INTRODUCTION

One of the biomolecules discussed in the introduction is protein. An extraordinary

number of different proteins, each with a different function, exist in the human body. A typical
human cell contains 9000 different kinds of proteins, and the human body contains 100,000
different proteins. Proteins are needed for the synthesis of enzymes, certain hormones, and
some blood components; for the maintenance and repair of existing tissues; for the synthesis of
new tissues; and sometimes for energy. (Stoker, 2011)

Lesson 1. Definition of Proteins, Structure and Classification of Proteins

Activity 1.
1. Given the following words, come up with your own definition of protein.
Organic, amino acids, carbon, body-building, macromolecule, hydrogen, oxygen, tissue
repair, nitrogen, large amount, building blocks

.
2. Illustrate the structure of amino acid and label each component.

Proteins are organic compounds of high molecular weight made up of alpha-amino

acids joined by means of peptide linkage. They are the most important of all biological
substances, being the fundamental constituent of the protoplasm of the cells. Proteins was
derived from the Greek word “proteios” which means “of first importance” or “to take first place.”

Proteins supply the body not only with heat and energy, but also provide materials for
building and repair. They are the “working molecules” which perform operational functions in
living systems (e.g. enzymes, antibodies, hormones, etc.) and composed of amino acids as
building blocks.

Amino Acids: The Building Blocks of Proteins

Amino acid is an organic compound that contains both an amino (-NH2) group and a
carboxyl (-COOH) group. AA in proteins are always the α-amino acids. The different
components of the amino acid structure have corresponding functions.
C (alpha carbon) is tetrahedral, chiral, asymmetric and covalently linked to both an
amino
group and a carboxyl side chain. Bonded to it is a H atom and a variable R group.
R side chain varies and give the amino acid its characteristics. The nature of this side
chain distinguishes a-amino acids from each other. Side chains vary in size, shape, charge,
acidity, functional groups present, hydrogen-bonding ability, and chemical reactivity.
 NH2 or amino group exists as –NH3 + in neutral solutions.
COOH or the carboxyl group exist as –COO- in neutral solution.

CLASSIFICATION OF AMINO ACIDS

1. Non-polar amino acids
• Hydrophobic amino acids. When incorporated into a protein, they are not attracted to
water molecules. They are generally found in the interior of proteins, where there is
limited contact with water.
• Include all those with alkyl side chain R groups
o Alanine
o Valine
o Leucine
o Isoleucine
• Cyclic structure
o proline
• sulfur containing amino acids
o cysteine and methionine
• Aromatic amino acids
o phenylalanine and tryptophan

2. Polar, uncharged amino acids

• Can form hydrogen bonds with water except glycine
• Usually more soluble in water than the non-polar amino acids
• Good hydrogen-bond forming moieties
o The amide groups of asparagine and
glutamine
o The hydroxyl groups of tyrosine, threonine and
serine
o Sulfhydryl group of cysteine
• Glycine – simplest amino acid
o Has only a single hydrogen for an R group and is not a good hydrogen bond
former
3. Acidic amino acids
• R groups contain a carboxyl group
o Aspartic acid
o Glutamic acid
• They have net negative charge at pH 7
• Play an important role in protein functions
• Proteins that bond metal ions for structural or functional purposes possess metal
binding sites containing one or more aspartate and glutamate side chains

4. Basic amino acids

• With net positive charge at neutral pH
o Histidine
o Arginine
o lysine
• Histidine side chains play important roles as proton donors and acceptors in many
enzyme reactions
• Histidine containing peptides are important biological buffers

5. *other amino acids

• Occur only rarely in proteins
• Found mainly in the collagen and gelatin proteins
o Hydroxylysine
o hydroxyproline
 • Found only in a light-driven proton-pumping protein call bacteriorhodopsin
o Pyroglutamic acid

TYPES OF AMINO ACIDS

1. Alpha amino acids – the amino acid found in proteins where the amino group is attached
to the 1st carbon atom adjacent to the carboxyl group
2. Beta amino acids – this type of amino acids is names as such because the amino group
is attached to the 2nd carbon atom adjacent to the carboxyl group
3. Gamma amino acids – the amino acid where the amino group is attached to the 3 rd
carbon atom adjacent to the carboxyl group

