Cells - Structure and Function

Important Events in the Discovery of Cells

1665 - Robert Hooke looks at cork under a microscope. Calls the chambers he
see "cells"
1665 - 75 Anton van Leeuwenhoek, the inventor of the microscope, studies
organisms living in pond water (like you did in lab). He calls them
"Animalcules."
1830 - German scientists Schleiden and Schawann summarize the findings of
many scientists and conclude that all living organisms are made of cells. This
forms the basis of the Cell Theory of Biology

The Cell Theory of Biology

All organisms are composed of cells

The cell is the structural unit of life - units smaller than cells are not alive
Cells arise by division of preexisting cells - spontaneous generation does not
exist
Cells can be cultured to produce more cells
o in vitro = outside organism or cell
o in vivo = inside organism or cell

Properties of Cells
Cells are complex and highly organized

They contain numerous internal structures

Some are membrane bound (organelles) while others do not

Cells contain a genetic blueprint and machinery to use it

Genes are instructions for cells to create specific proteins

All cells use the same types of information
o The genetic code is universal
o The machinery used for synthesis is interchangeable
However, for this to function properly, information transfer must be error free

Errors are called mutations

Cells arise from the division of other cells

Daughter cells inherit the genes from the mother cells

Mitosis - the genetic complement of each daughter cell is identical to the other
and to the mother cell. This is asexual reproduction
Meiosis - the genetic complement of each daughter cell is reduced by half and
each daughter cell is genetically unique. This is used in sexual reproduction
Daughter cells inherit cytoplasm and organelles from the mother cells
o Asexual - organelles from mother cell
o Sexual - organelles predominately from one parent
In eukaryotes, the chloroplasts and mitochondria come from the
egg cell
This can be used to trace the evolutionary origin of the organism

Cells aquire and utilize energy

Plant cells undergo photosynthesis

o convert light energy and CO2 to chemical energy (ATP and glucose)
Most cells respire
o release energy found in organic compounds
o convert organic compounds to CO2 and O2
o make ATP

Cells can perform a variety of chemical reactions

o
o
o

Transform simple organic molecules into complex molecules

(anabolism)
Breakdown complex molecules to release energy (catabolism)
Metabolism = all reactions performed by cells

Cells can engage in mechanical activities

o
o
o

Cells can move

Organelles can move
Cells can respond to stimuli
chemotaxis - movement towards chemicals
phototaxis - movement towards light
hormone responses
touch responses

Cells can regulate activities

o
o
o

Cells control DNA synthesis and cell division

Gene regulation - cells make specific proteins only when needed
Turn on and off metabolic pathways

Cells all contain the following structures:

o
o
o

Plasma membrane - separates the cell from the external environment

Cytoplasm - fluid-filled cell interior
Nuclear material - genetic information stored as DNA

Types of Cells
Prokaryotes
o
o
o
o

Pro = before; karyon = nucleus

relatively small - 5 to 10 um
lack membrane-bound organelles
earliest cell type

Eukaryotes
o
o
o
o

Eu = true; karyon = nucleus

contain membrane-bound organelles
Evolved from prokaryotes by endosymbiotic association of two or more
prokaryotes
Include Protists, Fungi, Animals, and Plants

Features of Prokaryotic Cells

o
o

Capsule - outer sticky

protective layer
Cell Wall - rigid
structure which helps
the bacterium maintain
its shape
this is in NO
way the same
as the cell wall
of a plant cell
Plasma membrane separates the cell from
the environment
Mesosome - infolding
of plasma membrane
to aid in
compartmentalization
Nucleoid - region
where nakedDNA is
found
Cytoplasm
semi-fluid cell
interior
no membranebound
organelles
location for
metabolic
enzymes
location of
ribosomes for
protein
synthesis

Properties of Eukaryotic Cells

Features shared with Prokaryotic cells

Rigid cell wall
Plant cells, some Fungi, some Protists
Animal cells lack cell wall
Plasma membrane
Cytoplasm with ribosomes
Cytoskeleton - flexible tubular scaffold of microfilaments
maintains cell shape and provides support
anchors organelles & enzymes to specific regions of the cell

contractility and movement (amoeboid movement)

intracellular transport - tracks for vesicle and organelle movement
by motor proteins
Cytoskeleton components
Microfilaments
solid protein (actin) which is assembled at one end and
disassembled at the other end
Intermediate filaments - rope-like fibrous proteins
provide structural reinforcement
anchor organelles
keep nucleus in place
Microtubules - hollow tubes of tubulin (a globular protein>
maintains cell shape
anchor organelles
movement of organelles
track for motor proteins
Cilia and Flagella - involved in cellular movement
composed of microtubules
cilia - short, numerous, complex
flagella - longer, fewer, less complex
both arranged in a 9+2 pattern with dynein arms projecting
outward
Nucleus
Double membrane with pores
Outer membrane continuous with ER
Nuclear matrix - protein-containing fibrilar network
Nucleoplasm - the fluid substance in which the solutes of the
nucleus are dissolved
Chromosomes - protein and DNA complexes
Nucleolus - involved in the synthesis and assembly of ribosomes
Endomembrane System
Endoplasmic Reticulum - an extinsive membranous network
continuous with the outer nuclear membrane.
Rough ER - has ribosomes and is involved in secreted
protein synthesis
Smooth ER - lacks ribosomes and is involved in membrane
lipid synthesis
Golgi Apparatus
Flattened vesicles in stacks which receive protein from ER
Form secretory vesicles to transport proteins to different
parts of the cell (vacuole, lysosome, etc) or for secretion

cis face - "receiving" side of Golgi apparatus

trans face - "shipping" side of Golgi apparatus
Lysosome
found only in animal cells
contain enzymes for use in the hydrolytic breakdown of
macromolecules
Plant Central Vacuole - major storage space in center of plant
cell with many functions
Digestive - break down of macromolecules
Storage - ions, sugars, amino acids, toxic waste
Maintain cell rigidity - high ionic concentration generates
high water potential

Images of Vesicle Transport Between Endomembrane Organelles

o

Ribosomes
The "factories" of the cell - involved in protein synthesis
Facilitate the specific coupling of tRNA anticodons with mRNA
codons during protein synthesis
May either be free or bound to ER
Made up of two subunits, the large and the small subunit
Both subunits are constructed out of protein and RNA (called
rRNA)
The ribosomes of prokaryotes and eukaryotes vary slightly with
regard to size and shape
Mitochondria
Found in ALL eukaryotic cells (yes, even in plant cells)
Site of aerobic respiration
sugars + O2 - - > ATP + CO2 + H2O
Contain DNA which codes for mitochondrial proteins, ribosomes,
etc.
Divide by a process similar to binary fission when cell divides
Enclosed in a double membrane system
Inner Membrane forms the Cristae (invaginations into
interior region)
Site of energy generation
Matrix is the soluble portion of the mitochondira
Site of carbon metabolism
Location of mDNA
Site of mitochondrial protein synthesis
Chloroplasts

Found only in plant cells

Site of photosynthesis
conversion of solar energy to chemical energy in the form
of ATP and sugars
Contain DNA which codes for chloroplast proteins, ribosomes,
etc.
Divide when plant cell divides
Enclosed in a double membrane envelope that does not invaginate
into the chloroplast
Thylakoid is a third internal membrane system
contains membrane-bound photosynthetic pigments
site of photochemistry (the conversion of light energy to
ATP)
site of O2 generation
Stroma is soluable portion of chloroplast
site of CO2 fixation
site of sugar synthesis (carbon metabolism)
location of cpDNA
site of chloroplast protein synthesis

Endosymbiotic Origin of Chloroplasts and Mitochondria

Free-living prokaryote eaten by host

Genes transferred to host nucleus
Some genes retained but most lost - can no longer survive outside of host
Symbiotic relationship
o photosynthetic symbiont provides sugar - degenerates to form
chloroplast
o aerobic symbiont provides a more efficient energy generation system degenerates to form mitochondria
o host provides stable environment, nutrients, energy, and most proteins

Evidence for Endosymbiotic Theory

o Chloroplasts and mitochondria have DNA
does not code for all proteins
some genes in nucleus
proteins imported rom cytoplasm

o
o

Organelle proteins similar to bacterial form

Ribosome structure and metabolic enzymes more similar to bacterial
forms

There are over 200 different types of cells within the human body. These cells all vary in
size, shape and diameter. Most cells range between ten and fifteen micrometers in
diameter, however, some cells, such as the human egg cell, are much larger than this,
with a diameter of roughly 100 micrometers. The human eggs cell is just barely visible to
the naked eye. Some of the longest cells include nerve cells, which can be as long as a
meter, but are so thin, they are invisible to the naked eye. Cells, although they range in
size and shape, cannot become too large, or they may become unable to support their
own functions, or could burst.
Cells are found in many different shapes and sizes. Some of the most common cell
shapes include; squamous, cuboidal, columnar, polygonal, spheroid, discoid, fusiform
and fibrous.
Squamous
Squamous cells are thin and flat, with a slight bulge where the nucleus lies. These cells
are commonly compared to the appearance of a fried egg. These cells are most abundant
in the skin and the lining of the esophagus.
Polygonal
Polygonal cells, much like their name implies, are polygonal in shape, with five or more
sides. Sometimes these sides are elongated in such a manner that they form a stellate, or
star-like shape.
Cuboidal
Cuboidal cells are square-like in shape and are typically as tall as they are wide. This
type of cell is commonly found in the liver.
Columnar
Columnar cells are similar to cuboidal cells, however, they are taller than they are wide.
This type of cell is commonly found in the lining of the intestines.
Spheroid
Spheroid cells, sometimes referred to as ovoid cells, range from circular to ovular.
Examples of spheroid and ovoid cells include fat cells and human egg cells.

Discoid
Discoid cells are shaped much like a disc, or a frisbee. An example of a discoid cell
includes red blood cells.
Fusiform
Fusiform cells are often thought of as spindle-shaped; thick in the center and tapered at
the ends. These cells make up the smooth muscles.
Fibrous
Fibrous cells are long and thread like, without any area larger than another. The skeletal
muscles are composed of fibrous cells.

Eukaryotic Cell vs Prokaryotic Cell

Diffen
Science
Biology
Microbiology
All cells can be classfied into prokaryotes and eukaryotes. Prokaryotes were the only form of life
on Earth for millions of years until more complicated eukaryotic cells came into being through the
process of evolution.

