Proteins

By: Roselita O. Natividad

Nat. Science – Ateneo de Zamboanga University

Be able to:
1. Enumerate the functions and characteristics of proteins.
2. Classify the different amino acids according to the side-chain functional group.
3. Describe what happens to the amino acid molecule in the zwitterion form.
4. Demonstrate peptide bond formation between amino acids.
5. Identify the amino acid residues present in some oligopeptides.
6. Describe the different classes of proteins and give examples for each.
7. Describe each level of protein structure.
8. Explain the mechanism of protein denaturation by different agents.

• chief constituents of all cells of the body

• more complex than either the carbohydrates or fats
• derived from the Greek word “proteios” which means of first importance
• all proteins contain the elements carbon, hydrogen, oxygen, and nitrogen
• most proteins also contain sulfur, some contain phosphorus, and a few, such as hemoglobin, contain
some other elements
• have a very high molecular weights
• a polymer of amino acid
• also an energy source (4 kcal/gram)
• necessary for the formation of the various enzymes and hormones found in the body

Questions:
1. What are the elements found in all protein molecules?
2. Proteins are to be polymers. Why is that?
3. What is the repeating unit of proteins?
1) Source

– through nitrogen cycle

Air nitrogen

nitrogen-fixing bacteria denitrifying bacteria

Soluble cpds in soil Soil nitrates

synthesis synthesis
decay

Animal waste product Plant protein

metabpolism digestion and

synthesis
Animal protein

2. Functions
• building of new cells
• maintenance of existing cells
• replacement of old cells

3. Specific functions
• structure – skin, bones, hair, nails (collagen and keratin)
• catalysis – enzymes
• transport – hemoglobin
• movement – myosin and actin in muscles
• hormones – insulin (maintenance of blood sugar)
• protection – antibodies
• storage
• regulation

4. Major types
1) fibrous protein – for structural purposes
2) globular proteins
5. Composition
• are made up of amino acids
• the general formula is as follows

COOH

H ⎯ C ⎯ NH2

R

• the amino group has a lone pair of electrons which impart basic characteristics
• the COOH possesses an acidic hydrogen as a result of the pi electron delocalization
• the carbon has a weakly acidic hydrogen as a result of the electron attracting inductive effect of the
nitrogen of the NH2 group
• the carbon is called alpha carbon as well as asymmetric carbon
• depending on the amino acid, the R group can be varied:
– acidic side chains
– basic side chains
– non-polar side chains
– polar but neutral side chains

• also, the R groups of the amino acid can give rise to specific interactions especially if these are imagined
as extending out of the of the protein chain, that is, the groups will determine both the structure and the
function of
each protein molecule
– structure – chief constituents of skin, bones, hair, and fingernails. Two important structural
proteins are collagen and keratin
– catalysis – called enzymes which catalyze reactions
– movement – muscle expansion and contraction are involved in every movement.
Muscles are made up of protein molecules called myosin and actin
– transport – hormones
– protection – storage
– regulation

• the body can synthesize some but not all of the amino acids that it needs
• those that cannot synthesize but are needed by the body are called essential amino acids
• the essential amino acids are:
– arginine (can be synthesized but too slow to be of practical value)
– histidine (only required during childhood)
– isoleucine
– leucine
– lysine ─ threonine
– methionine ─ tryptophan
– phenylalanine ─ valine

Question:
1. What are the non-essential amino acids?
6. Structure
• just as we make thousands of words from the 26 letters of the alphabet, nature makes molecules that
serve thousands of different functions from just 20 amino acids
• consist of many amino acids joined together what is called a peptide linkage
• 50 units of amino acids or less is called polypeptides, over 50 units are called proteins
• on hydrolysis, proteins will yield proteoses, peptones, polypeptides, tripeptides, dipeptides, and finally
amino acids

7. The three dimensional structure of proteins

• the structure of protein is finely tuned to achieve a specific function

• primary structure
– refers to the number and sequence of amino acids
– held together by peptide bond
– the sequence of the amino acids determines the protein’s three-dimensional structure and
suggests its functional role and relationship to other proteins

• secondary structure
• way amino acids orient along its axis
• consists of several repeating patterns
• refers to the arrangement of the amino acids such as coiled and pleated shape
• held together by peptide bonds, H-bonds disulfide bonds
• can be in the form of an -helix and  -pleated sheet
 the -helix
• the chain of the -helix are twisted in such a manner that its shape
resembles a right handed coiled spring
• the shape is maintained by numerous intramolecular hydrogen bonds that
exist between the backbone of the N–H of the amino acid and the −C=O of
the amino acid four residues away while the R groups extend outward from
the helix
• the slight elasticity of hair and wool is due to the helical structure of keratin
• but because of the several structural constraints, certain amino acids do not
foster −helix formation such as amino acid sequences with large numbers
of charged amino acids and bulky R groups are incompatible with −helix
structures

the −pleated sheet

• the chain of the −pleated sheet is maintained by intermolecular hydrogen
bonds. The hydrogen bonding is between backbone −C=O and H−N groups
• consists of parallel and antiparallel
• in parallel −pleated sheet, the polypeptide chains are arranged in the same
direction while antiparallel chains run in opposite directions
• antiparallel are more stable than the parallel because fully collinear hydrogen
bonds form
• the softness of silk are related to the ease with which the neighboring sheets
slides past around each other (only having to overcome H-bond to move)

