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WEEK 10
BIOCHEMISTRY
Biochemistry is the study of those compounds produced and used by living organisms.
ELEMENTAL COMPOSITION
Most organisms are composed of only 16 chemical elements. Hydrogen, oxygen,
nitrogen and carbon comprise more than 99% of living matter. They are the smallest
atoms that can attain stable electronic configurations by sharing one, two, three, and four
electrons, respectively. All these elements form very stable covalent bonds. Oxygen,
nitrogen and carbon form stable multiple bonds. Carbon can bond to oxygen by a double
bond either within a biological compound or in the gaseous substance carbon dioxide, a
waste product produced by many living organisms. Carbon also bonds very stably to
hydrogen and to nitrogen.
O2 is vital to many living organisms. Two oxygens are joined by a double bond. Oxygen
is extremely electronegative and easily accepts electrons in energy yielding biochemical
reactions. This property satisfies the energy needs of many cells.
The action of phosphorous and sulfur in organisms is also related to cellular energy. In
the presence of water, large amounts of energy are required for these elements to form
bonds. Their formation is a means of carrying energy. When these bonds (high-energy
bonds), are hydrolyzed, large quantities of energy are released. ATP and coenzyme A
which contain sulfur act as energy carriers in living organisms.
Sodium is the major positive ion in extracellular fluids. Within cells, potassium is the
major positive ion. Both are involved in the actions of muscles and the nervous system.
Magnesium is necessary for the actions of enzymes and is found in cells. Chloride is the
major negative ion in the body. Approximately two-thirds of the negative ions in the
body are chloride ions. They are mainly in extracellular fluids. Calcium ions in the
forms of calcium phosphate and calcium hydroxide are important components of bone
and teeth. Calcium initiates the blood-clotting process; it also functions in the actions of
nerves and of muscles and is important to the activity of some enzymes and hormones.
Manganese, copper, and zinc act as parts of enzyme systems. The proper functioning of
nerves and the development of strong bones require manganese. Copper is important for
the synthesis of collagen, a connective-tissue protein. Besides being present in some
enzymes, zinc is also present in bone. The oxygen-carrying ability of blood is dependent
on iron.
Copper and iron also play an important role in respiration. They participate in a series of
oxidation and reduction reactions known as the electron-transport chain.
MOLECULAR COMPOSITION
The most abundant molecule found in living organisms is water. It has two properties
that qualify water as the basis of life: polarity and its formation of intermolecular
hydrogen bonds. Because it is polar, water easily dissolves other polar substances;
therefore, it provides an excellent transport system for nutrients. The extensive hydrogen
bonding between molecules of water causes its specific heat, heat of vaporization, and
heat of fusion to be unusually high in comparison to those of many other substances.
Because approximately 70% of the mass of an adult human is water, a considerable
amount of heat can be absorbed or released from a body without much change in body
temperature. An animal loses small quantities of body fluids in ridding itself of large
amounts of heat by surface evaporation. Organisms are protected against freezing,
because large quantities of heat must be removed before freezing occurs.
AMINO ACIDS
Twenty amino acids are the building blocks of proteins. These may be divided into
essential and nonessential amino acids. The nonessential ones can be synthesized by an
adult human from intermediates of carbohydrate breakdown. Humans, however, cannot
synthesize the essential amino acids, which therefore must be included in the diet.
AROMATIC BASES
The genetic code that governs growth and reproduction of cells is directed by nucleic
acids. Two groups of nucleic acids are ribonucleic acids (RNA) and deoxyribonucleic
acids (DNA).
SUGARS
Glucose is the building block for the carbohydrate compounds of cellulose, starch and
glycogen. It is also the primary carbohydrate that circulates in the blood. Oxidation of
glucose yields energy for cellular needs. The sugar ribose forms part of the structure of
RNA. In the form of deoxyribose (ribose minus one oxygen) it is found in DNA.
PHOSPHOLIPIDS
Membranes that surround both procaryotic cells and eucaryotic cells are composed of
phospholipids and proteins. The phospholipids are formed from the fatty acid, palmitic
acid, the alcohol, glycerol and the amine choline.