Comparison chart
All attributes Differences Similarities

Eukaryotic Cell

Prokaryotic Cell

Eukaryotic Cell

Prokaryotic Cell

Nucleus:

Present

Absent

Number of
chromosomes:

More than one

One--but not true

chromosome:Plasmids

Cell Type:

Multicellular

Unicellular

True Membranebound
Nucleus:

Present

Absent

Example:

Animals and Plants

Bacteria and Archaea

Telomeres:

Present (Linear DNA)

Circular DNA doesn't need

telemeres

Genetic Recombination:

Mitosis and fusion of gametes

Partial, undirectional transfers

DNA

Lysosomes and
peroxisomes:

Present

Absent

Microtubules:

Present

Absent or rare

Endoplasmic reticulum:

Present

Absent

Mitochondria:

Present

Absent

Cytoskeleton:

Present

May be absent

DNA wrapping on
proteins.:

Yes

No

Ribosomes:

larger

smaller

Vesicles:

Present

Present

Eukaryotic Cell

Prokaryotic Cell

Golgi apparatus:

Present

Absent

Mitosis:

Yes

No---but has binary fission

Chloroplasts:

Present (in plants)

Absent; chlorophyll scattered in the

cytoplasm

Flagella:

Microscopic in
size; membranebound; usually
arranged as nine doublets
surrounding two singlets

Submicroscopic in size, composed

of only one fiber

Permeability of
NuclearMembrane:

Selective

not present

Plasmamembrane with
steriod:

Yes

Usually no

Cell wall:

Only in plant cells (chemically

simpler)

Usually chemically complexed

Vacuoles:

Present

Present

Cell size:

10-100um

1-10um

Definition of eukaryotes and prokaryotes

Prokaryotes (pro-KAR-ee-ot-es) (from Old Greek pro- before + karyon nut or kernel, referring to
the cell nucleus, + suffix -otos, pl. -otes; also spelled "procaryotes") are organisms without a cell
nucleus (= karyon), or any othermembrane-bound organelles. Most are unicellular, but some
prokaryotes are multicellular).
Eukaryotes (IPA: [jukt]) are organisms whose cells are organized into complex structures by
internal membranes and a cytoskeleton. The most characteristic membrane bound structure is the
nucleus. This featuregives them their name, (also spelled "eucaryote,") which comes from the Greek
, meaning good/true, and , meaning nut, refering to the nucleus. Animals, plants, fungi, and
protists are eukaryotes.

Differences between eukaryotic and prokaryotic

cells
The difference between the structure of prokaryotes and eukaryotes is so great that it is considered to
be the most important distinction among groups of organisms.

Structure and contents of a typical Gram positive bacterial cell (a prokaryotic cell)

The most fundamental difference is that eukaryotes do have "true" nuclei containing their DNA,
whereas the genetic material in prokaryotes is not membrane-bound.
In eukaryotes, the mitochondria and chloroplasts performvarious metabolic processes and are
believed to have been derived from endosymbiotic bacteria. In prokaryotes similar processes
occur across the cell membrane; endosymbionts are extremely rare.
The cell walls of prokaryotes are generally formed of a different molecule (peptidoglycan) to
those of eukaryotes (manyeukaryotes do not have a cell wall at all).
Prokaryotes are usually much smaller than eukaryotic cells.
Prokaryotes also differ from eukaryotes in that they contain only a single loop of stable
chromosomal DNA stored in an area named the nucleoid, while eukaryote DNA is found on
tightly bound and organised chromosomes. Although someeukaryotes have satellite DNA
structures called plasmids, these are generally regarded as a prokaryote featureand many
important genes in prokaryotes are stored on plasmids.
Prokaryotes have a larger surface area to volume ratio giving them a higher metabolic rate, a
higher growth rate and consequently a shorter generation time compared to Eukaryotes.
Genes
Prokaryotes also differ from eukaryotes in the structure, packing, density, and arrangement
of their genes on the chromosome. Prokaryotes have incredibly compact genomes
compared to eukaryotes, mostly because prokaryote genes lack introns and large noncoding regions between each gene.
Whereas nearly 95% of the human genome does not code for proteins or RNA or includes a
gene promoter, nearly all of the prokaryote genome codes or controls something.

Prokaryote genes are also expressed in groups, known as operons, instead of individually,
as ineukaryotes.
In a prokaryote cell, all genes in an operon(three in the case of the famous lac operon) are
transcribed on the same piece of RNA and then made into separate proteins, whereas if
these genes were native toeukaryotes, they each would have their own promoter and be
transcribed on their own strand of mRNA. This lesser degree of control over gene
expression contributes to the simplicity of the prokaryotes as compared to the eukaryotes.

Proteins are very important molecules in our cells. They are involved in virtually all cell
functions. Each protein within the body has a specific function. Some proteins are
involved in structural support, while others are involved in bodily movement, or in
defense against germs. Proteins vary in structure as well as function. They are
constructed from a set of 20 amino acids and have distinct three-dimensional shapes.
Below is a list of several types of proteins and their functions.

Protein Functions

Antibodies - are specialized proteins involved in defending the body from antigens (foreign invaders).
They travel through the blood stream and are utilized by the immune system to identify and defend
against bacteria, viruses, and other foreign intruders. One way antibodies destroy antigens is by
immobilizing them so that they can be destroyed by white blood cells.

Contractile Proteins - are responsible for movement. Examples include actin and myosin. These proteins
are involved in muscle contraction and movement.

Enzymes - are proteins that facilitate biochemical reactions. They are often referred to as catalysts because
they speed up chemical reactions. Examples include the enzymes lactase and pepsin. Lactase breaks down
the sugar lactose found in milk. Pepsin is a digestive enzyme that works in the stomach to break down
proteins in food.

Hormonal Proteins - are messenger proteins which help to coordinate certain bodily activities. Examples
include insulin, oxytocin, and somatotropin. Insulin regulates glucose metabolism by controlling the bloodsugar concentration. Oxytocin stimulates contractions in females during childbirth. Somatotropin is a
growth hormone that stimulates protein production in muscle cells.

Structural Proteins - are fibrous and stringy and provide support. Examples include keratin, collagen, and
elastin. Keratins strengthen protective coverings such as hair, quills, feathers, horns, and beaks. Collagens
and elastin provide support for connective tissuessuch as tendons and ligaments.

Storage Proteins - store amino acids. Examples include ovalbumin and casein. Ovalbumin is found in egg
whites and casein is a milk-based protein.

Transport Proteins - are carrier proteins which move molecules from one place to another around the
body. Examples include hemoglobin and cytochromes. Hemoglobin transports oxygen through the blood.
Cytochromes operate in the electron transport chain as electron carrier proteins.

Protein Structure
Proteins serve various functions in the body. The structure of a protein determines its
function. For example, collagen has a super-coiled helical shape. It is long, stringy,

strong, and resembles a rope. This structure is great for providing support. Hemoglobin
on the other hand, is a globular protein that is folded and compact. Its spherical shape
is useful for maneuvering through blood vessels.
Organic Polymers
For information on other types of biological polymers, see:

The Role of Proteins in Humans: How Proteins Help Maintain

Life
written by: Emma Lloyd edited by: Leigh A. Zaykoski updated: 5/24/2011
Along with carbohydrates, fats and nucleic acids, proteins are the "building blocks of life." Learn
about the functions of protein in the human body to find out why these molecules are so important.

What does Protein Do?

Protein has a large number of important functions in the human bodyand in fact, the human body
is about 45% protein. Its an essential macromolecule without which our bodies would be unable to
repair, regulate, or protect themselves.

Functions of Protein

Protein has a range of essential functions in the body, including the

following:

Required for building and repair of body tissues (including muscle)

Enzymes, hormones, and many immune molecules are proteins
Essential body processes such as water balancing, nutrient transport, and muscle contractions require
protein to function.
Protein is a source of energy.
Protein helps keep skin, hair, and nails healthy.

Protein, like most other essential nutrients, is absolutely crucial for overall good health.

So what does protein do?

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Proteins are, in effect, the main actioners in cells and in an entire organism. Without proteins the
most basic functions of life could not be carried out. Respiration, for example, requires muscle
contractions, and muscle contractions require proteins.

Proteins as Enzymes
The function of proteins as enzymes is perhaps their best-known function. Enzymes are catalysts
they initiate a reaction between themselves and another protein, working on the molecule to change it
in some way.
The enzyme, however, is itself unchanged at the end of the reaction.
Enzymes are responsible for catalyzing reactions in processes such as metabolism, DNA replication,
and digestion.
In fact, enzymes are known to be involved in some 4,000 bodily reactions.

Proteins in Cellular Signaling and Molecular Transport

Cells signal one another for an enormous variety of reasons, the most basic of which is simply to
coordinate cellular activities. Signaling is how cells communicate with one another, allowing such
essential processes as the contraction of the heart muscle to take place.
Proteins are important in these processes due to their ability to bind other moleculesa protein
produced by one cell may bind to a molecule produced by another, thus providing a chemical signal
which allows the cells to provide information about their state. Proteins are also involved in
molecular transport.
A prime example of this is the protein called hemoglobin, which binds iron molecules and transports
them in the blood from the lungs to organs and tissues throughout the body.

Protein Composition

Proteins are organic macromolecules made up of linear chains of amino acids. However, while a
proteins basic structure is a linear amino acid chain, the final structure of a protein is not linear.
Instead, the proteins amino acid sequenceand the physical and chemical properties of the amino
acids and of the entire protein molecule - influences how it folds into a three dimensional shape.
The amino acid sequence of a protein is determined by the base pair sequence in the gene which
codes for the protein. There are twenty standard amino acids (along with one or two non-standard
proteins which are not coded for by DNA in the usual sense).

Structural Proteins
These are less active than those involved in catalyzing reactions, signaling cells, and transporting
molecules, but are no less important.

Structural proteins are those which confer strength and rigidity to biological components which would
otherwise be unable to support themselves.
Structural proteins tend to have very specific shapeslong, thin fibers or other shapes which, when
allowed to form polymers, provide strength and support.
Structural proteins are essential components of collagen, cartilage, nails and hair, feathers, hooves, and
other such components.
Structural proteins are also essential components of muscles, and are necessary to generate the force
which allows muscles to contract and move.

Additional Reading
Here is a list of some related reading covering a variety of topics on how protein plays a part in
health, protein function, protein sources, and more on the role protein plays in the human body.

What is Protein? Learn How Much Protein is Needed for a Balanced Diet
List of Foods High in Protein
Protein's Role in Muscle Building
De novo protein design (sequence design)
Protein Sequencing: Three Ways It Works
History of Protein and Protein in Living Organisms
Discovering How Proteins Function
Theory on the Function of Protein in How Cancer Spreads

Amino acids
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Amino acids are organic compounds that combine to form proteins. Amino acids and
proteins are the building blocks of life.
When proteins are digested or broken down, amino acids are left. The human body needs a
number of amino acids to:

Break down food

Grow
Repair body tissue
Perform many other body functions

Amino acids are classified into three groups:

Essential amino acids

Nonessential amino acids
Conditional amino acids

Essential amino acids

Essential amino acids cannot be made by the body. As a result, they must come
from food.
The nine essential amino acids are: histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, threonine, tryptophan, and valine.

Nonessential amino acids

"Nonessential" means that our bodies produce an amino acid, even if we don't get it
from the food we eat.
They include: alanine, asparagine, aspartic acid, and glutamic acid.

