
R: Hydrogen

Alanine (A, ala) Glutamine (Q, gln)

R: Methyl R: Carbamoyl + ethyl

Valine (V, val)

R: Isopropyl

Serine (S, ser)

Leucine (L, leu) R: Hydroxymethyl (a
R: Isobutyl primary alcohol group)

Threonine (T, thr)

R: 1-hydroxyethyl (a
Isoleucine (I, ile) secondary alcohol group)
R: sec-Butyl

Cysteine (C, cys)

R: Thiomethyl
C forms a disulfide
Methionine (M, met) linkage when reacted
R: Methylthioethyl with another C
molecule, the product
being named cystine.
Tyrosine (Y, tyr)
R: Phenol + methyl (or 4-
hydroxymethyl)
Phenylalanine (F, phe)
R: Benzyl (phenylmethyl)

CHARGED AMINO ACIDS

Further classified in to acidic and basic. The R groups
Tryptophan (W, trp) of acidic amino acids are negative in neutral pH;
R: Indole ring + methyl those of basic amino acids are positive in neutral pH.

ACIDIC AMINO ACIDS

Amino Acid Structure
Aspartic Acid (D, asp)
Proline (P, pro) R: Carboxyl + methyl
R: Propyl closing on the
alpha nitrogen to form a
pyrrolidone ring
Glutamic Acid (E, glu)
R: Carboxyl + ethyl
POLAR AMINO ACIDS
Amino Acid Structure

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 Arginine (R, arg)
BASIC AMINO ACIDS R: Guanidopropyl
Amino Acid Structure
Lysine (K, lys)
R: Aminobutyl

Histidine (H, his)

R: Imidazole ring +
methyl

Essential and Non-essential Amino Acids: A

Nutritional Classification One letter Amino acid
In the nutritional standpoint, amino acids have two abbreviation
main classifications based on whether they can be P P henylalanine
spontaneously produced by the body or not. V V aline
T T hreonine
Non-essential - can be produced by the body without W T ryptophan
requiring any additional dietary intake of food, and I I soleucine
therefore the food containing these are not essential M M ethionine
to assure that the body has sufficient amino acid H H istidine
count. A A rginine
Essential - can not be produced spontaneously by the L L eucine
body, and food that contain these are required for K L ysine
intake because lack of them will cause amino acid
The ten essential amino acids.
deficiency in the body. There are ten essential amino
acids, given a common acronym PVT TIM HALL

PART 3: TITRATIONS AND THE ISOELECTRIC PH

Titratable group any part of the amino acid that can be protonated (or deprotonated) at a given pH.
Recall that protonation happens when there are many protons around AKA acidic pH.
Isoelectric pH (abbreviated IpH or PI) - pH at which the amino acid possesses no NET charge (aka their zwitterionic
form)

Amino acids can be titrated to achieve the zwitterionic form. At alterations in pH during titration, titratable groups
become protonated or deprotonated. For example, histidine has its carboxyl, amino and imidazole groups
protonated. As the pH increases, the carboxyl group becomes deprotonated, then the imidazole nitrogen, then
the amino group respectively.

At pH 1, the net charge is +2 because all three titratable groups are protonated (the nitrogen atom in the ring
possesses a positive charge). This charge becomes +1 then 0 and -1 as the pH increases. We should realize that
when charge becomes 0, zwitterionic form has been achieved.

The IpH can be computed by getting the average of the two pKa values that flank the 0 net charge. For histidine,
the IpH is 7.585 (IpH = (6.0 + 9.17) / 2).
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PART 4: PEPTIDE BOND FORMATION: ENTRY TO PROTEINS
Peptide bond bond between two amino acids. It is structurally an amide bond.
Condensation - a ANE reaction of two carboxylic acids to form the peptide bond. The carboxyl group of one
(nucleophile) attacks the amino group (electrophile) of the other, creating the peptide bond and releasing water.
Thus, end products are trans-dipeptide and water.

Condensation reaction between two amino acids, leading to a dipeptide and water.
N-terminal end of a peptide/protein with an exposed amino group.
C-terminal end of a peptide/protein with an exposed carboxyl group.
Residue R groups of the bonded amino acids in a peptide/protein.