MACROMOLECULES
Small precursor molecules join together to form macromolecules. They form 85% to
95% of the dry mass of most organisms. The major types of macromolecules are
proteins, carbohydrates, and nucleic acids. The formation of all three is based on
reactions between the precursor molecules, which involve the elimination of water
between reacting sites in the molecules.
These large macromolecules are broken apart by another reaction, hydrolysis. These
synthesis and degradation reactions also require the presence of enzymes which help to
properly align the molecules. Synthesis is an energy-requiring process and degradation is
an energy releasing process. A general name for the former is ENDERGONIC and for
the latter is EXERGONIC.
PROTEINS – Polymerization of amino acids produces these macromolecules.
They have many functions. They form structures within organisms, and exercise
control over cellular reactions.
POLYSACCHARIDES – Polymers of sugar molecules, mostly glucose, act
as storage forms of energy in plant, animal, and human organisms. When energy
is needed, polysaccharides are degraded back to the precursor molecules.
NUCLEIC ACIDS
DNA and RNA are responsible for the transmission of genetic information and
the synthesis of protein by a cell. DNA is found in the cell nucleus and RNA in
both the nucleus and the cytoplasm. Both are usually joined to proteins, forming
nucleoproteins. Each individual strand of a nucleic acid is a polymer of units
called nucleotides. Nucleotides are composed of purine or pyrimidine, ribose or
deoxyribose and Phosphoric acid.
An actual molecule of DNA consists of two chains of nucleotides coiled in a
double-helix structure. Within each molecule, there is a 1:1 relationship between
cytosine and guanine nucleotides and adenine and thymine nucleotides.
RNA molecules generally are single-stranded. They can form helical structures
because of intrastrand hydrogen bonding. The bases usually pair adenine-uracil
and guanine-cytosine.
METABOLISM
The study of all the enzymatically controlled reactions in a living cell is METABOLISM.
This process can be divided into two parts: catabolism and anabolism. CATABOLISM
is a breaking-down process and produces the precursor molecules used by cells and
chemical energy for cellular needs. ANABOLISM is a building-up process and forms the
macromolecules of proteins, carbohydrates, and nucleic acids. Lipids are also produced
by these synthetic processes, but are not considered macromolecules because they are not
polymers.
WEEK 10
PROTEINS
Their functions are both structural and dynamic. They along with lipids form biological
membranes. These proteins are responsible for many of the processes that the
membranes perform. Two of these processes are transport and communication. They act
as hormones and antibodies, form a portion of the oxygen-carrying molecules
hemoglobin and myoglobin, and as enzymes serve as regulators of all biological
reactions.
Proteins are the focal point of the work of the embalmer. The embalming operation
achieves preservation and disinfection of a body by cross-linking proteins – both the
proteins of the human remains and the proteins present in microorganisms.
PROTEIN COMPOSITION
All proteins contain the elements carbon, hydrogen, oxygen, and nitrogen. Many also
contain sulfur and some contain small amounts of phosphorus, iodine, copper,
manganese, magnesium, and zinc. Proteins are very large molecules. They are polymers
of amino acids. Upon hydrolysis, they form mixtures of amino acids. Hydrolyzing
agents are acids, bases, and enzymes called proteases.
AMINO ACIDS
Twenty amino acids are the building blocks of proteins. Amino acids contain two
functional groups. They are the carboxyl group (-COOH) and the amino group (-NH2).
All amino acids contain another carbon, another hydrogen, and a side chain called and R
group.
R
H2N
C
COOH
H
The amino group is always attached to the carbon next to the carboxyl group. In organic
chemistry terminology, this carbon is the alpha carbon. Amino acids of this form are
called alpha amino acids.
A specific amino acid is identified by its R group. The simplest is glycine, in which the
R group is a hydrogen. Structures for additional amino acids are written by changing the
R group.
Because all amino acids have both the carboxyl group and amino group, they may act as
both acids and bases. Some amino acids have additional ability to act as bases, because
they contain nitrogen atoms in the side chains. Two amino acids have a carboxyl group
in their side chains and are therefore, acidic amino acids. The major properties of the
amino acids are inherent in the properties of the carboxyl and amino groups. Two of
these properties are amphoterism and peptide bond formation.
