The document discusses the structure and function of amino acids and proteins, including their classification, absorption, and metabolic fate. It details the different types of proteins, their structural organization (primary, secondary, tertiary, and quaternary), and the bonds that stabilize these structures. Additionally, it covers biochemical tests like the Biuret test for detecting proteins and references various sources for further reading.

1. AMINO ACIDS & PROTEINS
Pradipta Banerjee, Ph.D.
Dept. of Biochemistry & Plant Physiology
CUTM, Paralakhemundi, Odisha

2. Amino Acids
Ester

3. Peptide Bond
1. Dipeptide bond links 2 amino acids
2. Strong bond
3. Rigid, planar structure, with partial
double bond character
4. Trans configuration
5. -NH and –CO are polar and can form
H-bonds

4. Nutritional Classification of amino acids
(NB: Essential amino acids cannot be synthesised by the organism and must form part of their diet)

5. **Proline: imino acid
**Tryptophan: indole
**Histidine: imidazole
**Tyrosine: phenol

8. Physical Properties
1. Most aa are soluble in water
2. Most of them melt above 200 degree
Centigrade
3. Sweet (Gly, Ala, Val), tasteless (Leu),
bitter (Arg, Ile) ..[Monosodium
glutamate in food]
4. All aa except glycine possesses optical
isomer due to presence of asymmetric
Carbon
5. Aa are ampholytes – contain both –
COOH and –NH2, ie, can donate a
proton or can accept a proton. They
can exist as dipolar or Zwitterion
Isoelectric pH (pI): pH at which molecule
exist in Zwitterionic form.

9. Chemical Properties

10. Chemical Properties

11. Chemical Properties

12. Amino Acid Absorption
I. Amino acids are absorbed in the small intestine
II. Amino acids are transported to the liver from the intestines via
the portal vein
III. In the liver, amino acids are
 Used to synthesize new proteins
 Converted to energy, glucose, or fat
 Released to the bloodstream and transported to cells
throughout the body
IV. Occasionally proteins are absorbed intact

13. Metabolic Fate of Amino Acids

14. PROTEINS

15. Classification on the basis of chemical nature and solubility
1. Simple proteins – consists of only amino acids
a) Globular proteins – oval shaped, soluble in water or other solvents and digestible
b) Fibrous proteins - These are fiber like in shape, insoluble in water and resistant to digestion. Albuminoids or
scleroproteins constitute the most predominant group of fibrous proteins.
2. Conjugated proteins
3. Derived proteins – formed at various stages of hydrolytic cleavage of simple and compound proteins (peptides,
peptones, etc.)
a) Primary derived proteins
b) Secondary derived proteins

16. Simple - Globular Simple - Fibrous

17. Collagen
1. Provides strength
2. It is found in connective tissue such as tendons, cartilage, organic matrix of bone, and cornea of eye.
3. Collagen helix is a unique secondary structure quite distinct from the α-helix. It is left-handed and has three amino acid residues per turn.
4. Collagen is also a coiled coil, but one with distinct tertiary and quaternary structures: three separate polypeptides, called chains (not to be
confused with α-helices), are supertwisted about each other.
5. Superhelical twisting is right-handed in collagen, opposite in sense to left-handed helix of the chains.
6. There are many types of vertebrate collagen.
7. Typically they contain about 35% Gly, 11% Ala, and 21% Pro and 4-Hyp (4-hydroxyproline, an uncommon amino acid).
8. Food product gelatin is derived from collagen; it has little nutritional value as a protein because collagen is extremely low in many amino
acids that are essential in the human diet.
9. The unusual amino acid content of collagen is related to structural constraints unique to the collagen helix.
10. The amino acid sequence in collagen is generally a repeating tripeptide unit, Gly–X–Y, where X is often Pro, and Y is often 4-Hyp.
11. Only Gly residues can be accommodated at the very tight junctions between the individual chains
12. Pro and 4-Hyp residues permit the sharp twisting of the collagen helix.
13. Amino acid sequence and supertwisted quaternary structure of collagen allow a very close packing of its three polypeptides.

18. Hemoglobin
1. Some gases like carbon dioxide, oxygen and nitrogen are non-polar
2. Water soluble transport protein (haemoglobin, myoglobin) facilitate transport of oxygen
3. Found in RBC
4. Deliver oxygen from lungs to tissue and transport carbon dioxide from tissue to lungs for excretion
5. Hemoglobin has 4 polypeptide subunits: 2 identical α chain and 2 identical β chain, all four held together by noncovalent interactions
(hydrophobic, ionic, H-bond)
6. Alpha chain carries 141 amino acids and beta chain carries 146 amino acids
7. Each α subunit is paired in an identical way with a β subunit within the structure of this multisubunit protein, so that haemoglobin can be
considered either a tetramer of four polypeptide subunits or a dimer of αβ protomers.
 Multisubunit proteins have two or more polypeptides
associated non-covalently.
 Individual polypeptide chains in a multisubunit protein may be
identical or different.
 If at least two are identical, protein is said to be oligomeric, and
identical units (consisting of one or more polypeptide chains)
are referred to as protomers.

