PROTEINS Amino Acids

- Proteins are macromolecules composed of

NOTE: You need to know this table
amino acids linked together through peptide
bonds.

Element components of proteins

- Major elements: C, H, O, N, S.
- Trace elements: P, Fe, Cu, Zn, I,

Amino Acids: The basic building blocks of proteins

- Only 20 types of amino acids are used for
protein synthesis in biological systems
- Amino Acid chain are all wound up
- The shapes of proteins are like a balled-up
piece of string

AMINO ACID STRUCTURE

Classification of Amino Acids

(1) non-polar, hydrophobic;

(2) polar, neutral;
(3) acidic;
(4) basic;
(5) aromatic.

Essential, Nonessential, and Conditional AA

Structure of amino acids - Essential – must be consumed in the diet
- Nonessential – can be synthesized in the body
- Conditionally essential – cannot be synthesized
due to illness or lack of necessary precursors
– Premature infants lack sufficient enzymes
needed to create arginine

TWENTY STANDARD AMINO ACIDS

Physical properties of amino acids

- Amino acids are mainly water soluble which is
explained by its polarity and the presence of
charged groups. They are soluble in polar
solvents but not soluble in non-polar solvents.
- They have a high melting point reflecting the
high energy needed to break the ionic forces
maintaining the crystal lattice.
Peptide Structure of Proteins
- A peptide is a compound of amino acids linked - Made up of chains of amino acids; classified by
together by peptide bonds. number of amino acids in a chain
- A peptide bond is a covalent bond formed ➢ Peptides: fewer than 50 amino acids
between the carboxyl group of one AA and the • Dipeptides: 2 amino acids
amino group of its next AA with the elimination • Tripeptides: 3 amino acids
of one H2O molecule. • Polypeptides: more than 10 amino acids
➢ Proteins: more than 50 amino acids
• Typically 100 to 10,000 amino acids linked
together
- Amino acids are composed of carbon, hydrogen,
oxygen, and nitrogen CHON

Basis in the Classification of Proteins

COMPOSITION
1. Simple proteins:
1. Albumins- lactalbumin, ovalbumin
2. Globulins- legumin
Examples of Oligopeptides 3. Albuminoids-elastin
4. Histones-globin in hemoglobin
2. Conjugated Protein
▪ Nucleoprotein- ex. ribosomes
▪ Glycoprotein- mucin
▪ Phosphoprotein- casein
▪ Chromoprotein- hemoglobin
▪ Lipoprotein- HDL
▪ Metalloprotein-ferritin

Shape
- Fibrous- molecules have elongated shape Ex.
Collagen, fibrin, elastin, keratin
Peptide Bonds Link Amino Acids
- Globular-molecules are folded to spherical in
- Form when the acid group (COOH) of one amino
shape Ex. Hemoglobin, albumin, insulin, globulin
acid joins with the amine group (NH 2 ) of a
second amino acid
FUNCTIONS
- Formed through condensation
− Structural- ex. Keratin − Regulation - insulin
- Broken through hydrolysis
− Movement - myosin − Catalytic - peps
− Storage - casein − Protection - gamma
Condensation and Hydrolytic Reactions
− Transport - lipoprotein globulin

Functions of Proteins
− They are the major structural molecules in living
things for growth and repair : muscles,
ligaments, tendons, bones, hair, skin, nails…IN
FACT ALL CELL MEMBRANES have protein in
them
✓ They make up antibodies in the immune system
✓ They make up enzymes for helping chemical
reactions
✓ They makeup non-steroid hormones
Muscles, ligaments, tendons, and bones Tertiary Structure
− Without these particular structural proteins, we − The tertiary structure is defined as the three-
would look more like this… dimensional arrangement of all atoms of a
protein.
Hair, Skin, and Nails
− KERATIN Quaternary Structure
− The quaternary structure is defined as the
Cell Membrane spatial arrangement of multiple subunits of a
− The cell membrane is made mostly of protein protein resulting from the interaction between
AND lipids. polypeptide chains.
− These subunits are associated through Hbonds,
Antibodies ionic interactions, and hydrophobic interactions.
− part of the immune system.
− When something enters the body that isn’t
supposed to be there, like certain bacteria,
antibodies find the invader and stick themselves
onto it.
− When a white blood cell finds the invader
covered with antibodies, it knows it doesn’t
belong there and kills it

Enzymes
− proteins that speed up chemical reactions.
− If you don’t have enzymes in your stomach to
speed up digestion, the food would rot in your
stomach because it would take so long

Hormones
− chemicals made in glands that are in one place
in the body and then put into the blood to be
used in another.