AMPHOTERISM
Amino acids contain the carboxyl group and the amino group, so they act as both acids
and bases. Carboxyl groups cause a compound to act as a weak acid because of the
following ionization:
O
O
C OH
O-
C
+ H2O
+ H3O+
Similarly, the presence of the amino group in a compound gives it basic properties.
NH2
+ H+
NH3+
Because both of these groups are found in each amino acid, an acid-base reaction may
occur within each molecule.
R
R
H2N
C
+
H3N
COOH
C
COO-
H
H
The product of this reaction is a dipolar ion. Another name for this ion is the Zwitterion.
In a neutral solution, the predominant form of amino acids has the carboxyl and amino
groups ionized. In a very acid solution (pH=1), the major form of amino acids is a
positively charge ion.
R
+
H3N
C
COOH
H
In a very basic solution (pH=12), the predominant form is a negatively charge ion.
R
H2N
C
H
COO-
Any compound that can act as both an acid and a base is called amphoteric.
Amphoterism is a property of all amino acids. Because of their amphoteric nature, amino
acids are buffers in solution. To buffer means “to protect”. BUFFERS are compounds
that protect a solution against changes in pH. By neutralizing either acids or bases that
enter a solution, amino acids keep the pH of a solution relatively constant within limits of
the added acid or base. Proteins are polymers of amino acids, so they can also function
as buffers.
PEPTIDE LINKAGE
By definition, a protein is a chain of alpha amino acids joined together by the peptide
linkage. If we draw the structures for any two amino acids with the carboxyl group of
one next to the amino group of another, their chemical linkage can be demonstrated. For
example:
H2N
R
O
C
C OH
H
R O
+ H N C
H
C OH
R O
R O
H2N C C N C C OH
H
H
+ H2O
H H
The group that joins the two amino acids is the peptide linkage. It contains the carbonyl
group and the imide group:
O
H
C
N
Carbonyl
Imide
The product of joining together two amino acids is a dipeptide. Two more amino acids
can be joined to it at its ends. We can continue to do this until we have formed a large
chain of amino acids, or a protein. It is important to see that no matter how large the
protein, on one end will always be a free carboxyl group and on the other a free amino
group.
Like amino acids, proteins are amphoteric and act as buffers. The buffering action of
proteins is enhanced by acidic and basic groups present in the R groups of the amino
acids. Proteins buffer the blood by donating protons if it becomes too basic and
accepting protons if it becomes too acidic.
CROSS-LINKING PROTEINS
During the embalming process, proteins are cross-linked to one another. This crosslinking results in the firmness of embalmed tissue. The most commonly used crosslinking agent is formaldehyde.
The basis of cross-linking is the chemical reactivity of aldehydes with different forms of
nitrogen. Three forms of nitrogen found in proteins are imide groups, amino groups, and
the peptide linkage:
C N
N
N
H
H
O H
Amino
group
Peptide
linkage
Imide
H
The reaction that occurs is basically the same for all three groups. Two protein chains are
linked together and a methylene group is inserted between the nitrogens. A molecule of
water is also formed.
N
N
H
H
N
+ CH2O
CH2
+ H2O
N
The formation of water in these reactions has the implication that embalming with
formaldehyde is dehydrating to the embalmed tissue.
The peptide linkage is the most important cross-linking site in a protein. Between every
two amino acids in the chain is a peptide linkage. Reaction with formaldehyde at these
linkages joins many protein chains together.
Another cross-linking agent is the dialdehyde glutaraldehyde. Unlike formaldehyde,
glutaraldehyde does not join every peptide linkage, as is possible with formaldehyde.
The size of the five-carbon glutaraldehyde sterically prevents a reaction at every peptide
site. Tissue embalmed with glutaraldehyde is not as hard as that embalmed with
formaldehyde. A possible explanation for this is the lesser number of cross-links that
result from cross-linking with glutaraldehyde.