3 GROUPS OF AMINO ACIDS

1. Neutral A.A. – when their molecules have the same number of amino and carboxyl groups
• Alanine (Ala) – non polar
• Aspargine (Asp) – polar
• Cysteine (Cys) – polar
• Glutamine (Gln) – polar
• Glycine (Gly) – non-polar
• Isoleucine* (Ile) – non-polar
• Leucine* (Leu) – non-polar
• Methionine* (Met) – non-polar
• Phenylalanine* (Phe) – non-polar
• Proline (Pro) – non-polar
• Serine (Ser) – polar
• Threonine* (Thr) – polar
• Tryptophan* (Trp) – non-polar
• Tyrosine (Tyr) – polar
• Valine* (Val) – non-polar
2. Acidic A.A. – when their molecules have more carboxyl groups than amino groups
• Aspartic acid (Asp)
• Glutamic acid (Glu)
3. Basic A.A. – when their molecules have more amino groups than carboxyl groups
• Lysine* (Lys)
• Arginine (Arg)
• Histidine (His)
*Essential amino acids
ESSENTIAL VS NON-ESSENTIAL
Essential Amino Acids – are those amino acids that cannot be synthesized by the body and
are required in the diet. Since the body is not capable of synthesizing them, they must be
supplied in our diet if we are to enjoy
normal health
Non-essential amino acids – are those amino acids that can be synthesized by the body
and need not be included in the diet

Lesson 2. Chemical Properties of Amino Acids

Previously, the definition of protein and amino acids, and their classifications was
discussed. This lesson will talk about the different chemical properties of amino acids? Recall
your discussion of inorganic ch emistry. Can you remember examples of chemical changes
and properties? A good memory on that particular topic will help you understand the different
tests for amino acids.
Reactions are dependent on the numerous different reactive groups that are present in
the same molecule. Aliphatic monoamino monocarboxylic acids give all the reactions expected
for carboxyl and amino groups. In addition, some reactions are characteristic of additional
groups that may be present. For instance, cysteine give the reactions characteristic of the
sulfhydryl
(-SH) group and tyrosine give the reaction characteristic of phenolic group. These
reactions may also be exhibited by the protein containing these amino acids.

Ninhydrin test
• General test for amino acids
• Reagent : triketohydrindene hydrate
• Product: CO2, ammonia and an aldehyde containing one less carbon than the amino acid
• Positive result: blue or purple color
o proline & hydroxyproline
gives yellow color
o Color is produced by the ammonia group
• Alpha amino acid + Ninhydrin = diketohydrindylidene-diketohydrindamine + CO2 +
aldehyde

Reactions of specific amino acids

1. Millon Reaction
• reagent: Millon’s reagent; Hg(NO3)2 in HNO3 w/ trace HNO2
• Positive result: red color
• Specific for Tyrosine
2. Sakaguchi test
• reagent: α-naphthol and sodium hypochlorite
• Positive result: red color
• Specific for: guanidine and arginine
3. Nitroprusside test
• Reagent: sodium nitroprusside [Na2(NO)5.2H2O] in dilute ammoniacal solution
• result: red color
• Specific for cysteine and proteins with free sulfhydryl group
4. Aldehyde reaction
• Indole derivative gives strongly colored products with a number of aromatic
aldehydes. With paradimethylaminobenzaldehyde in H2SO4, a red violet color is
obtained with tryptophan (Ehrlich reaction). This test can be used for the quantitative
estimation of tryptophan in proteins
5. Folin reaction
• In alkaline solution, amino acids give a deep red color with sodium 1,2-naphthoquinone-
4-sulfonate
• Used for rapid quantitative estimation of amino acids
6. Pauly reaction for Histidine and Tyrosine
• Histidine and tyrosine couple with diazotized sulfanilic acid in alkaline solution giving a
red color

Lesson 3. Peptides

A peptide is an unbranched chain of amino acids, each joined to the next by a peptide
bond. Since amino acids contain both amino and carbonyl groups, they combine with each
other to form amides, called peptides. The linkage that joins them is known as a peptide
linkage.
 The product is an amide made up of two amino acids joined together, called a dipeptide

CLASSIFICATION OF PEPTIDES
• If three amino acid units are included in a molecule, it is a tripeptide, if 4 a tetrapeptide,
if 5 a pentapeptide and so on
• Oligopeptides – peptide chain of more than 12 residues and less than about 20
• Polypeptides are peptides containing about 40 – 50 amino acid units in a chain
• Amino acid residues – units making up amino acids minus the elements of water
• Proteins – larger chains
of amino acids peptides
By convention, peptide formation is written with the free amino group on the left and the
COOH on the right. In linear peptides, one end of the chain has a free amino and the other
end a free carboxyl group . The amino group end is called the N terminal residue and the other
end the C terminal residue
The sequence of amino acids in a chain is numbered starting with the N-terminal residue,
which is written on the left, the C- terminal residue is written on the right Any segment of the
sequence that is not specifically known is placed in parenthesis.

