REVIEW PROPER 1: AMINO ACIDS

PART I: INTRODUCTION AND CHARACTERISTICS carboxyl groups. Thus, they are chiral. Based on
In the structural sense, amino acids are amino-group absolute stereochemistry:
containing carboxylic acids. 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 Non-polar amino acids are listed in the sequence
Further classified in to non-polar and polar. The R aliphatic (G to M), aromatic (F, W) and finally the only
groups of polar amino acids only have a tendency to secondary amino acid (P); polar amino acids are listed
be charged in the neutral pH. 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 Pitch the vertical distance in one turn
peptide chain and the number of amino (5.4 )
acids present There are 3.6 residues per turn (13
In other words, the linking of the amino atoms)
acids by peptide bonds already makes the Rise distance between amino acids
primary level (rise = pitch / 3.6)
Proline cannot be found in an -helix
2. SECONDARY LEVEL OF ORGANIZATION because its cyclic nature and absence of
Refers to the hydrogen-bonded arrangement hydrogen bonding ability causes a bend
of the polypeptide chain. that restricts rotation.
The proximity of side chains with similar
A) -helix charges causes electrostatic repulsion
A polypeptide chain forms hydrogen therefore causing a strain on the helix.
bonds with itself (intrapolypeptidal H- The proximity of bulky side chains to
bonding), forming a helix each other causes steric crowding
Helix may be right-handed or left- causing straining of the helix.
handed

B) -pleated Sheet

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 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
Parallel and anti-parallel sheets.
and temperature
Hydrogen bonding between one or two 2. Globular Proteins spherical or ellipsoid;
peptide chains (interpolypeptidal H-
somewhat soluble because of the exposed
bonding)
polar groups and unexposed, insoluble inner
May be parallel or anti-parallel core; affected by pH and temperature (ex.
1. Parallel - the C-terminals of the two enzymes)
chains are going the same direction
2. Antiparallel - the C-terminals of the Proteins, in nature, have a native conformation.
two chains are going different When they take on this conformation, they are
directions biologically ACTIVE.
-bulge - localized disruptions of the
polypeptide chain; non-repetitive, Chaperones - assist in the correct folding of
irregular motifs in the anti-parallel proteins (ex. Hsp70).
position
Glycine and Proline cause reverse turns Ways of disrupting protein organization
that change the direction of the Degrading protein structure may disable them of
polypeptide chain. the activities they perform in native conformation.
However, this can be used as advantage for
Supersecondary Structures practical purposes (ex. egg white becomes more
Combinations of -helices and -pleated edible and easier to digest as in cooked form, hair is
sheets (ex. , -hairpin, -meander, easier to style upon heating) and analytical
Greek Key, -barrell etc.) purposes.
Domains independently folded
structures 1) Denaturation - process by which a protein loses
Motifs repeated supersecondary its natural conformation by disruption of its
structures 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
In simplified form, helices a re d rawn as th ey are, while bonding
pleated sheets a re d rawn as arrows. Domains are the d) Mercaptoethanol - reduces disulfide bonds.
lines connecting helices o r sheets e) Large changes in pH alter electrostatic
attractions between side chains especially with
3. TERTIARY LEVEL OF ORGANIZATION
the acidic and basic amino acids.
3-D arrangement of all the atoms in the
polypeptide chain with the side chains, 2) Hydrolysis destruction of primary structure
determined by covalent and non-covalent through hydrolysis of peptide bonds.
interactions within the chain such as:
A. Electrostatic attractions and hydrogen
bonding between R groups
B. Disulfide linkages PART 4: PROTEINS IN VERTEBRATES
C. Metal-ion coordination 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 3. Hemoglobin
biochemical composition: Transport protein; globular
Complex of heme, an iron containing
1. Collagen prosthetic group, and globin, the protein
Structural protein; filamentous portion which prevents the oxidation of
Left-handed triple helix containing 3.3 Fe 2+ into Fe3+
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.
Heme. Each 5-membered ring with
Synthesis of Collagen: nitrogen at the four corners is called a
i. Translation into pyrrole ring.
preprocollagen.
ii. Hydroxylation: formation of
hydroxyproline and Tetramer made of four different protein
hyroxylysine subunits each wrapped around a heme
iii. Release from ribosomes molecule; It has two alpha chains and two
and addition of beta chains
endoplasmic reticulum Types of Hemoglobin:
sugars like galactose and
i. Hb + O2 Oxyhemoglobin
glucose at hydroxylysine.
iv. Formation of the triple ii. Hb O2 Deoxyhemoglobin
helix and folding of iii. Hb + CO2
globular domains; Carbaminohemoglobin
transformation into iv. Hb + CO Carboxyhemoglobin
procollagen v. Hb + Fe 3+
v. Secretion from cell Ferrihemoglobin/Methemoglobin
vi. Removal of N and C
terminal domains;
vi. Hb + Fe 2+ Ferrohemoglobin
transformation into
tropocollagen Heme is made of four pyrrole rings that
vii. Deamination of lysine to form one porphyrin ring. It is also known
form allysine as an iron protoporphyrin. Each pyrrole
viii. Cross-linkage of allysine
ring forms the first four coordination sites
with a Lysine residue, or
another allysine residue to
of heme.
form collagen. Histidine F8, or the proximal histidine,
binds the iron strongly to the heme. This
2. Elastin 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
Structural protein; filamentous sigmoidal because it exhibits positive
Found in ligaments and arterial blood cooperation. This means, the binding of
vessels one O2 molecule to one coordination site
Non-repetitive coil enhances the attachment of O 2 to the
Rich in G, A, V, P, but not in other sites.
hydroxyproline and hydroxylysine Hemoglobin is an allosteric protein. That
Four lysine residues (or four allysine means, it is affected by H+, CO2, and 2,3-
residues) (or two lysine and two allysine) bisphosphoglycerate which decreases O2
residues condense to form the desmosine affinity to hemoglobin.
or isodesmosine crosslink giving elastin HbA is the normal hemoglobin which
its rubbery characteristic has six glutamic acid residues on its beta