Levels of Structure of Proteins

Primary Structure Physical properties of protein

− The primary structure of proteins is defined as a − Form colloidal solution
linear sequence of amino acids joined together − Non-dialyzable
by peptide bonds.
− Hydrophilic (absorb water)
− Peptide bonds and disulfide bonds are
− Electrically charged (amphoteric)
responsible for maintaining the primary
structure.
Physical and Chemical Properties of Proteins

▪ Amphoteric - can act as either an acid or a

Secondary Structure
base
− The secondary structure of a protein is defined
as a local spatial structure of a certain peptide
segment, that is, the relative positions of
backbone atoms of this peptide segment.
− H-bonds are responsible for stabilizing the
secondary structure.
Physical and Chemical Properties of Proteins Alcohol
▪ isoelectric point (pI) - Alcohol-disrupts H-bonding
o The pH at which the protein has zero net-
charge is referred to as isoelectric point (pI)
o At solution pH that is above the pI, the
surface of the protein is predominantly
negatively charged, and therefore like-
charged molecules will exhibit repulsive
forces.
o The isoelectronic point or isoionic point is
the pH at which the amino acid does not
migrate in an electric field. Acids and bases
o This means it is the pH at which the amino - Disrupts salt bridges held together by ionic
acid is neutral, i.e. the zwitterion form is charges.
dominant

Colloidal property
- Hydration shell and electric repulsion make
proteins stable in solution

- Proteins are hydrolyzed by acid, alkalies, and

enzymes. They are broken down into simpler
forms, the end product being an amino acid. The
stages in the hydrolysis of proteins are:
Reducing agents
Proteins → proteoses → peptones → - Causes denaturation
→ polypeptides → peptides → amino acids

▪ Coagulation-The process of changing from a

liquid to a gel or solid state by a series of
chemical reaction
▪ Denaturation-Alteration of a protein shape
through some form of external stress in such a
way that it will no longer be able to carry out
its cellular functions

Heat Color Reactions for Qualitative analysis of protein

- disrupts the H-bonding and non polar ▪ Biuret test- specific test for protein test for
hydrophobic interaction the complete hydrolysis of protein. Violet
color . Rgt CuSO4 and NaOH
▪ Ninhydrin- general test for amino acid blue
violet, red violet . purple pro and HO-pro gives
yellow Asn gives brown

Xanthropoteic test- test for benzene ring Rgt. Conc.

HNO3, yellow to orange
- Millons test – test for phenolic group red color .
Tyr
- Hopkins cole test - test for indole group violet or
blue, trp
- Sakaguchi test - test for guanidyl group red to
orange, arg
- Lead acetate- test for sulfur black ppt,. Cys met
 ENZYMES
- Enzymes are proteins that catalyze chemical
reactions. They speed up chemical reactions by
lowering activation energies
- Enzymes are specific in the type of reactions
they catalyze.
➢ Absolute specificity acts only on one
substance.
➢ Relative specificity acts on structurally
related substances.
➢ Stereochemical specificity distinguishes ENZYME COFACTORS
between stereoisomers - Some enzymes require a second substance
present (cofactor) in order to be active, not a true
Enzyme activity can be regulated. prosthetic group (only weakly bound to the
- The cell controls rates of reactions. enzyme).
- The cell controls amount of any product formed - Cofactors can be a nonprotein molecule or ion.
by regulating the action of enzymes - If the cofactor is an organic molecule, it is called a
coenzyme.
CLASSIFYING AND NAMING ENZYMES - An apoenzyme is the catalytically inactive protein
- The earliest enzymes have names with –in to formed by the removal of the cofactor
indicate their protein composition.
- Examples: Apoenzyme + cofactor (coenzyme or inorganic ion)
➢ pepsin → active enzyme
➢ trypsin
➢ chymotrypsin - Coenzymes are often derived from vitamins.