1 2 3 4 5 6 7
Ala – pro – tyr – met – gly – lys – gly

N-terminal residue C-terminal residue peptides

NOMENCLATURE OF PEPTIDES
• Named as acyl derivatives of the C-terminal amino acid, the C-terminal unit keeping its
complete name
• The –ine ending of all but the C-terminal amino acid is changed to –yl
• Except tryptophan (tryptophyl), cysteine (cysteinyl), glutamine (glutaminyl), asparagine
(asparaginyl)
• They are listed in the order in which they appear, starting with the N-terminal amino acid
Example: Ala-Tyr-Gly is called
alanyltyrosylglycine
Arg-Glu-His-ala is arginylglutamylhistydylalanine
Lesson 4. Polypeptides

The amino acid sequence and chain length give a polypeptide its biological
effectiveness. The sequence of amino acid residues is essential for proper polypeptide
function. This sequence aligns the side-chain characteristics in the proper positions for a
specific polypeptide function.

PRIMARY STRUCTURES AND FUNCTIONS OF SOME BIOLOGICAL POLYPEPTIDES

Name Primary Structure General Biological Functions

Substance F Arg-Pro-Lys-pro-Gln-Gln-Phe- A pain producing agent

Phe-gly-leu-met-NH2
Bradykinin Arg-pro-pro-gly-phe-ser-pro-phe Affects tissue inflammation and blood pressure

Angiotensin II Asp-arg-val-tyr-val-hist-pro-phe Maintains water balance and blood pressure

Leu-enkephalin Tyr-gly-gly-phe-leu Relieves pain, produces sense of well being
Met-enkephalin Tyr-gly-gly-phe-met
Vasopressin Cys-tyr-phe-gln-asn-cys-pro-arg- Increases blood pressure, decreases kidney
gly-NH2 water excretion
 Oxytocin Cys-tyr-ile-gln-asn-cys-pro-arg- Initiates childbirth labor, causes mammary gland
gly-NH2 milk release, affects kidney excretion of water and
sodium
Thyrotopin- Glu-His-Pro Stimulates release of hormones from the pituitary
releasing gland
hormone
Orexin 33 amino acids long Causes wakefulness
Neuropeptide Phe-leu-phe-gln-pro-gln-arg-phe- Modulates pain sensation
FF NH2
Neurotensin Glu-leu-tyr-glu-asn-lys-pro-arg- Involved in brain memory functions
arg-pro-tyr-ile-leu
Neuropeptide Y 36 amino acids long Stimulates eating
Endomorphin Tyr-pro-trp-phe-NH2 Acts as a morphine-like analgesic

Structure of proteins
• Proteins contain the elements C, H, O, N and usually S (and P) plus traces of Fe, Cu, I,
Mn and Zn
• Amino acids – building blocks of proteins

3 Categories of Protein Structure

Primary Structure
• The sequence of amino acids which are joined together to form the polypeptide
• The main mode of linkage is the peptide bond
• Primary structure is the order of attachment of the AA to each other through peptide
bond. It is always the same regardless of where protein is found in an organism. i.e.
insulin
• It is the most important because it is the protein’s AA sequence that determines its
overall shape, function
and properties.
• Crucial that a change of only one AA can drastically alter its biological function.
o i.e. Sickle-cell anemia val is substituted by glu
Secondary Structure
• The peptide chains are folded regularly, the folding resulting from the linkage of the
carbonyl group of one peptide chain with the amine group of another chain by means of
hydrogen bonds
• This hydrogen bonding produces a regular coiled arrangement called Helix
• α-helix found in α-keratins that make up the hair, skin and nails of humans
• Secondary structure result primarily from hydrogen-bonding interactions b/n different
amide linkages along the protein backbone.
Tertiary structure
• The tertiary structure of the protein arises from the interactions that take place among
the side groups of polypeptide chains, interactions which cause bending and folding of
the protein molecule
• Maintained by covalent disulfide bonds
• Each type of protein molecule has in its native state, a characteristic 3-dimensional
shape referred to as conformation (refer to the combined 2o and 3o structure of the
peptide chain
• Tertiary forms the overall 3-dimensional shape of a protein that results from the
interaction between AA
side chains

2 major groups of proteins

• Globular proteins – named to describe the spherical shape into which their polypeptide
chains are bent and folded as a result of tertiary structure
o Perform the operational functions in the body by behaving as enzymes,
hormones, antibodies and transporters (Hgb and serum albumin)
 • Fibrous proteins – made up of polypeptide chains arranged in parallel fashion along
a single axis, to yield long fibers or sheets
• Basic structural unit of connective tissues
• Ex. Collagen of tendons and bone matrix, α-keratin of hair, horn, nails, Elastin
(elastic connective tissue)
• Some proteins (e.g. myosin and fibrinogen) are intermediate between fibrous and
the globular, Rod- like structures (like the fibrous) and like the globular, they are
soluble in aqueous salt solutions