Question: What intermolecular bonding is responsible for the

alpha-helix and beta-pleated sheet?

tertiary structure
• the shape is critical to its function and this is where the
3o structure of protein comes in
• refers to the specific folding and bending of the coils
into specific layers or fibers
• gives their specific biologic activity
• many polypeptides fold in such a fashion that amino
acid residues that are distant from each other in the
primary structure come into close proximity
 • the structures are stabilized by:
• covalent bond – are created by chemical reactions that alter a polypeptide’s structure
during or after its synthesis. This is referred to as posttranslational modifications. Most
prominent is the disulfide bonds found in many extracellular proteins which partly protect
protein structure from adverse changes in pH or salt concentration
• disulfide bond - found in many extracellular proteins which partly protect protein
structure from adverse changes in pH or salt concentration. Intracellular proteins do not
contain disulfide bridges because of high cytoplasmic concentrations of reducing agents
• hydrogen bonding between polar groups on side chains
• salt bridge between two amino acids with ionized side chains, that is, between an acidic
amino acid and a basic amino acid held together by simple ion-ion attraction – significant
only in regions of the protein where water is excluded because of the energy required to
remove water molecules from ionic groups near the surface
• hydrophobic interactions – as the polypeptide folds, the hydrophobic R groups are
brought into close proximity because they are excluded from water. Then the highly
ordered water molecules are released from the interior, increasing the disorder of the
water molecules.
– The favorable disorder change is a major driving force in protein folding

Quaternary structure
• Composed of several polypeptides chains
• Each chain is called sub-unit
• The sub-units may be identical or quite different
• Determines how the different subunits of the protein fit into an
organized whole
• Held together by hydrogen bonds, salt bridges, and
hydrophobic interactions
• Example is the hemoglobin

Reasons for common occurrence of multisub-unit proteins

1. Synthesis of separate subunits may be more efficient than substantially increasing the length of a single
polypeptide chain
2. In supermolecular complexes such as collagen fibers, replacement of smaller, worn-out or damaged
components can be managed effectively
3. The complex interactions of multiple subunits help regulate a protein’s biological function

Structure of proteins at different levels

 8. Classification
• simple proteins – on hydrolysis yield amino acids
• conjugated proteins – on hydrolysis would yield amino acids and some other types of compounds usually
a non-protein compound
• derived proteins – do not occur naturally but are partial hydrolysis products of protein molecules

Question: What do you call the bond that holds the amino acids in a polypeptide chain

9. Properties
• the amino acids in the protein molecule are linear
• the continuing pattern of peptide bonds is the backbone of the protein molecule, the R groups are called
the side chains
• the six atoms of the peptide backbone are rigid and lie in the same plane, and two adjacent peptide bonds
can rotate relative to one another about the C – N and C – C bonds
• colloidal in nature
• easily precipitated by:
– alcohol – concentrated inorganic acids
– salts (salting out) – radiation
– salts of heavy metals – amphoteric in nature
– Heat – possesses isoelectric point
– alkaloidal reagents – zwitterions

Zwitterions
• RCOOH does not exist as is but in the form of RCOO− and H+
• RNH2 also does not exist as is but as RNH3+
• And amino acid has both the COOH and the NH2 on the same molecule, and in solution, the COOH
donates the H+ (proton) to the NH2
H

R − C − COO−

NH3+
 • Compounds that have a positive charge on one side and a negative charge on the other are called
zwitterions
• Amino acids are not only zwitterions in water solution but in solid form as well
• Because of this characteristic, amino acids are considered to be internal salts and therefore ionic
compounds
• Ionic compounds have high melting points and fairly soluble in water and so are amino acids

Isoelectric point (pI)

• The pH where molecules have equal (+) and (−) charges. At pH lower than the pI, protein molecules have
a net of (+) charge; at pH higher than the pI, the protein molecules have a net of (−) charge. This explains
the buffer action of protein molecules
• Also, the water solubility of large molecules such as proteins often depends on the repulsive forces
between like charges on their surface
• When protein molecules are at the pH where net charges can either be (+) or (−), the protein molecules
repel each other but at the pI, this repulsion forces are smallest
• At pI, protein molecules tend to clump together to form aggregates, reducing their solubility, therefore,
proteins are least soluble in water at this point and can be precipitated from their solutions