Conditional amino acids

Conditional amino acids are usually not essential, except in times of illness and
stress.
They include: arginine, cysteine, glutamine, tyrosine, glycine, ornithine, proline, and
serine.

You do not need to eat essential and nonessential amino acids at every meal, but getting a
balance of them over the whole day is important.

Amino Acid General Structure

Amino Acid

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This is the general structure of an amino acid. This also shows the ionization of an amino acid at
pH = 7.4.
Todd Helmenstine

Amino acids are comprised of a functional group R attached to an amine group (NH2) and a carboxyl group
(COOH).

Classification - Amino acid classification according to reactive group or R

group
1. Aliphatic - Glycine, Alanine, Valine, Leucine, Isoleucine
2. Aromatic - Phenylalanine, Tyrosine, Tryptophan
3. OH- - Serine, Threonine
4. Acidic - Aspartic Acid, Glutamic Acid
5. Acid amide - Aspargine, Glutamine
6. Basic - Arginine, Lysine, Histidine
7. Sulphur - Cystine, Methionine
8. Cyclic - Proline
take the front letters of amino acid names
1. Aliphatic - GAVLI
2. Aromatic - Phe, Ty, Try
3. OH- - ST
4. Acidic - Asp, Glu
5. Acid amide - Aspargine, Glutamine
6. Basic - Arg, Lys, His
7. Sulphur - CM
8. Cyclic - P
If you remember one letter code of amino acid than
1. Aliphatic - GAVLI
2. Aromatic - FYW
3. OH- - ST
4. Acidic - DE
5. Acid amide - NQ
6. Basic - RKH
7. Sulphur - CM
8. Cyclic - P
Symbolic representation after reduction of letters
1.
2.
3.
4.
5.
6.
7.
8.

Ali
Aro
OH
COOH
CONH2
NH2
S
Cyclic

GAVLI
FYW
ST
DE
NQ
RKH
CM
P

Classification - Dependent in the property of R group

1. Non polar or Hydrophobic - Glycine, Alanine, Valine, Leucine, Isoleucine,

Proline
2. Polar uncharged - Phenylalanine, Tryptophane, Tyrosine
3. Polar charged - Serine, Threonine (OH-), Cystine, Methionine (sulphur),
Aspargine, Glutamine (acid amide)
4. Positively charged - Aspartic Acid, Glutamic Acid
5. Negatively charged - Arginine, Lysine, Histidine
Dependent in R group (Symbolic)
1. Non polar or Hydrophobic - GAVLIP
2. Polar uncharged - FWY
3. Polar charged - ST (OH-), CM (S), NQ (acid amide)
4. (+) charged - DE
5. (-) charged - RKH
Classification - dependent according to polarity of R group.
Amino acid can be divided into the above 3 groups (non-polar, flexible and polar)
and then subdivided by their chemical character.
Non-Polar -- 8 Amino Acids
Hydrocarbon
Ala Val Leu Ile Pro
Aromatic
Phe Trp
Thiol Ether
Met
Flexible -- 1 Amino Acids
Gly -- flexible because it has no side chain
Polar -- 11 Amino Acids
Alcohol
Ser Thr Tyr
Thiol
Cys
Amides
Asn Gln
Acids
Asp Glu
Bases
Lys Arg His

Amino Acids
The Amino Acids

Zwitterions

The Amino Acids Used to Synthesize

Proteins

The Acid-Base Chemistry of the

Amino Acids

The Amino Acids

Proteins are formed by polymerizing monomers that are known as amino

acids because they contain an amine (-NH2) and a carboxylic acid (-CO2H) functional
group. With the exception of the amino acid proline, which is a secondary amine, the
amino acids used to synthesize proteins are primary amines with the following generic
formula.

An amino acid

These compounds are known as -amino acids because the -NH2 group is on the
carbon atom next to the -CO2H group, the so-called carbon atom of the carboxylic
acid.

Zwitterions
The chemistry of amino acids is complicated by the fact that the -NH2 group is a base
and the -CO2H group is an acid. In aqueous solution, an H+ ion is therefore transferred
from one end of the molecule to the other to form a zwitterion (from the German
meaning mongrel ion, or hybrid ion).

Zwitterions are simultaneously electrically charged and electrically neutral. They

contain positive and negative charges, but the net charge on the molecule is zero.

The Amino Acids Used to Synthesize Proteins

More than 300 amino acids are listed in the Practical Handbook of Biochemistry and
Molecular Biology, but only the twenty amino acids in the table below are used to
synthesize proteins. Most of these amino acids differ only in the nature of
the R substituent. The standard amino acids are therefore classified on the basis of
these R groups. Amino acids with nonpolar substituents are said to
be hydrophobic (water-hating). Amino acids with polar R groups that form hydrogen
bonds to water are classified as hydrophilic (water-loving). The remaining amino

acids have substituents that carry either negative or positive charges in aqueous
solution at neutral pH and are therefore strongly hydrophilic.
The 20 Standard Amino Acids
STRUCTURE
(AT NEUTRAL pH)
Nonpolar (Hydrophobic) R Groups
NAME

Glycine (Gly)
Alanine (Ala)

Valine (Val)

Leucine (Leu)

Isoleucine (Ile)

Proline (Pro)

Methionine (Met)

Phenylalanine
(Phe)

Tryptophan (Trp)

Polar (Hydrophilic) R Groups

Serine
(ser)
Threonine
(Thr)

Tyrosine
(Tyr)

Cysteine
(Cys)

Asparagine
(Asn)

Glutamine
(Gln)

Negatively Charged R Groups

Aspartic acid (Asp)

Glutamic acid
(Glu)

Positively Charged R Groups

Lysine
(Lys)

Arginine
(Arg)

Histidine
(His)

Practice Problem 1:
Use the structures of the following amino acids in the table of standard amino acids to classify
these compounds as either nonpolar/hydrophobic, polar/hydrophilic, negatively
charged/hydrophilic, or positively charged/hydrophilic.
(a) Valine: R = -CH(CH3)2
(b) Serine: R = -CH2OH
(c) Aspartic acid: R = -CH2CO2(d) Lysine: R = -(CH2)4NH3+
Click here to check your answer to Practice Problem 1

Amino Acids as Stereoisomers

With the exception of glycine, the common amino acids all contain at least one chiral
carbon atom. These amino acids therefore exist as pairs of stereoisomers. The
structures of the D and L isomers of alanine are shown in the figure below. Although
D amino acids can be found in nature, only the L isomers are used to form proteins.
The D isomers are most often found attached to the cell walls of bacteria and in
antibiotics that attack bacteria. The presence of these D isomers protects the bacteria
from enzymes the host organism uses to protect itself from bacterial infection by
hydrolyzing the proteins in the bacterial cell wall.

D-Alanine

L-Alanine

A few biologically important derivatives of the standard amino acids are shown in the
figure below. Anyone who has used an "anti-histamine" to alleviate the symptoms of
exposure to an allergen can appreciate the role that histamine a decarboxylated
derivative of histidine
plays in mediating the body's response to allergic reactions.
L-DOPA, which is a derivative of tyrosine, has been used to treat Parkinson's disease.
This compound received notoriety a few years ago in the film Awakening, which
documented it's use as a treatment for other neurological disorders. Thyroxine, which
is an iodinated ether of tyrosine, is a hormone that acts on the thyroid gland to
stimulate the rate of metabolism.

LDOPA

Histamine

Thyroxine

The Acid-Base Chemistry of the Amino Acids

Acetic acid and ammonia often play an important role in the discussion of the
chemistry of acids and bases. One of these compounds is a weak acid; the other is a
weak base.

Thus, it is not surprising that an H+ ion is transferred from one end of the molecule to
the other when an amino acid dissolves in water.

The zwitterion is the dominant species in aqueous solutions at physiological pH

(pH
7). The zwitterion can undergo acid-base reactions, howeer, if we add either a
strong acid or a strong base to the solution.
Imagine what would happen if we add a strong acid to a neutral solution of an amino
acid in water. In the presence of a strong acid, the -CO2- end of this molecule picks up
an H+ ion to form a molecule with a net positive charge.

In the presence of a strong base, the -NH3+ end of the molecule loses an H+ ion to
form a molecule with a net negative charge.

The figure below shows what happens to the pH of an acidic solution of glycine when
this amino acid is titrated with a strong base, such as NaOH.

In order to understand this titration curve, let's start with the equation that describes
the acid-dissociation equilibrium constant expression for an acid, HA.

Let's now rearrange the Ka expression,

take the log to the base 10 of both sides of this equation,

and then multiply both sides of the equation by -1.

By definition, the term on the left side of this equation is the pH of the solution and
the first term on the right side is the pKa of the acid.

The negative sign on this right side of this equation is often viewed as "inconvenient."
The derivation therefore continues by taking advantage of the following feature of
logarithmic mathematics

to give the following form of this equation.

This equation is known as the Henderson-Hasselbach equation, and it can be used to

calculate the pH of the solution at any point in the titration curve.
The following occurs as we go from left to right across this titration curve.

The pH initially increases as we add base to the solution because the base
deprotonates some of the positively charged H3N+CH2CO2H ions that were
present in the strongly acidic solution.
The pH then levels off because we form a buffer solution in which we have
reasonable concentrations of both an acid, H3N+CH2CO2H, and its conjugate
base, H3N+CH2CO2-.
When virtually all of the H3N+CH2CO2H molecules have been deprotonated,
we no longer have a buffer solution and the pH rises rapidly when more NaOH
is added to the solution.
The pH then levels off as some of the neutral H3N+CH2CO2- molecules lose
protons to form negatively charged H2NCH2CO2- ions. When these ions are
formed, we once again get a buffer solution in which the pH remains relatively
constant until essentially all of the H3N+CH2CO2H molecules have been
converted into H2NCH2CO2- ions.
At this point, the pH rises rapidly until it reaches the value observed for a
strong base.

The pH titration curve tells us the volume of base required to titrate the positively
charged H3N+CH2CO2H molecule to the H3N+CH2CO2- zwitterion. If we only add half
as much base, only half of the positive ions would be titrated to zwitterions. In other
words, the concentration of the H3N+CH2CO2H and H3N+CH2CO2- ions would be the
same. Or, using the symbolism in the Henderson-Hasselbach equation:
[HA] = [A-]
Because the concentrations of these ions is the same, the logarithm of the ratio of their
concentrations is zero.

Thus, at this particular point in the titration curve, the Henderson-Hasselbach equation
gives the following equality.
pH = pKa
We can therefore determine the pKa of an acid by measuring the pH of a solution in
which the acid has been half-titrated.
Because there are two titratable groups in glycine, we get two points at which the
amino acid is half-titrated. The first occurs when half of the positive H3N+CH2CO2H
molecules have been converted to neutral H3N+CH2CO2- ions. The second occurs
when half of the H3N+CH2CO2- zwitterions have been converted to negatively charged
H2NCH2CO2- ions.
The following results are obtained when this technique is applied to glycine.