Quaternary/quaternary protein structures

• Protein organization is produced by fitting together separate coiled and folded
structures to form an aggregate functional structure
• Ex. Isoenzymes (enzymes with similar but not identical catalytic powers)-
LDH, hexokinase, phosphatases
• Oligomeric protein – proteins with more than one chain
o Protomers – component chain
o Ex. Hemoglobin – made up of 4 polypeptide chain (2α and 2β), it contains 140
amino acids in
each chain
Lesson 5. Proteins and Their Biochemical Functions

Proteins and their Biochemical Functions

1.Structure– for animals, it is structural proteins which are the chief constituents of skin, bones, hair and
fingernails
• Collagen – found in bones – provides the support for calcium to bind and form the solid structure of
the bones
• Keratin – hair and nails

2. Catalysis– virtually all the reactions that take place in the living organisms are catalyzed by proteins called
enzymes. The enzyme trypsin – catalyzes hydrolysis of proteins; sucrose – catalyzes the hydrolysis of
sucrose; and dehydrogenase – converts ethanol to acetaldehyde. Without enzymes, the reactions would take
place so slowly as to be useless.

3. Movement and Contraction– every time we crook our finger, climb stairs, or blink an eye, we use our
muscles. Muscles expansion and contraction are involved in every movement we make. Muscles are made up
of protein molecules called myosin and actin

4. Elasticity– the protein elastin, which possesses unique elasticity and strength, enables the skin, ligament
and blood vessels to stretch and rebound

5. Transport– a large number of proteins fall into this

category
• Albumin – transports bilirubin, calcium, fatty acids, some drugs
• Ferritin and transferrin – transport iron
• Transcortin – cortisol (cortisol-binding protein)
• Hemoglobin – carries oxygen
• Lipoproteins – cholesterol and triglycerides

6. Hormones– Many hormones are proteins, among them insulin, oxytocin, human growth hormone and
endomorphines.

7. Protection– when a protein from an outside source or other substance (called antigen) enters the body, the
body makes its own proteins (called antibodies) to counteract the foreign protein. This is the major
mechanism the body used to fight diseases. Blood clotting is another protective device carried out by proteins
called fibrinogen. Without clotting, we would bleed to death from any small wound

8. Storage– some proteins are used to store materials, in the way that starch and glycogen store energy.
Examples are casein in milk and ovalbumin in eggs, which store nutrients for newborn m ammals and
birds. Ferritin, a protein in the liver, stores iron

9. Regulation – some proteins control the expression of genes are thereby regulate the kind of proteins
manufactured in a particular cell, and control when such manufacture takes place

Classification of Proteins on a Nutritional Basis

Complete proteins
• Proteins that supply all the essential amino acids needed by the human body for normal health
• They are derived from animal sources
• They are also referred to as high biological value protein.
Incomplete proteins
• Are the proteins that are deficient in one or more essential amino acids
• Many proteins especially those from vegetable sources, are incomplete thus the other name which
low biological value protein.
• For example, protein from corn (maize) is deficient in lysine
TWO MAJOR GROUPS OF PROTEIN:
SOME COMMON FIBROUS AND GLOBULAR
PROTEINS
NAME OCCURRENCE AND FUNCTION
Fibrous Proteins (Insoluble)
Keratins Found in wool, feathers, hooves, silk, and fingernails
Collagens Found in tendons, bone, and other connective
Elastins tissues Found in blood vessels and ligaments
Myosins Found in muscle tissue
Fibrin Found in blood clots
Globular Proteins (Soluble)
Insulin Regulatory hormone for controlling glucose metabolism
Myoglobin Involved in oxygen storage in muscles
Hemoglobin Involved in oxygen transport in blood
Transferrin Involved in iron transport in blood
Immunoglobulins Involved in immune system responses
Source: Biochemistry, Stoker, 2011.