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 chain. HbS - hemoglobin of patients with Released when blood glucose levels are
sickle cell anemia. below normal; hyperglycemic hormone
In sickle cell anemia, valine replaces
glutamic acid. The ionic interactions of 7. Immunoglobulins
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
Also known as antibodies
Found mainly in the muscles
Defense proteins
Composed of only one protein chain with
a heme prosthetic group in the center Secreted by B-lymphocytes
Composed of two light chains and two
Myoglobins oxygen binding curve is
hyperbolic instead of sigmoidal 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:
Comparisons in structure and oxygen binding curves
of myoglobin and hemoglobin. 1. SOLUBILITY/POLARITY
a. Isoelectric Precipitation adjusting the
5. Insulin
pH of the solution until the protein
Regulatory protein/ hormone reaches its IpH and becomes insoluble
Produced from the -cells of the Islets of b. Salting Out removing water from
Langerhans proteins by adding an excess amount of a
Composed of two polypeptide chains, salt; makes the protein less soluble
containing 51 amino acid residues, linked (salting in increases the solubility of
together by intermolecular and proteins in water by adding a sufficient
intramolecular disulfide linkages amount of a salt)
Also known as hypoglycemic hormone c. Normal and Reversed Phase
Breaks down glucose in a process known Chromatography protein separation
as glycolysis to synthesize pyruvate then based on affinities with the mobile or
ATP stationary phases; in normal phase
Converts glucose into glycogen, to be chromatography, the polar proteins are
stored in the liver, through a process the last to be eluted
known as glycogenesis d. High Performance Liquid
Aids in fatty acid synthesis and protein Chromatography (HPLC) uses a column
synthesis pre-packed with the stationary phase and
is pressurized
6. Glucagon
Also a regulatory protein/hormone 2. MOLECULAR SIZE/WEIGHT
Brings about glycogenolysis a. Dialysis movement of particles through
Produced from the -cells of the Islets of a semi-permeable membrane; large MW
Langerhans proteins will remain inside the dialysis bag
Single polypeptide chain composed of 29 b. Ultrafiltration filtration using a vacuum
amino acid residues c. Ultracentrifugation centrifugating a
solution at various speeds will separate its
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 molecular components; high MW proteins i. Aminopeptidase cleaves off the N-
will settle at a high speed, while low MW terminal amino acid
proteins need a higher speed to settle at ii. Carboxypeptidase cleaves off the C-
the bottom terminal amino acid
d. Gel Filtration Chromatography (or Size b. Endopeptidases cleave peptide chains
Exclusion or Molecular Sieve) uses from the inside
porous gel beads such as agarose i. Trypsin cleave off the carboxyl side
(Sepharose) or dextran (Sephadex) which of basic amino acids lysine and
trap smaller molecules leaving the larger arginine
molecules to be eluted first ii. Chymotrypsin cleave off the
e. SDS-PAGE (Sodium Dodecyl Sulfate carboxyl side of aromatic amino acids
Polyacrylamide Gel Electrophoresis) phenylalanine, tyrosine, and
utilizes a detergent, SDS, which imparts a tryptophan
negative charge to the proteins; smaller
protein molecules treated with the Ex. Give the correct sequence of a nonapeptide
detergent move faster towards the containing arg, ser, asp, gly, trp, met, ala, phe,
positively charged anode cys.
Edman Reagent: G + PTH
3. CHARGE Cyanogen bromide: pentapeptide: M, F, D, S, G
a. Electrophoresis separates the proteins tetrapeptide: W, C, R, A
(or amino acids) based on their attraction Aminopeptidase: G
towards the negatively charged cathode Carboxypeptidase: A
or the positively charged anode at a Trypsin: octapeptide: D, M, W, C, G, S, R, F
specific pH single alanine residue
b. Ion Exchange Chromatography uses Chymotrypsin: tripeptide: F, G, S
cation or anion exchangers (resins); when tripeptide: M, W, D
a cation exchanger is used, positively tripeptide: C-R-A
charged amino acids are eluted last Sequence: _ _ _ _ _ _ _ _ _