PROTEIN STRUCTURE
To understand protein structure, we discuss it at different levels, referred to as primary,
secondary, tertiary, and quaternary. Only proteins with more than one chain have a
quaternary structure.
Primary Structure
This is the sequence of amino acids in a protein. For example, are the amino acids
arranged glycine, glycine, alanine or are they arranged alanine, glycine, glycine.
The primary structure also tells if there are disulfide bridges in the protein. These are
covalent bonds between sulfur atoms in two different cysteine amino acids.
The –SH groups on the cysteines can be oxidized to form a covalent bond called a
disulfide bridge.
The biological activity of a protein is related to its primary structure. A change of one
amino acid in the sequence can alter biological function. Sickle cell anemia is caused by
glutamic acid being replace by valine in one of the protein chains that make up
hemoglobin.
Secondary structure
Proteins are chains of alpha amino acids. The chains take certain shapes, because of
electrostatic interactions between atoms within one chain or between chains. Three
common shapes of proteins are the alpha helix, the beta pleated sheet, and the triplestranded helix of collagen. Fundamental to these shapes is hydrogen bonding. A
hydrogen atom attached to a nitrogen in one peptide linkage is attracted to an oxygen
atom in another peptide linkage. This attraction or hydrogen bond holds the protein in a
definite shape. Therefore, the secondary structure of a protein is its shape due to
hydrogen bonding between the hydrogen on a nitrogen of one peptide linkage and the
oxygen on the carbon of another peptide.
In the alpha helix, hydrogen bonding occurs between the carbonyl group of each amino
acid and the –NH group of the amino acid that is four amino acids ahead in the same
chain. In a beta pleated sheet hydrogen bonding occurs between the carbonyl groups and
the –NH groups in adjacent polypeptide chains. The triple-stranded helix of collagen is
due to the coiling of the polypeptide chains to minimize repulsions. This causes each
chain to form a helix to minimize repulsions on the side chains. The three chains then
wrap around each other and form hydrogen bonds between the different chains.
Tertiary structure
Interactions between the R groups on the amino acids are responsible for folding of the
protein molecules, often into very compact shapes. This folding is referred to as the
tertiary structure. Several types of bonds that influence the tertiary structure of a protein
are hydrogen bonds, electrostatic interactions often referred to as salt bridges, disulfide
bridges, and hydrophobic-hydrophilic interactions. Hydrophobic literally means “water
fearing” and hydrophilic means “water loving.” Those amino acids with nonpolar
hydrocarbon side chains are hydrophobic. Hydrophilic amino acids have polar regions in
their side chains. Proteins in an aqueous environment will assume a shape that minimizes
the contact between the hydrophobic side chains and water, and maximizes the contact of
the hydrophilic groups with water.
Quaternary structure
Those proteins with more than one polypeptide chain have another level of structure
called quaternary. The polypeptide chains in these proteins are called subunits. A
protein’s quaternary structure tells how the subunits are arranged. Hemoglobin has four
polypeptide chains. These four chains arrange themselves tetrahedrally in one
hemoglobin molecule. The quaternary structure of hemoglobin, therefore, is four
subunits in a tetrahedral shape.
HEMOGLOBIN
One molecule of hemoglobin contains a protein part, globin, and a nonprotein part, heme.
Every molecule of hemoglobin contains four polypeptide chains and four hemes. Two of
the subunits, designated as alpha chains, each contain 141 amino acids. The other two,
which are beta chains, each contain 146 amino acids. Nestled within the folds of each
subunit is a molecule of heme. Each heme portion contains an atom of iron. The iron is
responsible for carrying oxygen. Hemoglobin carries oxygen in blood. A similar
function is performed by myoglobin in muscle.
BREAKDOWN OF HEMOGLOBIN
Red blood cells have a lifespan of about 120 days. Their degradation occurs in the
spleen. The protein portion of hemoglobin is metabolized to amino acids. Heme is
degraded in a stepwise procedure to several compounds of interest to the embalmer. In
the first step, heme is converted to biliverdin, a green compound. A molecule of carbon
dioxide is also formed. The second step is the reduction of biliverdin to bilirubin, a
yellow compound. Both reactions require certain enzymes and coenzymes present in a
living organisms. The bilirubin is then carried by serum albumin to the liver, from which
it is secreted as bile.