Lesson 6. Classification of Proteins

CLASSIFICATION OF PROTEINS
I. SIMPLE PROTEINS- true proteins found abundantly in both plants and animals. On hydrolysis with enzymes,
they yield α-amino acids and their derivatives
a. Albumins- soluble in water & dil. Neutral salt solution
- coagulated by heat & precipitated by full saturation with (NH4)2SO4 but not with NaCl except in the presence
of acid
- Members: serum albumin – blood
Lactalbumin- milk
Ovalbumin- egg white

b. Globulins- soluble in neutral dil. Salt solution but NOT water

-coagulated by heat and can be precipitated from their solutions by half saturation with (NH 4)2SO4 and
complete saturation with NaCl
-Members: Ovoglobulin- egg white
Edestin- hempseed
Legumin- peas
Myosinogen- muscles
Serum globulin

c. Glutelins- soluble in dil. Acids and alkalies but insoluble in neutral solvents
- Glutenin- wheat
- Oryzenin- rice

d. Prolamines- insoluble in ordinary solv ent but soluble in 70% alcohol

- not coagulable by heat
- present in plants (e.g. Gliadin- wheat, Zein- corn, Hordein- barley)

e. Histones- soluble in water, dilute acids & alkalies but not in dil. Ammonia
- not readily coagulated by heat
- strongly basic and occur in tissues in the form of salt - combinations with acid substances like heme of
the hemoglobin molecule
Globin- hemoglobin
Histone- thymus
Scombrone- mackerel

f. Protamines- contain small number of amino acids

- soluble in water & dilute acids and alkalies and are not coagulated by heat
- strongly basic; form soluble salts with strong mineral acids
Salmin- salmon sperm
* Prolamine vs. prolamine – protamine has low MW that are rich in arginine and are found associated
especially with DNA in place of histone in the sperm cells of various animals (as fish)
g.Scleroproteins (Albuminoids)- insoluble in water and neutral solvents
Keratin- epidermal tissues
Elastin- ligaments
Collagen- hides, bones & cartilages

II. CONJUGATED PROTEINS- made up of protein molecules combined with non-protein groups
a. Nucleoproteins- combinations of histones and protamines with nucleic acid
- soluble in dilute solutions of NaCl and can be extracted from the tissues by the use of this solvent;
precipitated by acidification
Chromatin
Products obtained from glandular tissues
Germ of grains

b. Glycoproteins- proteins with a carbohydrate component

utilized for lubricating purposes in view of their slimy nature
Mucin- saliva
Tendomucoid- tendons
Osseomucoid- bones
- Glycoproteins are not digested by the enzymes of the gastro-intestinal tract, thus help in protecting
the membranes of the tract against digestion.

c. Phosphoproteins- prosthetic group (H3PO4) joined to the protein molecule

Casein- milk
Vitellin- egg yolk

d. Chromoproteins- protein compounds with hematin or similar pigments

Hemoglobin, cytochromes, rhodopsin

e. Lipoproteins- fatty substances combined with their molecules like lecithin, cephalin,
etc. blood serum, brain tissues, cell nuclei,
egg yolk and milk

TYPES OF CONJUGATED PROTEINS

Class Prosthetic Group Specific Example Function of Example
Hemoproteins Heme unit Hemoglobin Carrier of O2 in blood
myoglobin Oxygen binder in muscles
Lipoproteins Lipid Low-density lipoprotein Lipid carrier
(LDL)
High-density lipoprotein Lipid carrier
(HDL)
Glycoproteins Carbohydrate Gamma globulin Antibody
Mucin Lubricant in mucous secretion
Antiviral protection
Interferon
Phosphoproteins Phosphate group Glycogen phosphorylase Enzyme in glycogen phosphorylation
Nucleoproteins Nucleic acid Ribosomes Site for protein synthesis in cells
Self-replicating, infectious complex
viruses
Metalloproteins Metal ion Iron-ferritin Storage complex for iron
Zinc-alcohol DH Enzyme in alcohol oxidation
Source: Biochemistry, Stoker, 2011.

III. DERIVED PROTEINS- substances formed from simple and conjugated proteins
A. Primary Protein derivatives- synonymous with denatured proteins; have undergone slight
intramolecular rearrangement through the hydrolytic action of certain physical and chemical agents
• Proteans- insoluble substances resulting from the preliminary action of water, dilute acids or enzymes
Ex. Myosan (from Myosin)
 Edestan (from Edestin)

• Metaproteans- products of further hydrolysis; soluble in weak acids and alkalies

Ex. Acid metaproteans (acid albuminate)
Alkali metaproteans (alkali albuminate)

• Coagulated proteins- insoluble products resulting from either the action of heat, alcohol, UVR or
even simple mechanical shaking
Ex. Cooked egg albumin, cooked meat

B. Secondary Protein derivatives- products of more extensive

hydrolysis mixtures of fragments of original protein varying in composition
and size
- exhibit common properties (e.g. solubility in water and non-coagulability by heat)

1. Primary proteoses- soluble in water, precipitated by conc. HNO3 & half saturation with (NH4)2SO4 or
ZnSO4;
not coagulated by heat