Let's compare these values with the pKa's of acetic acid and the ammonium ion.
CH3CO2H
NH4+

pKa = 4.74
pKa = 9.24

The acid/base properties of the -amino group in an amino acid are very similar to the
properties of ammonia and the ammonium ion. The -amine, however, has a

significant effect on the acidity of the carboxylic acid. The -amine increases the value
of Ka for the carboxylic acid by a factor of about 100.
The inductive effect of the -amine can only be felt at the -CO2H group. If we look
at the chemistry of glutamic acid, for example, the -CO2H group on the R substituent
has an acidity that is close to that of acetic acid.

When we titrate an amino acid from the low end of the pH scale (pH
1) to the high
end (pH 13), we start with an ion that has a net positive charge and end up with an
ion that has a net negative charge.

Somewhere between these extremes, we have to find a situation in which the vast
majority of the amino acids are present as the zwitterion
with no net electric
charge. This point is called the isoelectric point (pI) of the amino acid.
For simple amino acids, in which the R group doesn't contain any titratable groups,
the isoelectric point can be calculated by averaging the pKa values for the carboxylic acid and -amino groups. Glycine, for example, has a pI of about 6.
pI = 2.35 + 9.78 = 6.1
2
At pH
6, more than 99.98% of the glycine molecules in this solution are present as
the neutral H3N+CH2CO2H zwitterion.
When calculating the pI of an amino acid that has a titratable group on the R side
chain, it is useful to start by writing the structure of the amino acid at physiological
pH (pH
7). Lysine, for example, could be represented by the following diagram.

At physiological pH, lysine has a net positive charge. Thus, we have to increase the
pH of the solution to remove positive charge in order to reach the isoelectric point.
The pI for lysine is simply the average of the pKa's of the two -NH3+ groups.
pI = 9.18 + 10.79
2

10.0

At this pH, all of the carboxylic acid groups are present as -CO2- ions and the total
population of the -NH3+ groups is equal to one. Thus, the net charge on the molecule
at this pH is zero.
If we apply the same technique to the pKa data for glutamic acid, given above, we get
a pI of about 3.1. The three amino acids in this section therefore have very different
pI values.
Glutamic acid (R = -CH2CH2CO2-):
pI = 3.1
Glycine
(R = -H):
pI = 6.1
+
Lysine
(R = -CH2CH2CH2CH2NH3 ): pI = 10.0

Thus, it isn't surprising that a common technique for separating amino acids (or the
proteins they form) involves placing a mixture in the center of a gel and then applying
a strong voltage across this gel. This technique, which is known as gel
electrophoresis, is based on the fact that amino acids or proteins that carry a net
positive charge at the pH at which the separation is done will move toward the
negative electrode, whereas those with a net negative charge will move toward the
positive electrode.

All living things are water-based systems, which means that they depend heavily on
aqueous equilibria, especially acid-base equilibria. Therefore, all the acid-base and pH
concepts we have discussed so far are extremely important to biochemistry, which is
the study of the chemistry of biological systems.
Reasons why we should be concerned about pH in biological systems:

It gives a qualitative measure for many problems in cell biology and related
fields
Proton dissociable groups are found in macromolecules (such as proteins) as
well as the small molecules we have discussed already
The cell environment is always buffered at approximately pH 7
Experiments such as biological enzymatic assays require a certain pH

Just as in other acid-base systems, biological macromolecules act as acids and bases
by donating and accepting protons. However, due to the size of these molecules, they
often contain several different groups that accept or donate protons instead of just one
such group. Thus, we talk about macromolecules as having acidic and basic
groups rather than as being acids and bases. These acidic and basic groups act as
weak acids and bases, with Ka values which determine the extent of dissociation of
the group depending on the pH of the system. Therefore, changes in the pH around the
macromolecule will determine which groups are protonated and which are not, which
in turn determines properties of the molecule. This is especially important
for enzymes, which are proteins that act as catalysts for important biological
reactions. Most enzymes only work within a certain pH range.
For example, consider an enzyme with a carboxyl group. The structure of that group
will depend on the pH:

If the enzyme needs to be protonated in order to be active, then the enzyme will only
work in the pH range in which the majority of the enzyme molecules have their
carboxyl group protonated. In this way, pH determines which enzymes are active and
thus which biochemical reactions can occur.

Buffer Systems in Living Organisms

Learning Goal 41
Describe the buffer action that controls the pH of the cytoplasm and blood

Because all biological processes are dependent on pH, cells and organisms must
maintain a specific and constant pH in order to keep their enzymes in the optimum
state of protonation.
1. Cytoplasm controlled by the phosphate buffer system:
H2PO4

H+ + HPO42

This system provides the maximum buffering capacity near pH 6.86 (the pKa
of H2PO4 ). It provides the buffering effect in intracellular fluid, and is
important in urine.
2. Blood must be maintained at pH ~7.4 (pH < 6.9 and >7.6 are life-threatening).
The pH of the blood is controlled by the bicarbonate buffer system:
CO2(g)

CO2(aq) + H2O(l)

H2CO3(aq)

H+(aq) + HCO3(aq)

The tissues release CO2 into the blood, where it is converted to HCO3 . In this
form, it is carried to the lungs, where it is converted back to gaseous CO2 for
exhalation. pH depends on [H2CO3] and [HCO3 ], and [H2CO3] depends on the
CO2 dissolved in the blood.

Breathing:
Tissues release H+ into blood:

[H+] increases
[H2CO3] increases
[CO2] dissolved in blood increases
pressure of CO2 in lungs increases
exhalation occurs, restoring equilibrium

Tissues release OH into blood (or remove H+ from blood):

[H+] decreases
[H2CO3] decreases
[CO2] dissolved in blood decreases
pressure of CO2 in lungs decreases
inhalation occurs, restoring equilibrium

Major Buffer Systems of the Human Body

Bicarbonate buffer CO2 + H2O
Hemoglobin
Phosphate buffer
Protein

Hb-H
H2PO4
Pr-H

H2CO3

H+ + HCO3 In blood plasma

Hb + H+
H+ + HPO42
Pr + H+

Interior of red blood cells

Most important in urine
Intracellular fluid

Amino Acids
Amino acids are small molecules with both an amino group and a carboxyl group.
Since each of these groups can be either protonated or deprotonated, the structure of

an amino acid depends on the pH of the solution it is in. At pH 7, amino acids have
the following structure:

There are twenty different amino acids that are commonly found in living systems.
For the most part, these twenty amino acids differ only in their side chains:
Glycine (Gly)

Alanine (Ala)

Valine (Val)

Leucine (Leu)

Isoleucine (Ile)

Proline (Pro)

Serine (Ser)

Threonine (Thr)

Cysteine (Cys)

Phenylalanine (Phe)

Methionine (Met) Asparagine (Asn) Glutamine (Gln)

Tyrosine (Tyr)

Tryptophan (Trp)

Lysine (Lys)

Arginine (Arg)

Histidine (His)

Aspartate (Asp)

Glutamate (Glu)

Amino acids can be classified into categories based on their side chains:

Aliphatic: Gly, Ala, Val, Leu, Ile, Pro

Polar: Ser, Thr, Cys, Met, Asn, Gln
Aromatic: Phe, Tyr, Trp
Positively charged (basic): Lys, Arg, His
Negatively charged (acidic): Asp, Glu

Amino acids can be joined together through condensation or dehydration reactions

between the carboxyl group of one amino acid and the amino group of the next. The
result is a new bond between the carboxyl carbon and the amino nitrogen. This bond
is called a peptide bond.

Two amino acids joined together in this manner are referred to as a dipeptide. Note
that two different amino acids can be joined in either of two ways: either through the
amino group of the first and the carboxyl group of the second or through the carboxyl
group of the first and the amino group of the second. Consider a dipeptide made up of
threonine and histidine:

We can avoid ambiguity by stating which amino acid residue is the amino terminus,
the one with the free amino group, and which is the carboxyl terminus, the one with
the free carboxyl group.
Similar principles apply to peptides consisting of more than two amino acids. Three
amino acids make a tripeptide, four make a tetrapeptide, five make a pentapeptide,
and so on. (Note that the numerical prefix refers to the number of amino acids, not the
number of peptide bonds.) When several amino acids are joined, the resulting
structure is referred to as an oligopeptide, and when thousands are joined, it is called
a polypeptide, orprotein.
Proteins are extremely important to the body, acting as structural components,
enzymes, hormones, and more. Thus, extreme diversity is required for proteins. This
diversity comes from the fact that there are 20 different amino acids found commonly
in living things. Since each position in a polypeptide can be occupied by any of these
20 amino acids, the number of possible polypeptide molecules with a length
of n amino acids is 20n.(Why?) This is a huge number, allowing for the necessary
diversity.

Acid-Base Properties of Amino Acids

Learning Goal 42
Describe how amino acids act as acids and bases and what information about them can be obtained
from their titration curves

When in an aqueous solution, amino acids can act as both acids and bases they are
amphoteric.

To fully describe the ionization state of an amino acid, both the individual charges and
the net charge must be considered:
If only positive charges or only negative charges are present, the molecule is
described as either a cation or an anion respectively. However, both positive and
negative charges can be present at the same time. When this happens, the molecule is
called a dipolar ion or zwitterion. All amino acids exist as zwitterions at pH 7.0.
Depending on whether the side chain is positively-charged, negatively-charged or
uncharged, an amino acid zwitterion may or may not have a net charge. Note that
there is no pH at which both groups are electrically neutral.
The net charge on an amino acid is the sum of all the individual charges. The net
charge can be described as monopositive, dipositive, mononegative, dinegative or
isoelectric. Isoelectric means that the molecule has both positive and negative charges
but no net charge, as opposed to neutral which means that no charges are present.
The ionization state of an amino acid can be described completely and unambiguously
by describing both the net charge and the individual charges (for example, the
mononegative zwitterion form of aspartate). However, for a given amino acid, there is
only one form that has a given net charge, so the net charge alone can be used to
describe the amino acid. (For example, the mononegative form of aspartate is by
definition a zwitterion, whereas the mononegative form of lysine is an anion) This
method is much less wordy, and will be used throughout this tutorial.
The titration of an amino acid is simply a polyprotic acid titration, with the pH values
at the midpoints giving the pKa's of the dissociable groups. Here is the curve for an
amino acid with a non-dissociable side chain. (Note that one equivalent of NaOH is a
volume of NaOH equal to the volume of amino acid being titrated.)

1.

pH < pKaC

Almost all monopositive

form

Avg. net charge just

below +1

2.

pH = pKaC

Half monopositive, half

isoelectric

Avg. net charge = +0.5

3.

pH = 1/2(pKaC + pKaN)

All isoelectric form

Avg. net charge = 0

4.

pH = pKaN

Half isoelectric, half

mononegative

Avg. net charge = 0.5

5.

pH > pKaN

Almost all
mononegative

Avg. net charge just

above 1

Each amino acid has different pKaC and pKaN values, and those with dissociable side
chains also have a pKaR.
Click here or on the
button on the menu bar for a table of pKa and pI values (see
below) for the twenty common amino acids.