Bonds in peptides
Psi ( ) bond between the alpha carbon and the carboxyl carbon
Phi () bond between the alpha carbon and the amino nitrogen
Omega () - AKA peptide bond

The bonds existing within peptides.

PART 5: NAMING POLYPEPTIDES

Peptides are classified according to the number of amino acid residues they possess (dipeptide if two AA,
tetrapeptide if 4 AA). A polypeptide refers to a chain of amino acids. An oligopeptide refers to a chain of 30-50
amino acid residues. A protein refers to a chain containing more than 50 residues.

Condensing amino acids also changes their names. The ine suffix is changed into yl for most of the amino acids.
There are exceptions, namely: cysteinyl, tryptophanyl/ tryptophyl, asparagyl, glutaminyl (from glutamine;
glutamyl is used for glutamate), and aspartyl. For example, the nonapeptide CHEMISTRY is also known as
cysteinylhistidylglutamylmethionylisoleucylserylthreonylarginyltyrosine.

PART 6: TITRATING POLYPEPTIDES

Polypeptides, like amino acids, can be titrated as well. Most amino acids have only two titratable groups, the
amino and carboxyl groups. But some amino acids R groups are titratable as well ( ONLY for D, E, H, C, Y, K, and R).
When the pH goes beyond the pKa of a titratable group, it becomes deprotonated.

In tripeptide STC, there are three titratable groups, the amino group from serine, and the carboxyl and thiol group
from cysteine. They all become deprotonated at different pH values.

At pH 1, all titratable groups are protonated. This gives the tripeptide a net charge of +1. At pH 1.71, the carboxyl
group loses its protonation giving it a negative charge. This gives the tripeptide a net charge of 0, and the
zwitterionic form is achieved.

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At pH 8.33, the thiol group loses its protonation. This gives the tripeptide a net charge of -1. At pH 9.15, the amino
group is deprotonated. This gives this tripeptide a net charge of -2 from the charged carboxyl and thiol groups.
- The IpH of the tripeptide is 5.02.

REVIEW PROPER 2: PROTEIN STRUCTURE AND CHARACTERIZATION

PART I: INTRODUCTION
Proteins serve different biological functions.
Examples of which are: catalytic proteins or
enzymes; regulatory proteins like the hormones
insulin and glucagon; transport proteins like
myoglobin and hemoglobin; structural proteins like
collagen and elastin; and defense proteins known
as the immunoglobulins.

Before we enumerate several useful proteins, its

better to review first their structure.

PART 2: LEVELS OF ORGANIZATION OF PROTEINS

1. PRIMARY LEVEL OF ORGANIZATION The alpha helix.

The order/sequence of amino acids of the
peptide chain and the number of amino
acids present
In other words, the linking of the amino
acids by peptide bonds already makes the
primary level
2. SECONDARY LEVEL OF ORGANIZATION
Refers to the hydrogen-bonded arrangement
of the polypeptide chain.
A) -helix
A polypeptide chain forms hydrogen
bonds with itself (intrapolypeptidal H-
bonding), forming a helix
Helix may be right-handed or left-
handed
Pitch the vertical distance in one turn
(5.4 )
There are 3.6 residues per turn (13
atoms)
Rise distance between amino acids
(rise = pitch / 3.6)
Proline cannot be found in an -helix
because its cyclic nature and absence of
hydrogen bonding ability causes a bend
that restricts rotation.
The proximity of side chains with similar
charges causes electrostatic repulsion
therefore causing a strain on the helix.
The proximity of bulky side chains to
each other causes steric crowding
causing straining of the helix.