2. Secondary proteoses- precipitated by complete saturation with (NH4)2SO4

3. Peptones- soluble in water; precipitated by saturation with certain alkaloidal reagents (e.g.
Phosphotungstic acid, Tannic acid)

4. Peptides- combinations of 2 or more amino acids, the carboxyl group of one being united with the
amino group of the other

Lesson 7. Physical and Chemical Properties of Protein

PHYSICAL AND CHEMICAL PROPERTIES OF PROTEINS

The number, kind and arrangement of the various amino acids within the protein molecule are
responsible for the variety of complex proteins. However, protein substances share certain COMMON
properties which serve to identify them as a distinct class of biological substances.
- when pure, proteins are generally tasteless
- colorless, amorphous compounds
- insoluble in fat solvents & present varied degrees of solubility in water, salt solution, dilute acids
and alkalies
- due to high MW, form non-diffusible colloid solutions of the emulsoid type
- proteins are amphoteric; they are labile, readily modified in solution when subjected to alterations in
pH, UVR, heat and organic solvents
- very reactive & highly specific due to the presence of side chains & active NH 2 groups

SOLUBILITY
Various kinds of proteins differ markedly in their solubility; utilized for group separation and most often
for purification of individual proteins.

Factors Affecting Solubility:

1. Neutral salt- low concentration of neutral salts increases the solubility of proteins (“salting in” effect) while
an increase in concentration decreases solubility (“salting out” effect).
The solubility of any substance depends upon the relative affinity of solute molecules for each other and
for the solvent. Any factor which decreases the interaction of solute molecules will tend to increase solubility.

2. Effect of pH- solubility is influenced by pH because of their amphoteric nature; solubility is minimum
at isoelectric point and increases with increasing acidity or alkalinity

3. Effect of Organic solvents

 When an organic solvent (which is miscible in water—e.g. methanol, ethanol, acetone) is added to
aqueous solution of proteins, it lowers the dielectric constant, so that the electrical forces between the
charged
particles in the solution are increased, thus diminishing the solubility of proteins.
 Selective separation of the constituent proteins of a mixture can be achieved by proper adjustment of
pH, temperature, alcohol concentration, protein concentration and salt concentration kept at low level.
E.g. Fractionation of plasma with this procedure produces fractions which are of clinical
significance: Fibrin film & foam – used for surgery
Albumin – treatment of shock, nephrosis, cirrhosis
Globulin – passive immunization
Agglutinins – for blood typing

A C T IO N O F H E A T
- when burned, proteins decompose and liberate a characteristic odor of burned hair or
feather
Denaturation- the process by which solution of proteins undergo slight intramolecular rearrangements when
they are heated between 38 to 60 degrees Celsius, giving rise to changes in chemical, physical and biologic
properties.
• Believed to be due to the unfolding of the characteristic folded structure of polypeptide chain.
• Reversible process; the unfolded molecule will return to its native form when conditions become
favorable
(Renaturation, Refolding or Annealing)
• Renders the protein insoluble at isoelectric point, causing it to precipitate (Flocculation)

Coagulation- If flocculated protein is heated further, the clumped chains become matted together in a
mass which is insoluble, not only at isoelectric point but also over the entire pH range
• a process involving the linkage of adjacent protein molecules by means of side chain Hydrogen bonds
• coagulated protein is hard to dissolve & when dissolved, it differs from the original protein from which
it is derived; process is irreversible.

Other Factors which bring about Denaturation :

1. Low temperature
2. high pressure
3. UVR – operates very similarly to the action of heat (i.e. sunburning)
4. surface action
5. mechanical shaking or violent whipping – causes molecules in globular shapes to extend to
longer lengths, which then entangle (i.e. beating egg with white into meringue)
6. chemical agents (e.g. acids, alkalies, organic solvents, alcohol, etc.).
a. strong acids and bases disrupt hydrogen bonds and salt bridges; prolonged action leads to
actual hydrolysis of peptide bonds
b. organic solvents interfere with R-group interactions because these solvents also can form
hydrogen bonds; quickly denature proteins in bacteria, killing them

PRECIPIT ATION
By Acids- due to presence of the NH2 group in their molecules, amino acids have basic properties and
form insoluble salts with acids.
Organic acids- Trichloroacetic, Phosphomolybdic, Phosphotungstic, Picric, Tannic acids are used
as precipitants
• Tannic acid & Picric acid are used for treating burns because they produce astringent effect
on the tissues, diminish secretion of mucous membranes and prevent absorption of toxins.
They are also administered in some forms of gastro-intestinal irritation to relieve diarrhea.
• Inorganic acids also precipitate protein. Nitric acid is used for detecting the presence of
proteins in the urine (Heller’s test).