Isoelectric Point and Average Molecular Charge

Learning Goal 43
Calculate the isoelectric point for an amino acid and the average net charge on an amino acid at a given
pH

The isoelectric point (pI) of a molecule such as an amino acid, peptide or protein is
the pH at which it has a net charge of zero. We saw above that an amino acid with a
non-dissociable side chain had a net charge of zero when the pH was halfway between
the two pKa values. Thus, for an amino acid with a non-dissociable side chain,
pI = 1/2(pKaC + pKaN)
EXAMPLE

Calculate the pI of methionine.

Methionine has Ka values pKaC = 2.1 and pKaN = 9.3.
pI = 1/2(pKaC + pKaN)
pI = 1/2(2.1 + 9.3)
pI = 5.7
For amino acids with acidic and basic side chains, the pI is halfway between the pKa
values on either side of the isoelectric form.
EXAMPLE

Write equations for the dissociation of aspartate and calculate its pI.

The isoelectric form is found after the first dissociation, between pKaC and pKaR
pI = 1/2(pKaC + pKaR)
pI = 1/2(2.1 + 3.9)
pI = 3.0

Looking at these two examples, we can see a connection between the net charge on
the amino acid and the relative values of the pH and the pI:

If the pH is less than the pI, the amino acid will have a net positive charge.
If the pH is greater than the pI, the amino acid will have a net negative charge.
If the pH equals the pI, the amino acid will have no net charge (this is the
definition of pI.)

At certain pH's, the amino acid will have an integral charge, but most pH's are
between these points and give fractional charges. There is, in fact, a continuous
(though not uniform) charge gradient as the pH is changed. However, in discussing
the charge on amino acids, we must remember that each individual molecule has an
integral charge, not a fractional charge. The fractional charge comes from the
proportions of two integrally-charged species in equilibrium, giving an average net
fractional charge to the solution.
For example, in a 3:1 mixture of mononegative to isoelectric forms, 3 out of every 4
molecules (75%) has a charge of 1, and 1 out of every 4 (25%) is neutral. The
average net charge is then
qnet = (0.75)(1) + (0.25)(0) = 0.75
Remember from Section 13 (Buffers) that the ratio between a conjugate acid and base
can be obtained from the Henderson-Hasselbalch equation
pH = pKa + log ([base]/[acid])
At any given pH, there will be at most two forms of an amino acid present in
significant quantities, and they will be a conjugate acid-base pair. This means that
their ratio can be calculated with the Henderson-Hasselbalch equation, and the
average net charge on the amino acid can be calculated.
EXAMPLE

Find the average net charge on a solution of asparagine at pH 3.0

First, identify the major species present:
pH 3.0 is between pKaC, and the pI, which means that the major species
present are the monopositive and isoelectric forms.
Next, calculate the ratio of isoelectric form to monopositive form:

pH = pKaC + log([base]/[acid])
3.0 = 2.1 + log([base]/[acid])
0.9 = log([base]/[acid])
100.9 = [base]/[acid]
7.94 = [base]/[acid]
The isoelectric form is the conjugate base and the monopositive form is the
conjugate acid, so the ratio of isoelectric form to monopositive form is 7.94:1.
Finally, calculate the average net charge.
7.94 out of 8.94 molecules (89%) are isoelectric, and 1 out of 8.94 (11%) is
monopositive.
qnet = (0.89)(0) + (0.11)(+1) = +0.11

Electrophoresis
Learning Goal 44
Explain the ideas behind electrophoresis and how different types of electrophoresis can be used to
identify amino acids and measure their pI.

We can use the idea of charge variation with pH change to understand a technique
called electrophoresis, which can be used to separate and identify amino acids. In this
technique, a strip of paper is soaked in a buffer and attached to the terminals of a
battery to generate an electric field along it. Amino acids are then placed in the centre
of the paper and their migration is observed. Just like all charged objects, they will
move towards the region of opposite charge. The amount of migration will be
dependent on the magnitude of the charge, which is in turn dependent on the
difference between the pH and the pI. If the identities of the amino acids are known, a
buffer can be chosen with a pH that will cause the amino acids to migrate differently,
allowing them to be separated. If not, the results of running the electrophoresis at
several different pH values allows the amino acids to be identified.
Here we see the result of running a mixture of lysine and glutamine on an
electrophoresis gel at a pH of 7.6

pILys = 1/2(9.1 + 10.5) = 9.8

pILys > pH, so lysine is positively charged and migrates toward the cathode.
pIGln = 1/2(2.2 + 9.1) = 5.65
pIGln < pH, so glutamine is negatively charged and migrates toward the anode.
The isoelectric point of any amino acid, peptide or protein can be measured using
another type of electrophoresis called isoelectric focusing (IEF). In IEF, the
molecules in question are placed on a gel with a stable pH gradient along its length.
Since the pH of the surrounding medium determines the charge on the molecules, that
charge will change as the molecules migrate through the pH gradient. When each
molecule reaches a pH equal to its pI, it will be electrically neutral and will no longer
migrate in the electric field. Thus, every molecule subjected to IEF will move to a pH
equal to its pI, so by looking at the pH at which a molecule stops, its pI can be
measured.

Amino Acids, Peptides, and Proteins

The Amino Acids
The general structure of the amino acids found in proteins could be depicted as:
R
C
C
H2N H
O OH
As their name implies, they all contain a carboxylic acid group and an amino group.
They are called
alpha () amino acids based upon an archaic system of nomenclature. In this system
the carbon
adjacent to a carboxyl carbon is designated the carbon. Since the naturally
occurring amino acids
have the amino group on the a carbon they are amino acids.
The carboxylic acid group is a weak acid with a pKa
in the range of 1.7 to 2.6. The carboxylic acid
groups of the amino acids are stronger acids than simple organic acids. The amino
group is a
weak base having a pKa
in the range of 8.8 to 10.8.
Since the carboxyl group is an acid and the amino group is a base, in solution amino
acids self
neutralize / autoneutralize. The structure given previously for the naturally occurring
amino acids is

incorrect, the correct structure for an amino acid in solution is:

R
C
C
H3N H
OO
This DIPOLAR IONIC form exists when the amino acids are in solution at neutral pH
and when
they are in the solid state. The dipolar ionic form explains many of the properties of
the amino
acids. For example their melting points, boiling points, and solubilities more closely
resemble those
of ionic salts rather than organic acids. The dipolar ionic form of an amino acid is
called a
ZWITTER ION. Any molecular compound that contains a mixture of positive and
negative charges
is a zwitter ion.
pH Properties of the Amino Acids
The ionic state of the amino acids depends upon the pH of the solution in which they
are dissolved.
At low pH (pH ~ 1.0) the amino acid is in the cationic form and if placed in an electric
field they will
migrate toward the cathode. As the pH is increased the carboxylic acid group ionizes.
When the pH
is equal to the pKa
the amino acid exists as a 50:50 mixture of the cationic and zwitter ionic forms.

Adding more base results in continued ionization of the carboxylic acid group until
the zwitter ionic
form is the predominant form of the amino acid in solution. By the addition of more
base, the pKa
of the amino group is reached and at this point the amino acid exists as a 50:50
mixture of the zwitter
ionic form and the anionic form. As the pH is increased further the amino group
continues loses its proton and ultimately, at high pH (pH ~ 12.0), the anionic form is
the predominant form in
solution. The anionic form will migrate toward the anode if placed in an electric field.
This titration
curve demonstrates that the a amino acids never exist in an uncharged form.
As the amino acid is titrated, there is a solution pH along the titration curve where the
amount of
positive charge on the molecule is exactly balanced by the amount of negative charge.
At this pH the
amino acid in solution has no net charge. The amount of positive charge is exactly
balanced by an
equal amount of negative charge. The solution pH at which the opposite charges
exactly balance one
another is called the ISOELECTRIC POINT (pI) of the amino acid.
R
C
C
H3 N H
OO
R

C
C
H3N H
O OH
Forms present at pKa
(1)
R
C
C
H3N H
OO
R
C
C
H2N H
OO
Forms present at pKa
(2)
R
C
C
H3

NH
O OH
R
C
C
H3N H
OO
R
C
C
H2N H
OO
Dominant form
at pH 1
Dominant form
at pI
Dominant form
at pH 12At the isoelectric point an amino acid will not migrate in an electric field
since it has no net charge.
At pHs below the isoelectric point, the amino acid has a net positive charge and it
will migrate
toward the cathode; above the isoelectric point the molecule has a net negative charge
and it will

migrate toward the anode.

The R Groups - The Amino Acid Side Chains
The different R groups, the different amino acid side chains give each of the 20 amino
acids different
chemical and physical properties. Different combinations of the amino acids within a
protein give
the protein its unique chemical and physical properties. The unique sequence of
amino acids defines
the structure and function of the protein.
Its time to examine the structure of the side chains of the 20 naturally occurring amino
acids
The amino acids can be divided into two major groups: Nonpolar (Hydrophobic)
Amino Acids and
Polar (Hydrophilic) Amino Acids. The Polar Amino Acids can be divided into three
subgroups:
Polar But Not Charged Amino Acids, Amino Acids with Negatively Charged Side
Chains (Acidic
Amino Acids), and Amino Acids with Positively Charged Side Chains (Basic Amino
Acids).
The Hydrophobic (Nonpolar) Amino Acids are
H
C
C
H3N H
CH3
C

C
H3N H
CH
C
C
H3N H
H3
C CH3
CH2
C
C
H3N H
CH
H3C CH3
HC
C
C
H3N H
CH2
CH3
H2
N

H2C
CH2
CH
H2C
C
O
O
CH2
C
C
H3N H
CH2
S
CH3
H3N C H
CH2
NH
C
H3N C H
CH2
C
CH3

Glycine (Gly, G)
Alanine (Ala, A)
Valine (Val, V)
Leucine (Leu, L)
Isoleucine (Ile, I)
Proline (Pro, P)
Methionine (Met, M) Tryptophan (Trp, W)
Phenylalanine (Phe, F)
OOOOOOOOOO
OOOOOO
Note 1: The amino acids Tryptophan (Trp) and Glycine (Gly) are sometimes included
in the polar
but not charged group of amino acids. Tryptophan is sometimes included in this
group because the nitrogen in the ring system makes it slightly polar. However, the
complex ring system is fairly nonpolar and overall Trp behaves most often like a
nonpolar amino acid. Glycine is included because its
side chain is the small H and because its solubility in water resembles that of the other
polar amino
acids.
The Polar But Not Charged Amino Acids are:
H3N C H
CH2
OH
C