B) -pleated Sheet

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Parallel and anti-parallel sheets.
Hydrogen bonding between one or two
peptide chains (interpolypeptidal H-
bonding)
May be parallel or anti-parallel
1. Parallel - the C-terminals of the two
chains are going the same direction
2. Antiparallel - the C-terminals of the
two chains are going different
directions
-bulge - localized disruptions of the
polypeptide chain; non-repetitive,
irregular motifs in the anti-parallel
position
Glycine and Proline cause reverse turns
that change the direction of the
polypeptide chain.
Supersecondary Structures
Combinations of -helices and -pleated
sheets (ex. , -hairpin, -meander,
Greek Key, -barrell etc.)
Domains independently folded
structures
Motifs repeated supersecondary
structures
In simplified form, helices a re d rawn as th ey are, while
pleated sheets a re d rawn as arrows. Domains are the
lines connecting helices o r sheets
3. TERTIARY LEVEL OF ORGANIZATION
3-D arrangement of all the atoms in the
polypeptide chain with the side chains,
determined by covalent and non-covalent
interactions within the chain such as:
A. Electrostatic attractions and hydrogen
bonding between R groups
B. Disulfide linkages
C. Metal-ion coordination
D. - complexation interaction of
aromatic molecules with each other
(stacking)
4. QUATERNARY LEVEL OF ORGANIZATION
Same interactions occur but between two
or more polypeptide chains.
PART 3: PROTEIN CONFORMATION AND
DENATURATION
1. Fibrous Proteins rod-like; insoluble because
of high molecular weights; unaffected by pH
and temperature
2. Globular Proteins spherical or ellipsoid;
somewhat soluble because of the exposed
polar groups and unexposed, insoluble inner
core; affected by pH and temperature (ex.
enzymes)
Proteins, in nature, have a native conformation.
When they take on this conformation, they are
biologically ACTIVE.
Chaperones - assist in the correct folding of
proteins (ex. Hsp70).
Ways of disrupting protein organization
Degrading protein structure may disable them of
the activities they perform in native conformation.
However, this can be used as advantage for
practical purposes (ex. egg white becomes more
edible and easier to digest as in cooked form, hair is
easier to style upon heating) and analytical
purposes.
1) Denaturation - process by which a protein loses
its natural conformation by disruption of its
structural order be it quaternary, tertiary, or
secondary, but never primary.
Denaturation may be reversible or irreversible.
Denaturing agents include:
a) Heat - disrupt hydrophobic interactions
b) Detergents such as sodium dodecyl sulfate -
disrupt hydrophobic interactions as well
c) Urea or guanidine - disrupt hydrogen
bonding
d) Mercaptoethanol - reduces disulfide bonds.
e) Large changes in pH alter electrostatic
attractions between side chains especially with
the acidic and basic amino acids.
2) Hydrolysis destruction of primary structure
through hydrolysis of peptide bonds.
PART 4: PROTEINS IN VERTEBRATES
While it is now known that thousands and
thousands of different proteins exist in living
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systems such as our human bodies, those below
comprise very large percentages of our bodily
biochemical composition:
1. Collagen
Structural protein; filamentous
Left-handed triple helix containing 3.3
residues per turn
Contains 800 residues
Distinctive peptide chains containing a
proline or hydroxyproline residue bound
between a glycine residue and another
amino acid (x pro gly or x hyp gly)
Proline is converted into hyroxyproline by
an enzyme known as hydroxylase which
is catalyzed by Vitamin C.
Synthesis of Collagen:
i. Translation into
preprocollagen.
ii. Hydroxylation: formation of
hydroxyproline and
hyroxylysine
iii. Release from ribosomes
and addition of
endoplasmic reticulum
sugars like galactose and
glucose at hydroxylysine.
iv. Formation of the triple
helix and folding of
globular domains;
transformation into
procollagen
v. Secretion from cell
vi. Removal of N and C
terminal domains;
transformation into
tropocollagen
vii. Deamination of lysine to
form allysine
viii. Cross-linkage of allysine
with a Lysine residue, or
another allysine residue to
form collagen.
2. Elastin
Structural protein; filamentous
Found in ligaments and arterial blood