By salts of Heavy metals (alkali metals)- Hg, Ag, Pb

proteins behave like acids by virtue of the carboxyl radical; form insoluble salts with alkaline metals
• In metallic poisoning, antidotes like egg white and milk are given. They form insoluble
precipitates which can be removed subsequently by means of a stomach tube.
By Alcohol- Maximum precipitation occurs at isoelectric point (when proteins are electrically neutral).
This accounts for the antiseptic property of alcohol.
* 95% alcohol has less germicidal property compared to 70% alcohol. It readily coagulates proteins
and coagulum forms a protective coating on the surface of bacteria preventing further penetration of the
alcohol.

Lesson 1. Protein Metabolism

Activity 1. Given the following figure, answer the question that follows:

Stoker, H. Stephen. Biochemistry, 1st Philippine Reprint 2011.

Based on the information in the previous figure, determine the location within the body where each of
the following aspects of protein digestion occurs:

1. The enzyme aminopeptidase is active

2. Amino acids are produced
3. Peptide bonds are hydrolyzed under the action of trypsin
4. Large polypeptides are produced
5. Protein is denatured by HCl

PROTEIN METABOLISM
Protein forms the primary constituent not only of the cell but also of such regulatory agents as
hormones and enzymes. It confers upon the tissue a sort of biological specificity; that is, the protein of a
particular tissue is distinct and different from those of other tissues. This is so because complex proteins are
made up of varying
proportions of essential and non-essential amino acids depending upon the tissue, organ or specie.

ESSENTIAL OR INDISPENSABLE AMINO ACIDS:

- Methionine - Leucine
- Threonine - Phenylalanine
- Lysine - Tryptophan
- Valine - Histidine
- Isoleucine - Arginine

The term essential and non-essential relates only to dietary requirements, not in relation to importance
in metabolism.
The study of protein metabolism is centered on the metabolism of amino acids, mostly concerned with
the fate of the amino nitrogen and those of the non-nitrogenous residues.
The amino nitrogen enters in the formation of urea while the non-nitrogenous residues participate
either in the carbohydrate or lipid metabolism.

ABSORPTION OF AMINO ACIDS

After a complete hydrolysis in the gastro-intestinal tract, the proteins are split into their component
amino acids. These amino acids are absorbed mainly in the small intestines through the portal circulation, and
only slightly through the lymphatics. The absorption of amino acids is very rapid so that the maximum
concentration
in the blood is attained 30 to 50 minutes after eating.

There is rapid removal of the absorbed amino acids from the blood. The amino acids are quickly
disposed and appear in all tissues and organs of the body. This is evident from the fact that the amino acid
nitrogen level is kept more or less constant between 4-8 mg/100 ml of blood plasma.

FACTORS THAT AFFECT DISTRIBUTION OF AA IN METABOLIC REACTIONS

1. Convalescing or growing individuals – for constructions of new proteins
2. The availability of new carbohydrate and lipid - influence the amount of total caloric requirement which
must be supplied by amino acids
3. The pattern of amino acid mixture supplied to the tissues
4. The influence of hormones in modifying the direction and the rate of certain metabolic reactions
 Three sources of AA in the AA pool:
1. Dietary protein
2. Protein turnover – repetitive
degradation and resynthesis of
protein (degradation is from disease,
injury and wear and tear)
3. Biosynthesis of AA in the liver

Metabolic pathways of absorbed Amino acids

1. Synthesized into new tissue proteins including enzymes and hormones
2. Synthesized into plasma proteins
• Liver is the site for biosynthesis of plasma proteins, albumin, globulin and fibrinogen
Synthesis is dependent upon the presence of indispensable amino acids and is increased when there
is generous supply of dispensable AA.
3. Synthesized into liver proteins
4. Synthesized into non-protein nitrogenous components of various cells
• Purine, pyrimidine, porphyrins, glutathione, creatine, thyroxin, nicotinic acid, etc.
5. Decarboxylated with formation of physiologically active amines
6. Deaminated, with the subsequent formation of urea and oxidation of α-ketoacid or its conversion
into carbohydrate or fat
7. Take part in the glucose-alanine cycle
• Prevents the fluctuations in the blood glucose level between meals
• Functions
o To carry amino groups from skeletal muscles to the liver to be converted into urea for
excretion
o To provide the working muscles with blood glucose made by the liver form the carbon
structure
of alanine

Lesson 2. Nitrogen Metabolism

DYNAMIC ASPECTS OF NITROGEN METABOLISM

NITROGEN BALANCE- quantitative difference between the nitrogen intake (from food) and the nitrogen
output
(represented by nitrogen lost in the urine and feces)
POSITIVE NITROGEN BALANCE- intake exceeds the output; this is found whenever new tissues are
being synthesized as in growing young, convalescence, pregnancy, etc.