CH2
C
C
H3N H
SH
CH2
C
C
H3N H
OH
H3N C H
HO CH
CH3
C
H3N C H
CH2
C
O
H2N
C
H3N C H
CH2

CH2
C
C
H2N O
Cysteine (Cys, C)
Serine(Ser, S) Threonine (Thr, T)
Asparagine (Asn, N)
Glutamine (Gln, Q)
Tyrosine (Tyr, Y)
OOOOOO
OOOOOO
Note 2: The side chains of cysteine and tyrosine have some acid / base properties.
The -SH group on
cysteine has a pKa
of 8.3 and the -OH group on the tyrosine side chain has a pKa
of 10.1. These
pKa
s can be measured in vitro (in a test tube). At physiological pH, pH 7.4, these side
chains are for
the most part unionized.
Note 3: Phenylalanine, Tyrosine and Tryptophan are called the aromatic amino acids
because they
all contain an aromatic ring as part of their side chains. The aromatic groups on the
side chains of

Phenylalanine, Tyrosine and Tryptophan absorb light at the ultraviolet region of the
spectrum, at
280 nm. This property is employed by biochemists to detect and quantify proteins in
solutions and /
or biological fluids.
The Amino Acids with Negatively Charged Side Chains (Acidic Amino Acids)
are:H3N C H
CH2
CH2
C
H3N C H
CH2
C
CC
OO
OO
OO
Aspartate (Asp, D) O O
[Aspartic Acid]
Glutamate (Glu, E)
[Glutamic Acid]
The side chain carboxyl group of aspartate (aspartic acid) has a pKa
of 3.9 and the side chain

carboxyl group of glutamate (glutamic acid) has a pKa

of 4.3. These values are closer the pKa
s of
simple organic acids.
The Basic Amino Acids are:
H3N C H
CH2
CH2
CH2
CH2
NH3
H3N C H
CH2
NH
HN
CH
CH2
CH2
CH2
NH
C
NH3

HN
CCC
OOOOOO
Lysine (Lys, K)
Histidine (His, H)
Arginine (Arg, R)
H3N
The imidazole ring of histidine has a pKa
of 6.0, the side chain amino group of lysine has a pKa
of
10.5, and the guanidinium group of arginine has a pKa
of 12.5.
Chiral Carbon
When the R group (side chain) of an amino acid is an organic group other than a
hydrogen (H)
atom, the carbon of the molecule is an asymmetric carbon; a chiral carbon (a
tetrahedral
stereocenter). A chiral carbon in the molecule means that the molecule can exists as a
pair of
stereoisomers. These stereoisomers have mirror image configurations; they exist as a
pair of
enantiomers. The pair of enantiomers for an amino acid have the following
configurations:R
C

C
H3N H
OO
R
C
C
H NH3
OO
L Form D Form
In nature, when two enantiomers exist, usually only one is synthesized and used by
cells. In the case
of the amino acids, only the L configuration exists in nature and is used by cells.
Some bacteria and
fungi synthesize and use D configuration amino acids. The molecules synthesized
with these D-form
amino acids are often toxic to other living organisms. With these few minor
exceptions all of the
naturally occurring amino acids are in the L-form.
Calculation of Amino Acid Isoelectric Point
For the nonpolar and polar amino acids with two pKa
s, the isoelectric point is calculated by taking
the numerical average of the carboxyl group pKa
and the -amino group pKa
.

The titration curves for seven of the amino acids (Cys, Try, Glu, Asp, His, Lys, &
Arg) demonstrate
three inflection points, three pKa
s. One for the -amino group, one for the carboxyl group (carbon
1), and one for the ionizable side chain. The isoelectric point for these amino acids in
calculated by
taking the numerical average of the pKa
s of the groups with like charge when ionized. For example
to calculate the isoelectric point of Glu, the pKa
s of the two carboxyl groups are averaged. To
calculate the isoelectric point of Arg, the pKa
s of the -amino group and the guanidinium group are
averaged. Remember, at the isoelectric point the amino acid has no net charge. For
the amino acids
with three ionizable groups, the total charge on the groups with like charge must equal
one (1) so
that it can be balanced by the one (1) opposite charge present on the molecule.
Proteins: Their Primary Structure and Biological Functions
The word protein comes from the Greek PROTEIOS which means first or primary.
Proteins are of
primary importance to cells and organisms. Proteins can be compared to words of the
English
language. The words that are read, written, and spoken are composed of the 26 letters
of the
alphabet. The sequence of letters is the primary structure of the word. From the 26
letters of the

alphabet an infinite number of words can be made. Some of these words have
meaning, others
are just gibberish. For proteins the alphabet is the 20 amino acids. The sequence of
amino acids is
the primary structure of the polypeptide. From these 20 amino acids an infinite
number of
polypeptides can be made, each with a different primary structure. Many of these
polypeptides are
just gibberish. The cell only synthesizes proteins that have meaning.
Peptide Bonds
H3N C C
H
R
O
O
NCC
R
H
O
O
H
H
H
+ H3N C C N C C

H
R
O
H
R
H
O
O
+
H
O
HAmino acids are linked in proteins in a head to tail manner by a condensation
reaction between the
carboxyl group of one amino acid and the amino group of the second. The back
bone of a
protein consists of the repeating sequence -N-C-C-. The amino acid side chains of
the amino acids
project perpendicularly from the back bone of the molecule.
The bond between amino acids in a protein is an amide bond. Since this amide bond
holds peptides
and proteins together it is called a PEPTIDE BOND. This amide linkage, the peptide
bond, has no
acid or base properties. It will neither donate nor accept a proton. However, the
peptide bond is
very polar.

Physical chemical studies have shown that the peptide bond exists in two resonance
forms:
CN
O
H
CN
O
H
C
C
C
C
The real nature of the peptide bond lies somewhere between these two extremes, it has
partial double
bond character. The partial double bond character of the peptide bond restricts free
rotation about
this bond limiting the possible number of conformations that the peptide or protein
can assume. It
also places the six atoms of the peptide bond in the same plane.
Peptide / Protein Terminology
The unique sequence of amino acids in a peptide or protein is termed the Primary (1)
Structure of
the Protein. A gene contains the information necessary for the synthesis of a protein,
for the

assembly of the primary structure. The primary structure contains the information
necessary for the
protein to fold into its final three dimensional conformation and once correctly folded
to assume its
cellular function. If the 1 structure of a protein is changed very often the final shape
changes
resulting in a nonfunctional polypeptide.
A molecule containing two amino acids joined by a peptide bond is a Dipeptide; one
with three
amino acids held together by two peptide bonds is a Tripeptide; four is a Tetrapeptide;
etc. In
general, Peptides contain 12 or fewer amino acid residues. An Oligopeptide contains
between 12 and
20 amino acids and a Polypeptide contains greater than 20 amino acids. Twenty
appears to be a
magic number with respect to peptide/protein structure. Oligopeptides with 20 or
fewer amino acids
do not fold into, do assume a single low energy conformation, rather they exist in
numerous
random shapes. Molecules with greater than 20 amino acids very often fold into a
single stable
conformation, a single low energy conformation. The terms polypeptide and protein
are often used
interchangeably. However, the term protein is often reserved for molecules that
perform some
cellular function.
Monomeric Proteins contain a single polypeptide chain. Some proteins are
supramolecular

complexes composed of more than one polypeptide chain. These proteins are called
Multimeric
Proteins. Homomultimeric Proteins are composed of several polypeptides all with the
same sequence
of amino acids, all with the same primary structure. Heteromultimeric Proteins are
composed of
several different polypeptides; polypeptides with different primary structures.
Sequence ConventionH3N
CH
C
H
N
CH
C
N
H
CH
C
H
N
CH
C
N

H
CH
C
H
N
CH
C
N
H
CH
C
H
N
CH
C
O
HO
CH3 O CH2
O CH2
O CH2
O CH2
O

O
CH2 O
CH2
C
OO
CH2
CH2
CH2
NH2
CH2
CH2
NH
C
NH2
NH
CH2
C
H2N O
C
H2
NO
CH2

OH
gly-ala-glu-lys-arg-gln-asn-ser
A protein is a long linear sequence of amino acids joined by peptide bonds. One end
of the molecule
has a free amino group, an amino group not involved in a peptide bond. This is the
Amino
Terminus or N-Terminus of the protein. The opposite end of the molecule has a free
carboxyl
group, a carboxyl group not involved in a peptide bond. This end is the Carboxy
Terminus,
Carboxyl Terminus or C-Terminus of the protein.
When biochemists write the sequence of a protein they use either the three letter or the
one letter
abbreviation for the amino acids, usually separated by hyphens. The amino terminus
is always the
Left most amino acid and the carboxyl terminus is always the Right most amino acid.
Biological Functions of Proteins
Within the cell / organism proteins serve a wide range of important biological
functions.
Enzymes are the biological catalysts of the chemical reactions that occur within the
cell. Enzymes will
be examined in detail shortly.
Regulatory Proteins regulate the activities of the cell and the ability of other proteins
to carry out
their cellular function. The peptide and protein hormones are regulatory proteins.
They play a role in

regulating overall metabolism, growth, development, and maintenance of the

organism. Allosteric
enzymes can be considered regulatory proteins since they control key cellular
reactions. Gene
inducers and gene repressors are also considered regulatory proteins. Gene inducers
stimulate gene
expression, they turn genes on. Gene repressors inhibit gene expression, they turn
genes off.
Transport Proteins carry specific substances from one place to another. Membrane
transporters
carry polar molecules across cell membranes. Hemoglobin transports oxygen from
the lungs to the
tissues; serum albumin carries a wide variety of drugs and metabolites through out the
body.Storage Proteins provide a reservoir of an essential nutrient. Myoglobin stores
oxygen in skeletal
muscle tissue; ferritin stores iron in the liver and bone marrow; the protein ovalbumin
serves as a
storage depot of amino acids for developing birds; and casein, the major protein in
mammalian milk
stores amino acids for growing infants.
Structural proteins provide strength, support, and form to cells, tissues, and organisms.
Collagen is
the major protein of bone, tendons, and cartilage. a-Keratin is the protein of hair,
horns, hooves,
and fingernails. There are cytoskeletal protein fibers adjacent to the cell membrane
that give the cell
shape and support. Tubulin, actin, and spectrin are some of the proteins that make-up
the
cytoskeleton.

Contractile and Motile Proteins provide the cell / organism with motion. Examples
include actin and
myosin of muscle cells and tubulin of cilia and flagella. The proteins dynein and
kinesin drive the
movement of vesicles and organelles along cytoskeletal tracks within the cell. A
single protein can
serve several functions within a cell. For example actin and tubulin can be classified
as structural
proteins or motile proteins depending upon their location within the cell.
Scaffold Proteins act as bridges by binding to and localizing specific proteins to
specific sites within
the cell. They act as a site upon which supramolecular complexes are formed.
Protective Proteins play an active role in cell defense or in defense of the organism.
Antibodies of the
immune system protect the organism from foreign invaders. The hemostasis cascade
of proteins
protects the organism from excessive blood loss. Toxins, such as snake venoms, also
fall into this
class of proteins.
Exotic Proteins display functions that do not fit the other classifications. One example
is the glue
protein secreted by mussels. This protein allows the mussels to anchor to hard
surfaces.
Conjugated Proteins
Conjugated proteins contain nonprotein components integral to their structure and
necessary for
their function. The nonprotein part is called a Prosthetic Group. Conjugated proteins
include:

Glycoproteins - the prosthetic groups are carbohydrates.