Nondegraded hemoglobin is the cause of postmortem stain. Approximately 6 hours after
death, because of hemolysis of red blood cells, hemoglobin may seep from the capillaries
to the tissues. The pigment discolors the tissues, a condition known as postmortem stain.
The breakdown products of hemoglobin, bilirubin and biliverdin, cause another cosmetic
problem for the embalmer. This situation occurs in the embalming of jaundiced remains.
Jaundice is caused by an elevated level of bilirubin in the blood. The resulting yellow
color is known as jaundice. At one time, it was thought that reaction of formaldehyde
with the excess bilirubin converted it to biliverdin, resulting in a greenish discoloration of
the embalmed body. However, because the conversion of bilirubin to biliverdin is an
oxidation reaction and formaldehyde is a reducing agent, formaldehyde cannot be the
cause of these color changes. Some other chemical reaction must be responsible.
DENATURATION
A protein is denatured when its structure is changed in a way that modifies its properties.
The extreme result of denaturation of a protein is loss of biological activity. In an
aqueous solution, denaturation is generally observed by precipitation of the protein.
Denaturing agents interfere with the secondary and tertiary structures of a protein.
Coagulation is a type of denaturation. Coagulation is the process of converting soluble
protein to insoluble protein by heating or by contact with a chemical such as an alcohol or
an aldehyde.
Denaturation may have both bad and good effects. Loss of biological activity by
enzymes that have been denatured may seriously affect an organism. On the other hand,
blood clotting, which is the result of protein precipitation, is a normal biological function.
PROTEIN BREAKDOWN
Protein breakdown into smaller molecules begins in the stomach. The reactions are all
catalyzed by enzymes. Here in an acid environment, the enzyme pepsin catalyzes the
hydrolysis of peptide linkages. The acid pH of the stomach also aids in the digestion by
denaturation of the protein. The partially digested proteins, which are now smaller
polypeptides, move to the small intestine, where enzymes that have been secreted by the
pancreas complete digestion. From the small intestine amino acids, the final products of
protein digestion are absorbed into the bloodstream.
In the blood, the amino acids are a source of protein material called the amino acid pool.
They may be removed from the pool to build new proteins for the body’s use or may
undergo oxidations. The final oxidation products of protein breakdown are carbon
dioxide, water, urea, and energy.
PUTREFACTION
The anaerobic decomposition of proteins brought about by the action of enzymes is
putrefaction. It begins after cellular death. The rate at which it occurs depends on several
factors, including temperature, humidity, moisture content of the body, and cause of
death. It is favored by moisture and the presence of bacteria and increases with
temperature. IMBIBITION may increase the rate of putrefaction. It is the swelling and
softening of tissues and organs as a result of absorbing moisture from adjacent sources.
Putrefaction involves three major chemical reactions: hydrolysis, deamination, and
decarboxylation.
Hydrolysis
This chemical reaction initiates the putrefactive process. Hydrolysis is a chemical
property of water. In this process, biological compounds are broken apart and the
constituents of water are incorporated into their structure. Enzymes are necessary for the
process to occur. As in digestion, amino acids are the final hydrolysis products.
Deamination
This reaction is the removal of the amino group from an amino acid. Each amino acid
undergoes a specific reaction, but in general ammonia and an organic acid are the
products. The formation of ammonia as a product of putrefaction is significant to the
embalming process. Formaldehyde reacts with ammonia, producing urotropin. If
extensive putrefaction has occurred before embalming, there will be a higher-than-normal
formaldehyde demand due to this production of ammonia.
Decarboxylation
By this reaction, the carboxyl group of an amino acid is removed. The products are
carbon dioxide, water, and an amine. The amines may be further broken down into
ammonia and various hydrocarbons.
Actually, during putrefaction, both deamination and decarboxylation occur
simultaneously. Overall, the decomposition of proteins produces hydrocarbons, organic
acids, amines, ammonia, and carbon dioxide.