NEGATIVE NITROGEN BALANCE- output exceeds the intake; this is found in:
1. inadequate intake of proteins as in fasting, malnutrition, diarrhea;
2. increased catabolism as in fevers, infections, wasting diseases; and
3. increased loss of body proteins as in lactation, albuminuria

UTILIZATION OF INORGANIC NITROGEN

The only form of inorganic nitrogen, which can be utilized by living cells is ammonia.

Fixation of ammonia
1. Glutamic Acid synthesis
a-ketoglutaric acid + NH3 + NADH + H+ Glutamic acid + NAD+ + H2O

Enzyme: Glutamic acid dehydrogenase

• The α-amino group of glutamic acid can be transferred to other keto-acids by transamination process
• Provides a mechanism for synthesis of other amino acids

TRANSAMINATION
Is a biochemical reaction that involves the interchange of the amino group of an a-AA with the keto
group of an a-keto acid.

2. Synthesis of Glutamine
Glutamic acid + NH3 + ATP Mg++ Glutamine + ADP + Pi + H2O
Enzyme: glutamine synthetase
• Glutamine serves as a means of presenting to an enzyme an unprotonated nitrogen atom at
reduction level of NH3
• Glutamine serves as a store of ammonia
3. Carbamoyl Phosphate synthesis
CO2 + NH3 + ATP + H2O Carbamoyl PO4 + ADP + Pi + 3 H+
Enzyme: Carbamoyl phosphate synthetase
• Carbamoyl PO4 becomes available to the cells for carbamylation of amino groups as in the
synthesis of citrulline from ornithine

FATE OF AMMONIA:
The ammonia liberated during the deamination process is disposed off in several ways:
1. It enters the general ammonia pool of the body and may be drawn upon either for anabolic or
catabolic purposes. It may be used in the reductive amination of keto acids derived from CHO to form new
amino acids.
2. Since ammonia is toxic in large concentrations, it is being detoxified by synthesis to glutamine.
3. Ammonia may be excreted directly into the urine.
4. Ammonia may enter the ornithine cycle to form urea.

UREA CYCLE:
Mammals utilize urea as the major vehicle for excretion of surplus nitrogen (ureotelic). On the basis
of their observations, Krebs and Henseleit proposed a cyclic mechanism for the synthesis of urea involving
the amino acids ornithine, citrulline and arginine in the presence of the enzyme arginase, which catalyzes
the irreversible hydrolysis of arginine to ornithine and urea.

1. Nitrogen enters the urea cycle in the form of ammonium ion.

2. A reaction between ammonium ion and CO 2 produces Carbamoyl phosphate in a reaction that requires
two molecules of ATP for each molecule of carbamoyl phosphate.
3. Carbamoyl phosphate reacts with Ornithine to form Citrulline. (ornithine transcarbamoylase)
• The reactions of the cycle at this point take place in the mitochondrion.
• Citrulline is then transported to the cytosol.
4. A second nitrogen enters the urea cycle when aspartate reacts with citrulline to form
argininosuccinate
(arginosuccinate synthase) in another reaction that requires ATP (AMP and Ppi are produced in this
reaction).
• The amino group of the aspartate is the source of the second nitrogen.
5. Argininosuccinate is split to produce arginine and fumarate. (argininosuccinate lyase)
6. Finally, arginine is hydrolyzed to give Urea (arginase) and to regenerate ornithine, which is transported
back to the mitochondrion.
7. The synthesis of fumarate is a link between the urea cycle and the citric acid cycle.
• Fumarate is an intermediate of the citric cycle, and it can be converted to oxaloacetate.
• A transamination reaction can convert oxaloacetate to aspartate, providing another link between the
two cycles

Degradation of Amino Acids for Energy Production

Five amino acids are degraded to Acetyl CoA by way of pyruvate
• Alanine
• Threonine
• Glycine
• Serine
 • Cysteine

Five amino acids are degraded to acetyl CoA without forming pyruvate
• Lysine
• Tyrosine
• Phenylalanine
• Tryptophan
• Leucine

Five amino acids forming α-ketoglutarate

• Glutamate
• Glutamine
• Arginine
• Proline
• Histidine

Amino acids forming succinyl-CoA

• Methionine
• Valine
• Isoleucine
• Aspartate and Asparagine are deaminated to oxaloacetate
• Aspartate + α-ketoglutarate oxaloacetate + glutamate
+
• Asparagine + H2O Aspartate + NH 4