- Many known enzymes created the need of a

systematic nomenclature system (Enzyme
Commission (EC) system), which:
o has six major classes based on type of
reaction catalyzed.
o names the specific substrate and functional
group acted upon as well as the type of
reaction catalyzed.
o ends the name in –ase.
- A substrate is the substance that undergoes a
chemical change catalyzed by an enzyme

Enzymes also have common names, which:

- are shorter than EC name. - All enzymes have an active site – the location on
- can be formed by one of the following methods: the enzyme where a substrate binds and catalysis
- adding –ase to the name of the substrate. occurs.
- adding –ase to a combination of the substrate - Enzymes complex with the substrate and the
name and type of reaction. chemical reaction proceeds.
- include examples, such as the enzyme for: enzyme
- the substrate urea, which has a common name of E + S ⇆ ES → E+ P
urease. Enzyme substrate enzyme-substrate product
- the substrate alcohol and a dehydrogenation Complex
reaction type, which has a common name of
alcohol dehydrogenase.
 ,

There are two main theories on active sites:

- Lock-and-key theory states that the substrate has
a shape that exactly fits the active site. This
explains enzyme specificity
- Induced-fit theory states that the conformation of ENZYME INHIBITION
the active site changes to accommodate an - Inhibitors decrease enzyme activity.
incoming substrate. - Irreversible inhibitors covalently bond with the
enzyme and render it inactive.
ENZYME ACTIVITY - Many poisons are irreversible inhibitors.
- Enzyme activity is the rate at which an enzyme Examples: CN- , Hg2+, and Pb2+
catalyzes a reaction. - Some antibiotics are irreversible inhibitors.
- Turnover number is the number of substrate o Examples: Sulfa drugs and penicillins inhibit
molecules acted on by one enzyme molecule per specific enzymes essential to the life
minute. processes of bacteria.
- Enzyme international unit is the quantity of o Penicillins interfere with transpeptidase, an
enzyme that catalyzes the conversion of 1 µmol of enzyme that is important in bacterial cell wall
substrate per minute. construction.
- An enzyme assay is an experiment that measures o Inability to form strong cell walls prevents
enzyme activity the bacteria from surviving

ENZYME INHIBITION (continued)

FACTORS AFFECTING ACTIVITY
- The more enzyme present, the higher the enzyme - The cyanide ion:
o is an irreversible enzyme inhibitor.
concentration and the faster substrate reacts
o is extremely toxic.
- Increasing substrate concentration increases the
o acts very rapidly.
reaction rate until enzymes become saturated
o interferes with the operation of an iron-
(Vmax)
containing enzyme (cytochrome oxidase) by
- Enzymes have an optimum temperature range
forming a very stable complex.
(usually 25- 40°C), above or below which they
o does not allow the enzyme to function
begin to denature
properly.
- Enzymes have optimum pH values (usually around
7), above and below which the rate decreases o stops cellular respiration.
o causes death in minutes
The cyanide poisoning antidote:
- must be administered quickly.
- can be sodium thiosulfate (same substance known
as “hypo” in developing photographic film),
which:
- converts the cyanide ion to a thiocyanate ion,
which:
- does not bind to the iron of cytochrome oxidase

ENZYME INHIBITION (continued)

- Heavy metal toxicity:
o is due to ability to render the protein part of
enzymes ineffective.
o occurs when metals combine with the –SH
groups found on many enzymes.
o causes nonspecific protein denaturation.
o Mercury and lead poisoning can cause
permanent neurological damage

- Heavy-metal poisoning treated by administering ENZYME REGULATION

chelating agents (substances that combine with - Zymogens or proenzymes are an inactive
the metal ions and hold them very tightly). precursor of an enzyme.
- An example of a chelating agent is - Some enzymes are stored as inactive zymogens.
ethylenediaminetetraacetic acid, EDTA , which: - They are released when needed and activated at
o chelates all heavy metals except mercury. the location where the reaction occurs
- The calcium salt of EDTA administered
intravenously.
- Calcium ions are displaced by heavy-metal ions
that bind to the chelate more tightly.
- The heavy metal-EDTA complex is soluble in body
fluids and is excreted in the urine.

Reversible inhibitors reversibly bind with enzymes.