4. BINDING AFFINITY 1. We start by placing G and A at the N and C-

a. Affinity Chromatography a ligand acts terminals respectively based on the
as the stationary phase to entrap the reactions with the Edman reagent,
protein of interest aminopeptidase and carboxypeptidase.
b. Precipitation by Antibodies G ___ __ _ _A
2. The reaction with chymotrypsin yielded a
PART VIII: PROTEIN CHARACTERIZATION tripeptide C-R-A.
TECHNIQUES G ___ __ CRA
Proteins may be characterized by the types of amino 3. The reaction with chymotrypsin yielded two
acids they possess. Complete hydrolysis, using strong other tripeptides containing F, G, S and M,
acids or bases, cleaves all peptide bonds leaving W, D respectively. We know that G is the N-
individual amino acids. Incomplete hydrolysis terminal AA so the tripeptide containing M,
involves reagents or enzymes which are more W, D must be somewhere in the middle. We
specific and cleave peptide bonds at certain places also know that chymotrypsin cleaves the
only. carboxyl side of W. We place it beside C.
1. Edman Reagent phenyl isothiocyanate G ___ _W CRA
(PTH) reagent; cleaves the carboxyl side of 4. The reaction with cyanogen bromide yielded
the N-terminal amino acid; the products are two peptide chains. We know that cyanogen
the N-terminal amino acid attached to bromide cleaves at the carboxyl side of M.
phenylthiohydantoin (PTH), and the We therefore place M beside W.
remaining peptide G ___ MW CRA
Ex. STC + Edman Reagent (PTH+S) + T-C 5. The reaction with chymotrypsin shows that
2. Cyanogen bromide cleaves the carboxyl the amino acids M, W, and D come as one
side of methionine tripeptide. We therefore place D beside M.
Ex. BIOCHEMISTRY + Cyanogen Bromide G __DM W CRA
BIOCHEM + ISTRY 6. Lastly, we know that chymotrypsin cleaves
3. Proteinases/Proteases/Proteolytic Enzymes the carboxyl side of F. We therefore place
a. Exopeptidases cleave off terminal the last two amino acids at their rightful
amino acids positions.
G SFDMW CRA

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