vessels
Non-repetitive coil
Rich in G, A, V, P, but not in
hydroxyproline and hydroxylysine
Four lysine residues (or four allysine
residues) (or two lysine and two allysine)
residues condense to form the desmosine
or isodesmosine crosslink giving elastin
its rubbery characteristic
3. Hemoglobin
Transport protein; globular
Complex of heme, an iron containing
prosthetic group, and globin, the protein
portion which prevents the oxidation of
Fe 2+ into Fe3+
Heme. Each 5-membered ring with
nitrogen at the four corners is called a
pyrrole ring.
Tetramer made of four different protein
subunits each wrapped around a heme
molecule; It has two alpha chains and two
beta chains
Types of Hemoglobin:
i. Hb + O2 Oxyhemoglobin
ii. Hb O2 Deoxyhemoglobin
iii. Hb + CO2
Carbaminohemoglobin
iv. Hb + CO Carboxyhemoglobin
v. Hb + Fe 3+
Ferrihemoglobin/Methemoglobin
vi. Hb + Fe 2+ Ferrohemoglobin
Heme is made of four pyrrole rings that
form one porphyrin ring. It is also known
as an iron protoporphyrin. Each pyrrole
ring forms the first four coordination sites
of heme.
Histidine F8, or the proximal histidine,
binds the iron strongly to the heme. This
forms the fifth coordination site under
the heme molecule.
Histidine E7 distal histidine, forms the
sixth coordination site, allows the
hydrogen bonding of oxygen or carbon
monoxide to the molecule.
Hemoglobins oxygen binding curve is
sigmoidal because it exhibits positive
cooperation. This means, the binding of
one O2 molecule to one coordination site
enhances the attachment of O 2 to the
other sites.
Hemoglobin is an allosteric protein. That
means, it is affected by H+, CO2, and 2,3-
bisphosphoglycerate which decreases O2
affinity to hemoglobin.
HbA is the normal hemoglobin which
has six glutamic acid residues on its beta
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 chain. HbS - hemoglobin of patients with
sickle cell anemia.
In sickle cell anemia, valine replaces
glutamic acid. The ionic interactions of
glutamic acid are replaced by
hydrophoblic interactions of valine. The
cells clump because of this and oxygen
flow gets blocked.
4. Myoglobin
Transport protein; globular
Found mainly in the muscles
Composed of only one protein chain with
a heme prosthetic group in the center
Myoglobins oxygen binding curve is
hyperbolic instead of sigmoidal
Comparisons in structure and oxygen binding curves
of myoglobin and hemoglobin.
5. Insulin
Regulatory protein/ hormone
Produced from the -cells of the Islets of
Langerhans
Composed of two polypeptide chains,
containing 51 amino acid residues, linked
together by intermolecular and
intramolecular disulfide linkages
Also known as hypoglycemic hormone
Breaks down glucose in a process known
as glycolysis to synthesize pyruvate then
ATP
Converts glucose into glycogen, to be
stored in the liver, through a process
known as glycogenesis
Aids in fatty acid synthesis and protein
synthesis
6. Glucagon
Also a regulatory protein/hormone
Brings about glycogenolysis
Produced from the -cells of the Islets of
Langerhans
Single polypeptide chain composed of 29
amino acid residues
Released when blood glucose levels are
below normal; hyperglycemic hormone
7. Immunoglobulins
Also known as antibodies
Defense proteins
Secreted by B-lymphocytes
Composed of two light chains and two
heavy chains with constant and variable
regions (ex. V H is the heavy variable
region, CH is the heavy constant region)
Variable regions bind to the antigens
Constant regions activate immunological
defenses
Typically Y-shaped; some are monomers
(IgD, IgG, IgE), others are dimeric (IgA)
and pentameric (IgM)
PART VII: PROTEIN PURIFICATION
Before we can characterize or describe proteins,
experimental procedures first require us to assure
that the sample we are getting is the pure protein.
Purification techniques below isolate proteins
based on the following properties they possess:
1. SOLUBILITY/POLARITY
a. Isoelectric Precipitation adjusting the
pH of the solution until the protein
reaches its IpH and becomes insoluble
b. Salting Out removing water from
proteins by adding an excess amount of a
salt; makes the protein less soluble
(salting in increases the solubility of
proteins in water by adding a sufficient
amount of a salt)
c. Normal and Reversed Phase
Chromatography protein separation
based on affinities with the mobile or