- Competitive reversible inhibitors compete with Allosteric regulation of allosteric enzyme activity is
substrate for binding at the active site. altered by the binding of a modulator.
- Action can be reversed by increasing substrate - Modulators can increase allosteric enzyme
concentration (LeChâtelier’s principle). activity (activator) or decrease it (inhibitor).
- Sulfa drug on bacteria is an example of - Feedback inhibition is an example of a modulator
competitive enzyme inhibition. decreasing the activity of an allosteric enzyme.
- Folic acid normally synthesized within the
bacteria by process that requires p-aminobenzoic The synthesis of isoleucine is a five-step process.
acid. - Threonine deaminase (enzyme for first step) is
- Sulfanilamide resembles p-aminobenzoic acid and subject to inhibition from isoleucine (final
competes with it for the active site of the product).
bacterial enzyme. - Isoleucine and threonine have very different
- Sulfanilamide can prevent bacterial growth. structures; therefore, this is an example of a
noncompetitive inhibitor.
Noncompetitive reversible inhibitors bind to the - Isoleucine binds to an allosteric site, not an active
enzyme at a location other than the active site. site
- Substrate concentration doesn’t affect inhibitor
action
Enzyme induction is the synthesis of an enzyme in
response to a cellular need.
- This is an example of genetic control.
- The synthesis of -galactosidase is an example of
enzyme induction

MEDICAL APPLICATIONS
- Changes in blood serum concentrations of specific
enzymes can be used to detect cell damage or
uncontrolled growth (cancer).
- The measurement of enzyme concentrations in blood
serum has become a major diagnostic tool, particularly
in diagnosing diseases of the heart, liver, pancreas,
prostate, and bones NINHYDRIN TEST

Principle:
• This test is a general test and thus given by all
amino acids.
• Ninhydrin is a powerful oxidizing agent and its
presence, amino acid undergo oxidative deamination
liberating ammonia, CO2, a corresponding aldehyde
and reduced form of ninhydrin (hydrindantin).
• The NH3 formed from an amino group reacts with
another molecule of ninhydrin and is reduced product
(hydrindatin) to give a blue substance diketohydrin
(Ruhemanns complex).
COLOR TEST FOR PROTEINS

BIURET TEST

Principle of Biuret test:

▪ Biuret test is a general test for compounds
having a peptide bond.
▪ The principle of biuret test is conveniently
used to detect the presence of proteins in
biological fluids.
▪ Alkaline CuSO4 reacts with compounds
containing two or more peptide bonds to give
a violet-colored product which is due to
formation of coordination complex of cupric
ions with un shared electron pairs of peptide
nitrogen and O2 of water.
Biuret reagents:

▪ Copper sulfate (CuSO4)

▪ Sodium hydroxide (NaOH)
XANTHOPROTEIC TEST

Principle:
• Xanthoproteic test is used to detect amino
acids containing an aromatic nucleus
(tyrosine, tryptophan and phenylalanine) in a
protein solution which gives yellow color nitro
derivatives on heating with conc. HNO3.
• The aromatic benzene ring undergoes
nitration to give yellow colored product.
• Phenylalanine gives negative or weakly
positive reaction though this amino acid
contains aromatic nucleus because it is
difficult to nitrate under normal condition.
Reagents:
➢ Nitric acid SAKAGUCHI TEST
➢ Conc NH4OH
Principle of Sakaguchi test:
▪ This test is specific for arginine because this
reaction is given by guanidinium compound.
▪ The arginine reacts with α – naphthol and an
oxidizing agent such as bromine water or sodium
hypochlorite/sodium hypobromite to give a red
colored product.
▪ The other guanidinium containing compounds
other than amino acid also give this reaction.
▪ Reagents:
o α naphthol
o Sodium hypochlorite or bromine water

MILLON’S TEST

Principle of Millon’s test:

• Compounds containing hydroxybenzene radical
react with Millon’s reagent to form red
complexes. The only amino acid having
hydroxybenzene ring is tyrosine.
• Tyrosine when reacted with acidified mercuric
sulphate solution gives yellow precipitate of
mercury-amino acid complex.
• Sodioum nitrate solution and heating, the yellow
complex of mercury-amino acid complex converts
to mercury phenolate which is in red color.
Reagents:
• Millon’s reagent (Acidified mercuric sulphate)
• sodium nitrate
LEAD ACETATE TEST
Principle of lead sulfide test:
▪ Specific test for amino acid cysteine
▪ When cysteine is heated with strong alkali like
NaOH, some of the sulfur is converted to sodium
sulfide (Na2S) which can be detected by
precipitation as lead sulfide (PbS) from alkaline
solution.
PAULY'S TEST
▪ Reagents:
Principle:
• Lead acetate
▪ Test for detection of aromatic amino acid tyrosine
• NaOH
or histidine
▪ Diazotized sulfanilic acid couples with amines,
phenols and imidazole to form highly colored azo
compounds.
▪ Amino acids tyrosine or histidine coupled with
diazonium salt in alkaline condition to form red
colored azo dye.
▪ Reagents:
• sulfanilic acid
• sodium nitrite
• sodium carbonate
• ** Diazonium compound =Sulfanilic acid +
sodium nitrite + sodium carbonate

HOPKIN'S COLE TEST

Principle:
▪ Use to detect amino acid tryptophan present in
protein
▪ The indole group of tryptophan reacts with
glyoxylic acid in the presence of conc. H2SO4 to
give a purple-colored complex.
▪ Glyoxylic acid is prepared by reducing Oxalic acid
with magnesium powder or sodium amalgam.
Glacial acetic acid which has been exposed to the
sunlight also contains glyoxylic acid and can thus
be used for this test.
▪ Reagents:
• Formaldehyde Sterilization
• Mercuric sulfate - destroys the spores of various organisms present
• H2SO4 on surfaces, in liquids, in medication, or in
compounds such as biological culture media.
- Such “extreme” forms of decontamination are
needed during critical times like surgery, or in
environments like industrial, laboratory or
hospital
Cheese
- When producing (hard or semi-hard) cheese, the
cheese yield can be increased by subjecting part
of the cheese milk to a high temperature heat
treatment.
- Thermal denaturation of whey proteins changes
the protein structure so that part of the whey
remains in the curd during the cheese making
process

Detergent
- Detergents can be denaturing or non-denaturing
with respect to protein structure.
- Denaturing detergents can be anionic such as
sodium dodecyl sulfate (SDS) or cationic such as
ethyl trimethyl ammonium bromide.
- These detergents totally disrupt membranes and
denature proteins by breaking protein– protein
interactions

Cooking
- The heat coming from your stove denatures the
protein by disrupting some of its bonds that held
the molecule into shape.
- In the case of hard-boiled eggs, the proteins
clump together and solidify, causing the egg white
and yolk to harden
 VITAMINS biochemical function and deficiency
Definition - biochemical function
- organic substances , essential in the diet in small ＊Photographic substances in visual cell
amounts that are involved in fundamental ＊participating in the synthesis of glycoprotein
functions of the body and maintaining differentiation of epithelial cells
Classification - Deficiency
- lipid-soluble vitamin • night blindness, dry eyes dry skin
- water-soluble vitamin
Vitamin D
Chemical nature and properties
▪ types：
- VitD2（Ergocalciferol）
- VitD3（Cholecalciferol ）
▪ pro-VitD2：Ergosterol
▪ Pro-VitD3： 7-hydro-cholesterol
Ergosterol→VitD2
Lipid-soluble Vitamins cholesterol→7-hydro cholesterol→VitD3
Common features
- nonpolar (hydrophobic)
- poorly soluble in water，but soluble in fat and fat
solvents
Classification:
- VitA, VitD, VitE, VitK

Vitamin A
Chemical nature and properties
- natural form：A1（retinol）
A2（3-dehydro-retinol ）
biochemical function and deficiency
- active form ：retinol、retinal、retinoic acid
Biochemical function
- pro-vitamin A：β-carotene
 Targeting on intestinal mucous、kidney and renal
- storage : liver
tubular，
 Promoting absorbance of calcium and phosphorous
 Being beneficial to formation and calcification of
new bone
Deficiency
◼ children—— rickets
◼ adults——osteomalacia