Lipoproteins - the prosthetic groups are lipids.
Nucleoproteins - the prosthetic groups are nucleotides or nucleic acids.
Phosphoproteins - have phosphoryl (phosphate) groups in ester linkage to hydroxyl
groups on the
protein.
Metalloproteins - contain metal ions attached either by ionic interactions or by
coordinate covalent
bonds.

1
Amino Acids and Peptides
I. Structure
A. Definition: carboxylic acid with an amino group and R side chain extending from
the
-carbon.
H2N C
H
R
C
O
OH

Amino
Carboxyl
Side Chain
B. It is the R group which differentiates between the 20 available standard amino
acids.
1. Amino acids are grouped according to the distinguishing characteristic of the R
side chain at physiological pH:
a. aliphatic
b. aromatic
c. polar, uncharged
d. positively charged
e. negatively charged
2. Carbons in the R side chain are designated by Greek letters, such as , , , , etc.
as the carbons extend away from the -carbon.
II. Nomenclature
All of the amino acids have trivial or common names, which just simply must be
memorized. For example, many were named to the source from which they were first
isolated: asparagine from asparagus and tyrosine from cheese (tyros = cheese in
Greek).
Glycine was accordingly named simply for its sweet taste (glykos = sweet in Greek).
Note: On pg 78 of your text, you should MEMORIZE the 3-letter AND 1-letter
abbreviations for each of the amino acids listed. You will also be held responsible for
knowing which amino acids have an associated pKR

value, and, roughly speaking, whether

its value is greater than, less than, or approximately the same as physiological pH.
III. Backbone Charge
A. The amino group has a pK value in the range of 8.8 (Asn) to 11.0 (Pro). Which,
therefore, is the predominant form in the body, -NH3
+
or -NH2
?
B. The carboxyl group has a pK value in the range of 1.8 (Phe) to 2.4 (Trp). Which,
therefore, is the predominant form in the body, -COOH or -COO
?2
C. Amino acids are therefore considered zwitterions at neutral pH.
1. Zwitterions are defined as any molecule which bears opposite charges within its
structure (dipolar)
2. Zwitterions in general exhibit higher melting points than other organic compounds
of similar molecular weight, because their charges allow them to form crystal
lattices similar to NaCl.
3. Zwitterions are, of course, more soluble in polar solvents.
4. Zwitterions are electrically neutral, and therefore remain stationary in an electric
field.
IV. Acid / Base Characteristics

A. Amino acids are amphoteric or ampholytes, i.e. they can act as acids or bases at
physiological pH:
H3N C
H
R
C
O
O
+acid base
B. Titration Characteristics
1. Amino acids at low pH can be diprotic, or even triprotic if their R groups are
ionizable.
2. Consider the titration of glycine at very low pH with hydroxide ions:
pH
[OH] Equivalents
1
2
3
4
5
!

!
!
!
!
0 0.5 1 1.5 2
Glycine: pK1 = 2.34 ; pK2 = 9.60
Questions to Ponder:
(1) What is the significance of the pH
at points 2 and 4?
(2) What are the protonation state(s) of
glycine at each point?
(3) What is the average net charge of
glycine at each point?
(4) What is the significance of the pH
at point 3?
3. Problem: (a) Draw the step-wise ionization of glutamate, assuming pK1
=-2.19,
pK2
=9.67, and pKR
=4.25. (b) What is the net charge of each species? (c) What is the
value of pI for glutamate? What is its net charge when pH=pI?3
C. Proximity Effects

The pKa for acetic acid is 4.8. However, the highest pK1
value for an amino acid is
only 2.4? Why is the pK value for the same carboxyl group of an amino acid so much
lower than that of acetic acid, i.e. why is the hydrogen from the amino acids carboxyl
group so much more easily removed?
Consider the equilibria:
C COOH
H
H
H
C COO
H
H
H
+ H+
C COOH
NH3
+
H
H
C COO

NH3
+
H
H
+ H+
Notice that when glycine loses a hydrogen ion, the charge left behind can be partially
stabilized by the charge associated with the amino group. This species, therefore, is
preferentially stabilized vs. the deprotonated form of acetic acid, accounting for the
increased ease with which an amino acid is ionized. This is an example of a
proximity effect, i.e. the influence of other functional groups within a molecule on the
particular chemical attributes of another functional group. This effect similarly
explains why pK1
values between amino acids slightly differ, although the differences
are often not so easily correlated with structural features.
V. Classification of R Groups
*Amino acids are the constituent components of proteins. Their characteristics, then,
will
define the regional characteristics of proteins.
A. Aliphatic, Nonpolar
*Amino acids in this category can position themselves on the interior or exterior of
proteins, but, more often than not, are found on the interior where they can position

themselves away from the polar solvent.

1. Glycine ; Gly ; G
C COO
+H3N
H
H
a. Simplest of all amino acids, with the R group represented by a single hydrogen
atom.
b. Allows the most structural flexibility within a protein, via minimizing potential
steric complications.4
2. Alanine ; Ala ; A 3. Valine ; Val ; V
C COO
+H3N
H
CH
H
H
C COO
+H3N

H
C
H3C CH3
H
4. Leucine : Leu ; L 5. Isoleucine ; Ile ; I
C COO
+H3N
H
CH
C
H
H3C CH3
C COO
+H3N
H
C CH3
C
H
CH3
HH

6. Proline ; Pro ; P
+
H2N
H2C CH2
CH2
C
COO
H
a. Proline is the most rigid of all amino acids, with little flexibility available in
the cyclic R chain.
b. It contains a secondary, rather than primary, amino group, often referred to as
an imino acid.
c. It is responsible for forming bends in protein structure (-turns).
d. It is the only aliphatic amino acid that usually is solvent-exposed.
B. Polar, Uncharged
*Amino acids in this category are hydrophilic and therefore are often found in the
exterior folds of proteins, hydrogen bonding with water. However, they can also be
structurally or mechanistically important residues within the interior of proteins.
1. Serine ; Ser ; S 2. Threonine ; Thr ; T
C COO
+-

H3N
H
HCH
OH
C COO
+H3N
H
H C OH
CH3
pKR
= 13.60 pKR
= 13.60
pH < or > pKa pH < or > pKa
protonated or deprotonated protonated or deprotonated5
3. Cysteine ; Cys ; C
C COO
+H3N
H
HCH
SH

pKR
= 8.18
pH < or > pKa
protonated or deprotonated
a. Disulfide bridges can form when two cysteine side chains interact, providing a
very stabilizing influence on protein conformation:
b. Disulfide bonds can be broken in vitro through the use of reducing agents,
such as dithiothreitol (DTT) or -mercaptoethanol (BME).
c. The propensity to form disulfide bridges in vivo depends upon where the
proteins are located:
(1) Intracellular proteins are maintained in a reducing environment brought
about by the glutathione pool, itself maintained with NADPH via the
phosphogluconate pathway.
(2) Extracellular proteins are in an oxidizing environment, allowing disulfide
bridges to stabilize the conformation of the protein.
4. Methionine ; Met ; M 5. Asparagine ; Asn ; N 6. Glutamine ; Gln ; Q
C COO
+H3N
H
HCH
HCH

S
CH3
C COO
+H3N
H
HCH
C
H2 N O
C COO
+H3N
H
HCH
HCH
C
H2 N O
(thioether linkage) (amide group) (amide group)6
C. Aromatic
*Amino acids in this category are most likely to be found within the hydrophobic core
of proteins, escaping the polar solvent.
1. Phenylalanine ; Phe ; F

C COO
+H3N
H
HCH
a. Because of the phenyl component, this amino acid absorbs weakly in the deep
UV region of the spectrum. Its extinction coefficient at 280 nm is the lowest
of the aromatic amino acids. It also is blue-shifted relative to the other
aromatics, i.e. it absorbs at lower wavelengths. Peak abs = ______________.
b. It barely fluoesces, meaning it is capable of absorbing light at one wavelength,
decaying to a lower excited state, and reemitting the energy as light of a higher
wavelength.
2. Tyrosine ; Tyr ; Y
C COO
+H3N
H
HCH
OH
a. This amino acid is, like all aromatic residues, found predominately in the
interior of proteins, yet the polar hydroxyl group also provides it with
hydrogen bonding potential. Tyrosine, therefore, often plays a mechanistic

role in catalysis at an enzymes active site.

b. Like phenylalanine, tyrosine also absorbs in the deep UV (250-290nm), but
exhibits a greater extinction coefficient than Phe, and is red-shifted relative to
it. Peak abs = ______________.
c. Tyrosine is slightly more fluorescent than Phe.
d. Ionizability:
pKR
= 10.07
pH < or > pKa
protonated or deprotonated7
3. Tryptophan ; Trp ; W
C COO
+H3N
H
HCH
C CH
NH
a. This amino acid absorbs UV radiation very strongly. It exhibits an extinction
coefficient at 280 nm that is very high, relative to Phe and Tyr.
b. The absorption spectrum of this amino acid is red-shifted relative to that of
both phenylalanine and tyrosine (250-305 nm).

c. Tryptophan is the most important intrinsic fluorescent probe in biophysical

studies of proteins:
(1) It absorbs strongly, and exhibits a high quantum yield.
(2) Its properties are strongly influenced by its environment:
(lifetime, rotational characteristics, exposure to quenching)
(3) It is a rare amino acid, often only occurring 1-3 times per protein.
(4) It can be selectively excited at 300 nm.
D. Polar, Charged
1. Positively Charged Amino Acids
a. Lysine ; Lys ; K b. Arginine ; Arg ; R
C COO
+H3N
H
HCH
C
C
C
NH3
+
HH
HH

HH
C COO
+H3N
H
HCH
C
HCH
HH
N
C NH2
+
NH2
pKR
= 10.53 pKR
= 12.48
pH < or > pKa pH < or > pKa
protonated or deprotonated protonated or deprotonated8
c. Histidine ; His ; H
C COO
+H3N

H
HCH
C NH
CH
N
H
+
HC
pKR
= 6.00
pH < or > pKa
protonated or deprotonated
(1) Histidine is the only amino acid with physiologically relevant buffering
capacity near.
(2) This amino acid can shift in its ionization state in response to very small
variations in intracellular pH. This property makes histidine an important
residue in the mechanisms of enzyme-catalyzed reactions.
(3) Histidine is often found coordinated with heavy metals in proteins.
2. Negatively Charged Amino Acids
a. Aspartate ; Asp ; D b. Glutamate ; Glu ; E
C COO
+-