stationary phases; in normal phase
chromatography, the polar proteins are
the last to be eluted
d. High Performance Liquid
Chromatography (HPLC) uses a column
pre-packed with the stationary phase and
is pressurized
2. MOLECULAR SIZE/WEIGHT
a. Dialysis movement of particles through
a semi-permeable membrane; large MW
proteins will remain inside the dialysis bag
b. Ultrafiltration filtration using a vacuum
c. Ultracentrifugation centrifugating a
solution at various speeds will separate its
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 molecular components; high MW proteins
will settle at a high speed, while low MW
proteins need a higher speed to settle at
the bottom
d. Gel Filtration Chromatography (or Size
Exclusion or Molecular Sieve) uses
porous gel beads such as agarose
(Sepharose) or dextran (Sephadex) which
trap smaller molecules leaving the larger
molecules to be eluted first
e. SDS-PAGE (Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis)
utilizes a detergent, SDS, which imparts a
negative charge to the proteins; smaller
protein molecules treated with the
detergent move faster towards the
positively charged anode
3. CHARGE
a. Electrophoresis separates the proteins
(or amino acids) based on their attraction
towards the negatively charged cathode
or the positively charged anode at a
specific pH
b. Ion Exchange Chromatography uses
cation or anion exchangers (resins); when
a cation exchanger is used, positively
charged amino acids are eluted last
4. BINDING AFFINITY
a. Affinity Chromatography a ligand acts
as the stationary phase to entrap the
protein of interest
b. Precipitation by Antibodies
PART VIII: PROTEIN CHARACTERIZATION
TECHNIQUES
Proteins may be characterized by the types of amino
acids they possess. Complete hydrolysis, using strong
acids or bases, cleaves all peptide bonds leaving
individual amino acids. Incomplete hydrolysis
involves reagents or enzymes which are more
specific and cleave peptide bonds at certain places
only.
1. Edman Reagent phenyl isothiocyanate
(PTH) reagent; cleaves the carboxyl side of
the N-terminal amino acid; the products are
the N-terminal amino acid attached to
phenylthiohydantoin (PTH), and the
remaining peptide
Ex. STC + Edman Reagent (PTH+S) + T-C
2. Cyanogen bromide cleaves the carboxyl
side of methionine
Ex. BIOCHEMISTRY + Cyanogen Bromide
BIOCHEM + ISTRY
3. Proteinases/Proteases/Proteolytic Enzymes
a. Exopeptidases cleave off terminal
amino acids
i. Aminopeptidase cleaves off the N-
terminal amino acid
ii. Carboxypeptidase cleaves off the C-
terminal amino acid
b. Endopeptidases cleave peptide chains
from the inside
i. Trypsin cleave off the carboxyl side
of basic amino acids lysine and
arginine
ii. Chymotrypsin cleave off the
carboxyl side of aromatic amino acids
phenylalanine, tyrosine, and
tryptophan
Ex. Give the correct sequence of a nonapeptide
containing arg, ser, asp, gly, trp, met, ala, phe,
cys.
Edman Reagent: G + PTH
Cyanogen bromide: pentapeptide: M, F, D, S, G
tetrapeptide: W, C, R, A
Aminopeptidase: G
Carboxypeptidase: A
Trypsin: octapeptide: D, M, W, C, G, S, R, F
single alanine residue
Chymotrypsin: tripeptide: F, G, S
tripeptide: M, W, D
tripeptide: C-R-A
Sequence: _ _ _ _ _ _ _ _ _
1. We start by placing G and A at the N and C-
terminals respectively based on the
reactions with the Edman reagent,
aminopeptidase and carboxypeptidase.
G ___ __ _ _A
2. The reaction with chymotrypsin yielded a
tripeptide C-R-A.
G ___ __ CRA
3. The reaction with chymotrypsin yielded two
other tripeptides containing F, G, S and M,
W, D respectively. We know that G is the N-
terminal AA so the tripeptide containing M,
W, D must be somewhere in the middle. We
also know that chymotrypsin cleaves the
carboxyl side of W. We place it beside C.
G ___ _W CRA
4. The reaction with cyanogen bromide yielded
two peptide chains. We know that cyanogen
bromide cleaves at the carboxyl side of M.
We therefore place M beside W.
G ___ MW CRA
5. The reaction with chymotrypsin shows that
the amino acids M, W, and D come as one
tripeptide. We therefore place D beside M.
G __DM W CRA
6. Lastly, we know that chymotrypsin cleaves
the carboxyl side of F. We therefore place
the last two amino acids at their rightful
positions.
G SFDMW CRA
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