Denaturation
• Protein conformation are stabilized by the 2o and 3o structures and through aggregation of subunits in 4o
structure
• Any physical or chemical agent that destroys these structures changes the conformation of the protein
and the process is called denaturation
• Denaturation changes the 2o , 3o and 4o structures. It does not affect the 1o structure
• If denaturation occurs to a small extent, then it can be reversed
 1. Heat cleaves the H-bonding so boiling of protein solution destroys the -helix. In other proteins,
especially globular proteins, heat causes the unfolding of the polypeptide chains and because of
subsequent intermolecular protein–protein interaction, precipitation or coagulation takes place. This is
what happens to boiled egg.
2. Urea breaks the H–bonding and causes the unfolding of globular proteins
3. Detergents change protein conformation by opening up the hydrophobic regions
4. Acids, bases, affect the salt bridges as well as the H–bonding because they change the pH resulting in the
protonation of some protein side groups
5. Salt concentration – the solubility of proteins varies considerably. Fibrous
proteins, for example, are insoluble in water but the addition of small amount of salt, a process called
salting in, often improves solubility significantly. The binding of salt ions to the protein’s ionizable groups
decreases interaction between oppositely charged groups on the protein molecules. Water molecules
then can form solvation spheres around these groups.

When large amounts of salt are added to a protein in solution, a precipitate forms. The large number of salt ions
can effectively compete with the protein for water molecules, that is, the solvation spheres surrounding the
protein’s ionized groups are removed. As a result proteins will precipitate out. This process is called salting out is
usually reversible

6. Reducing agents can break the disulfide bonds reducing them to SH groups. The protein in keratin has a high
percentage of disulfide bonds and is responsible for the shape of the hair. In either the permanent wave or
straightening, the hair is first treated with reducing agent that cleaves some of the disulfide bonds. This allows the
molecules to loose their rigid orientations and become flexible. Then the hair is set in the desired shape and then
an oxidizing agent is applied. The oxidizing agent reverses the reaction, forming the new disulfide bonds

7. Heavy metals attack the disulfide bonds by forming salt bridges resulting to insoluble precipitate

8. Mechanical stress – stirring and grinding actions disrupt the delicate balance of forces that maintain protein
structure. The foam formed when egg white is beaten vigorously contains denatured protein
10. Tests
• Xanthoproteic test – means yellow, works only on proteins that consists of amino acids containing a
benzene ring, such as tyrosine or phenylalanine
• Biuret test – formation of a violet color of a solution, works for substances that contain two or more
peptide linkages; considered to be the general tests for proteins
• Millon’s test – formation of a white precipitate that on heating turns brick-red; specific for amino acid
tyrosine
• Hopkin’s-Cole test – purple color at the point of contact between two liquids; specific for the amino acid
tryptophan
• Ninhydrin test – production of blue-purple color, indicates the presence of free amino acids or peptide
groups

That is . . . . .
➢ All proteins contain the elements C, H, O, and N.
➢ A polymer of amino acid
➢ Each amino acid contains a central carbon called α-carbon, to which an amino group, carboxylate group, a
hydrogen atom and an R group are attached
➢ May consist of one or more polypeptide chains
➢ In addition to serving as structural materials, proteins are involved in metabolic regulation, transport,
defense, and catalysis
➢ According to their ability to interact with water, amino acids may be separated into 4 classes: nonpolar
and neutral, polar and neutral, acidic, and basic
➢ Proteins are also classified according to their shape and composition
➢ Fibrous proteins (such as collagen) are long, rod-shaped molecules that are insoluble in water and
physically tough
➢ Globular proteins (such as hemoglobin) are compact, spherical molecules that are usually water soluble
➢ There are 4 levels of protein structures: primary structure (determines the number and sequence of
amino acids); secondary structure (is the local folding pattern, the initial folding and bending) consist of
two types, the alpha-helix and the beta-pleated sheet; tertiary structure is the overall three-dimensional
shape (gives the protein its specific biologic activity)
➢ Proteins that consists of two or more polypeptides have quaternary structure
➢ Many physical and chemical conditions disrupt protein structure, specifically the secondary, tertiary and
quaternary but not the primary structure
➢ The biological activity of complex multisubunit proteins is often regulated by allosteric interactions
➢ Any change in the protein’s activity is due to changes in the interactions among protein’s subunits

Activity:
Case study paper: “Aspartame – Boon or Bane?”
Questions:
1. Distinguish between proteins, peptides and polypeptides
2. Define amphoterism.
3. Amino acids are said to be amphoteric. Why is that?
4. Biuret test is considered to be a general test for proteins. Why is that? Will amino acid give positive test
with Biuret?
5. List 3 factors that do not foster α-helix formation.
6. Denaturation is the loss of protein function from structural change or chemical reaction. At what level of
protein structure or through what chemical reaction does each of the following denaturation agents act?
a. Heat
b. Strong acid
c. Mechanical stress
7. What happens to the solubility of proteins at its isoelectric point?
8. Explain the action of egg white as an antidote in mercury poisoning.