Vitamin E （Tocopherol）
Chemical nature and properties
﹡types：Tocopherol ，Tocotrienols
﹡easy to be oxidized; protector of other substances
 Water-soluble Vitamins
Common features:
﹡water soluble，easy to be discharged through urine
Classification
- Vitamin B family
- Vitamin C
biochemical function and deficiency
▪ anti-oxidation Vitamin B1 (thiamine )
Chemical nature and properties
Vitamin E: antioxidant ﹡vitamin B1 : thiamine
ROO· + RH → ROOH + R·
（Peroxide free radical ） （polyunsaturated fatty acids ）（organic peroxide ）（organic free ﹡active form :Thiamine pyrophosphate (TPP):
radical）

R· + O2 → ROO· ROO·+Vit E-OH→ROOH+Vit E-O·

▪ Maintaining reproduction
▪ Promoting metabolism of Hemoglobin

Vitamin K
Chemical nature and properties
Natural form：K1、K2 (2-methyl-1-4
naphthoquinone)
Artificial synthesis ：K3、K4

biochemical function and deficiency

biochemical function
﹡ with effects in the nerve conduction，inhibiting
the cholinesterase activity deficiency
﹡beriberi，peripheral Neuritis

biochemical function and deficiency

biochemical function
- Maintaining the normal levels of coagulation
factor
- deficiency : hemorrhagic disease

Vitamin B2 (riboflavin)
Chemical nature and properties
﹡vitamin B2 : riboflavin
﹡ active form : mononucleotide (FMN)
flavin adenine dinucleotide (FAD):
 biochemical function and deficiency
biochemical function
﹡NAD+ and NADP+ : coenzyme of dehydrogenases
（Malate dehydrogenase, lactate dehydrogenase),
transfer of hydrogen 。
deficiency
﹡pellagra

biochemical function and deficiency Vitamin B6

biochemical function ： (pyridine derivatives)
- FMN and FAD are the prosthetic group of Chemical nature and properties
- oxidoreductases with function of transmitting
﹡vitamin B6 : pyridoxine
hydrogen
- deficiency：cheilosis ，glossitis, scrotitis etc pyridoxal
pyridoxamine
﹡active form : Pyridoxal-Phosphate
pyridoxamine-Phosphate

Vitamin B3 (nicotin acid, nicotinamide)

Chemical nature and properties
﹡vitamin B3: nicotinic acid nicotinamide
biochemical function and deficiency
﹡active form ﹡Pyridoxal-Phosphate
- Nicotinamide adenine dinucleotide (NAD+ ) - Coenzyme of amino acid aminotransferase,
- Nicotinamide adenine dinucleotide phosphate
decarboxyIase, and - amino-- levulinate
(NADP+ )
synthase （ALA synthase）
- Deficiency: seborrheic dermatitis

Vitamin B5 (pantothenic acid)

Chemical nature and properties
﹡pantothenic acid
﹡active form : CoA
4-phosphopantetheinyl : acyl carrier protein (ACP)
biochemical function and deficiency
﹡CoA and 4-phosphopantetheinyl are coenzyme of
acyl transferase ，transfer of acyl
 Vitamin B12 (cobalamin)
Chemical nature and properties
- vitamin B12: coholamine
- active form: coholamine
5 -deoxyadenosylcobalamin

biochemical function and deficiency

﹡biochemical function ：methyl transfer
﹡deficiency：Megaloblastic anemia , nerve disease ,
High blood level of homocysteine

Folic acid
Vitamin C (ascorbic acid)
Chemical nature and properties
Chemical nature and properties
﹡folic acid: Pteroylglutamic acid
﹡vitamin C: ascorbic acid
﹡active form: tetrahydrofolate (FH4 )

biochemical function and deficiency

﹡biochemical function：redox reaction,
biochemical function and deficiency
hydroxylation ,
biochemical function ：
synthesis of collagen protein
- FH4 : co-enzyme of transferase of one carbon unit
deficiency : Megaloblastic anemia ﹡deficiency：scurvy
- clinical application: Antitumor drug
biochemical function and deficiency
- cooperating with TPP to participate oxidative
decarboxylation of pyruvic acid 、α–keto acid;
coenzyme of lipoic acid acetyl transferase

- Vitamins are natural and essential nutrients,

required in small quantities and play a major role
in growth and development, repair and healing
wounds, maintaining healthy bones and tissues,
for the proper functioning of an immune system,
and other biological functions.
- These essential organic compounds have diverse
biochemical functions