H3N
H
HCH
CO
O
C COO
+H3N
H
HCH
HCH
CO
O
pKR
= 3.65 pKR
= 4.25
pH < or > pKa pH < or > pKa
protonated or deprotonated protonated or deprotonated9
F. Example Problems from Chapter 5
1. At a pH equal to its isoelectric point, the net charge on alanine (pK1

=2.34,
pK2
=9.69) is zero. Two structures can be drawn with a net charge of zero, but the
predominant one is zwitterionic:
H
C
CH3
+
H3N COO
vs.
H
C
CH3
H2N COOH
Zwitterionic Uncharged
What is the fraction of uncharged alanine present at its pI?
2. (a) Draw the step-wise ionization of histidine (pK1
=1.82, pK2
=9.17, and pKR
=6.00):
(b) What is the net charge of histidine at pH 1, 4, 8, and 12? For each pH, will

histidine migrate toward the anode (+) or cathode (-) when placed in an electric
field?10
VI. Stereochemistry
A. Definitions
1. Chiral Atom = an atom containing an asymmetric center, i.e. with four di erent
functional groups attached. All amino acids have a chiral center, except for
__________.
2. Stereoisomers = molecules which only differ in their spatial arrangements of
constituents.
3. Enantiomers = stereoisomers which are nonsuperimposable mirror images of
each other. Each chiral center gives rise to an enantiomeric pair.
4. Optically Active = capable of rotating plane-polarized light.
B. Nomenclature
1. Fischer Convention (D, L Method)
a. In this convention, all molecules are referenced to the simplest sugar to have an
asymmetric center, glyceraldehyde.
--glyceraldehyde has one chiral center, and therefore two enantiomers
--each enantiomer has been labeled either L or D
--the configuration of groups about other chiral centers are labeled according
to the groups chemical similarity to those of glyceraldehyde
b. Short-hand drawings:
(1) Geometric Formula
CHO

C
CH2OH
OH H
L
CHO
C
CH2OH
H OH
D
(2) Fischer Formula
CHO
C
CH2OH
OH H
CHO
C
CH2OH
H OH
L-glyceraldehyde D-glyceraldehyde
COOH
C
+

H3N H
R
COOH
C
+
H3N NH3
+
R
L-amino acid D-amino acid
*Amino acids in proteins are ______.
**Two amino acids have two chiral centers: ____________ and ____________.11
2. Cahn-Ingold-Prelog System (R, S Method)
a. In this system, groups around the chiral center are prioritized:
SH > OH > NH3
> CH2
-SH > COOH > CHO > CH2OH > C6H5
> CH3
To label an arrangement as either R or S:
(1) turn the lowest priority constituent group to the back, and
(2) if going from highest to lowest priority takes you clockwise, it is R.
(3) if going from highest to lowest priority takes you counterclockwise, it is S.
Examples: Are the following amino acids R or S as drawn:

NH3
+
HOOC C H
CH3
H
C
OOC NH3
+
C
C
HH
O
O
H
C
HCH
OH
COO
+H3N

________________ ________________ ________________

b. All L-amino acids with one chiral center are S-amino acids, except:
COO
C
+
H3N H
c. The two amino acids with 2 chiral centers each are still designated L, but
both L chiral centers are not necessarily S.
(1) What is the Cahn-Ingold-Prelog designation of L-isoleucine? _________.
COO
C
+
H3N H
C
C
H3C H
HH
CH3
(2) What is the Cahn-Ingold-Prelog designation of L-threonine? _________.
COO

C
+
H3N H
C
CH3
H OH12
VII. Peptides
A. Bond Characteristics
*Amino acids can join together end to end via a covalent amide linkage known as a
peptide bond. There are a variety of enzymes which mediate this process in vivo.
H
C
R1
H2N C
O
OH
H
C
R2
H2N C
O

+ OH + H2O
B. Reactions
*Amide bonds are subject to cleavage by strong acids or bases, although they are
stable to moderate pH changes generally encountered in vivo:
H
C
R1
H2N C
O
OH
H
C
R2
H2N C
O
+ OH
H2O
HCl or NaOH
C. Definitions
1. Peptide = two or more amino acids covalently joined by peptide bonds
2. Dipeptide = exactly two amino acids covalently joined by a peptide bond
3. Tripeptide = exactly three amino acids covalently joined by peptide bonds

4. Oligopeptide = general term for a peptide between 4-10 amino acids in length
5. Polypeptide = numerous amino acids covalently joined by peptide bonds,
usually greater than 10 in length
6. Protein = polypeptides generally greater than 40 amino acids in length
D. Nomenclature
There are three methods one can use to name a peptide. In each case, the peptide is
always drawn and named from the N-terminal amino acid (the amino acid with the
free amino group) to the C-terminal amino acid (the amino acid with the free
carboxyl group).
1. Link the names of the constituent amino acids together, replacing the suffix -ine
of each name with -yl in each amino acid except for the C-terminal amino acid,
which maintains its original name.
2. Link the 3-letter abbreviations of each constituent amino acid together with
dashes.
3. Link the 1-letter abbreviations of each constituent amino acid together with no
dashes.
Example: Draw the structure of the tripeptide of lysine, serine, and aspartatic acid as it
would appear at physiological pH, and give three different possible names for it:
Names: _________________________ or _______________ or ________13
E. Biological / Commercial Examples of Functional Peptides
1. Insulin : metabolic hormone occurring as a complex of two polypeptides, 30
and 21 amino acids in length.

2. Glucagon : metabolic hormone opposing the actions of insulin; exists as a

polypeptide of 29 amino acids
3. Glutathione : tripeptide maintaining an intracellular reducing environment
4. Nutrasweet : dipeptide serving as a commercial sweetener
5. Peptides are generally used in pharmaceutics as synthetic vaccines. Here the
strategy is to make a polypeptide with the same sequence as a viral antigen
to stimulate an immune response.

THE ACID-BASE BEHAVIOUR OF AMINO ACIDS

This page looks at what happens to amino acids as you change the pH by adding either
acids or alkalis to their solutions.
For simplicity, the page only looks at amino acids which contain a single -NH2 group
and a single -COOH group.

Amino acids as zwitterions

Zwitterions in simple amino acid solutions
An amino acid has both a basic amine group and an acidic carboxylic acid group.

There is an internal transfer of a hydrogen ion from the -COOH group to the -NH2 group
to leave an ion with both a negative charge and a positive charge.
This is called a zwitterion.

This is the form that amino acids exist in even in the solid state. If you dissolve the
amino acid in water, a simple solution also contains this ion.
A zwitterion is a compound with no overall electrical charge, but which contains
separate parts which are positively and negatively charged.

Adding an alkali to an amino acid solution

If you increase the pH of a solution of an amino acid by adding hydroxide ions, the
hydrogen ion is removed from the -NH3+ group.

You could show that the amino acid now existed as a negative ion
using electrophoresis.
In its simplest form, electrophoresis can just consist of a piece of moistened filter paper
on a microscope slide with a crocodile clip at each end attached to a battery. A drop of
amino acid solution is placed in the centre of the paper.
Although the amino acid solution is colourless, its position after a time can be found by
spraying it with a solution of ninhydrin. If the paper is allowed to dry and then heated
gently, the amino acid shows up as a coloured spot.
The amino acid would be found to travel towards the anode (the positive electrode).

Adding an acid to an amino acid solution

If you decrease the pH by adding an acid to a solution of an amino acid, the -COO- part
of the zwitterion picks up a hydrogen ion.

This time, during electrophoresis, the amino acid would move towards the cathode (the
negative electrode).

Shifting the pH from one extreme to the other

Suppose you start with the ion we've just produced under acidic conditions and slowly
add alkali to it.
That ion contains two acidic hydrogens - the one in the -COOH group and the one in the
-NH3+ group.
The more acidic of these is the one in the -COOH group, and so that is removed first and you get back to the zwitterion.

So when you have added just the right amount of alkali, the amino acid no longer has a
net positive or negative charge. That means that it wouldn't move towards either the
cathode or anode during electrophoresis.
The pH at which this lack of movement during electrophoresis happens is known as
the isoelectric point of the amino acid. This pH varies from amino acid to amino acid.
If you go on adding hydroxide ions, you will get the reaction we've already seen, in
which a hydrogen ion is removed from the -NH3+group.

Note: You might have expected the isoelectric point to be at pH 7 - when the solution is neither acidic
nor alkaline. In fact, the isoelectric point for many amino acids is about pH 6. Explaining why it isn't at pH
7 is quite long-winded and almost certainly beyond what you will need for UK A level (or its equivalent)
purposes. If you are interested, the problem is discussed at the bottom of this page.

You can, of course, reverse the whole process by adding an acid to the ion we've just
finished up with.
That ion contains two basic groups - the -NH2 group and the -COO- group. The NH2 group is the stronger base, and so picks up hydrogen ions first. That leads you
back to the zwitterion again.

. . . and, of course, you can keep going by then adding a hydrogen ion to the -COOgroup.

Why isn't the isoelectric point of an amino acid at pH 7?

Warning: If you are a studying a UK-based syllabus, you are extremely unlikely to need this for exam
purposes. It is included only for interest - you can probably safely ignore it. Check your syllabus and past
papers. If you haven't got these, follow this link to find out how to get hold of them.

When an amino acid dissolves in water, the situation is a little bit more complicated than
we tend to pretend at this level. The zwitterion interacts with water molecules - acting as
both an acid and a base.
As an acid:

The -NH3+ group is a weak acid and donates a hydrogen ion to a water molecule.
Because it is only a weak acid, the position of equilibrium will lie to the left.

As a base:

The -COO- group is a weak base and takes a hydrogen ion from a water molecule.
Again, the equilibrium lies to the left.
When you dissolve an amino acid in water, both of these reactions are happening.
But . . .
The positions of the two equilibria aren't identical - they vary depending on the influence
of the "R" group. In practice, for the simple amino acids we have been talking about, the
position of the first equilibrium lies a bit further to the right than the second one.
That means that there will be rather more of the negative ion from the amino acid in the
solution than the positive one.
In those circumstances, if you carried out electrophoresis on the unmodified solution,
there would be a slight drift of amino acid towards the positive electrode (the anode).
To stop that, you need to cut down the amount of the negative ion so that the
concentrations of the two ions are identical. You can do that by adding a very small
amount of acid to the solution, moving the position of the first equilibrium further to the
left.
Typically, the pH has to be lowered to about 6 to achieve this. For glycine, for example,
the isoelectric point is pH 6.07; for alanine, 6.11; and for serine, 5.68.
Note: I need to stress again that these figures (and the arguments) only hold where there is only one NH2 group and one -COOH group in the amino acid. The isoelectric points are quite different if the
molecule contains a second -NH2 or -COOH group.

Where would you like to go now?

To the amino acid and proteins menu . . .
To the menu of other organic compounds . . .
To Main Menu . . .

 Jim Clark 2004