19. Primary Derived Protein – denatured/coagulated/first hydrolysed product of protein
Secondary Derived Protein – These are progressive hydrolytic products of protein hydrolysis.
These include proteoses, peptones, polypeptides and peptides

20. Nutritional Classification of Proteins Functional Classification of Proteins
1. CATALYTIC: enzymes eg RUBISCO
2. COMMUNICATION: hormones (eg insulin) and
neurotransmitters
3. PROTECTIVE: eg immunoglobulin, fibrinogen, blood
clotting factors
4. STRUCTURAL: eg collagen, spiders silk
5. PIGMENTS: eg rhodopsin
6. STORAGE: eg ovalbumen (in eggs), casein (in milk)
7. TRANSPORT: eg hemoglobin
8. CONTRACTILE: eg actin, myosin
9. TOXINS: eg snake venom

21. Bonds responsible for
protein structure
1. Covalent Bond
a) Peptide bond
b) Disulphide bond
2. Noncovalent bond
a) H-bonding
b) Hydrophobic bond
c) Electrostatic bonds
d) Van der Waals forces
Secondary, tertiary
structure determination
of protein
1. X-ray crystallograpy
2. NMR spectra
Isolation and
purification of protein
1. Isolation
 Precipitation of protein by ammonium
sulphate (salting out).
 Fractionation of protein by
ultracentrifugation
2. Purification
a) Electrophoresis
b) Isoelectric focussing
c) Immuno-electrophoresis
d) Chromatography
 Ion-exchange
 Gel-filtration
 HPLC

22. Biologically important peptides (less than 10 aa)
 Glutathione
 Thyrotropin releasing hormone (TRH)
 Oxytocin
 Vassopressin (ADH)
 Angiotensin
 Methionine encephalin
 Bradykinin and kallidin
 Peptide antibiotics

23. Four levels of organization in the structure of a protein
 The amino acid sequence is known as primary structure of protein.
 Stretches of polypeptide chain that form α helices and β sheets constitute protein’s secondary structure.
 Full three-dimensional organization of a polypeptide chain is sometimes referred to as the protein’s tertiary structure,
 and if a particular protein molecule is formed as a complex of more than one polypeptide chain, the complete structure is
designated as the quaternary structure.

24.  Every protein has a three-dimensional structure that
reflects its function.
 Protein structure is stabilized by multiple weak
interactions. Hydrophobic interactions are major
contributors to stabilizing the globular form of most
soluble proteins; hydrogen bonds and ionic interactions
are optimized in the specific structures that are
thermodynamically most stable.
 Secondary structure is the regular arrangement of amino
acid residues in a segment of a polypeptide chain, in
which each residue is spatially related to its neighbors in
the same way.
 The most common secondary structures are α-helix, β-
conformation, and β-turns.

25. Secondary Structure of Protein: α-Helix
 If a polypeptide chain has a long block of Glu residues, this
segment of the chain will not form an helix at pH 7.0. The
negatively charged carboxyl groups of adjacent Glu residues
repel each other so strongly that they prevent formation of helix.
 For the same reason, if there are many adjacent Lys and/or Arg
residues, which have positively charged R groups at pH 7.0, they
will also repel each other and prevent formation of the helix.
 Bulk and shape of Asn, Ser, Thr, and Cys residues can also
destabilize an helix if they are close together in chain.
 A Pro residue introduces a destabilizing kink in an helix. Proline
is only rarely found within an helix.
 Glycine occurs infrequently in helices for a different reason:
it has more conformational flexibility than other amino acid
residues. Polymers of glycine tend to take up coiled structures quite
different from an helix.
Amino Acid Sequence Affects Helix Stability

26. Secondary Structure of Protein: β-Sheet
 β-conformation organizes polyptd chains to sheets.
 This is a more extended conformation of polypeptide chains, and its structure
has been confirmed by x-ray analysis.
 In conformation, the backbone of the polypeptide chain is extended into a
zigzag rather than helical structure.
 The zigzag polypeptide chains can be arranged side by side to form a structure
resembling a series of pleats.
 In this arrangement, hydrogen bonds are formed between adjacent segments
of polypeptide chain.
 Adjacent polypeptide chains in a sheet can be either parallel or antiparallel
(having the same or opposite amino-to-carboxyl orientations, respectively).
 The structures are somewhat similar, although the repeat period is shorter for
the parallel conformation (6.5 Å, versus 7 Å for antiparallel) and the hydrogen
bonding patterns are different.

28.  Tertiary structure is the complete three dimensional structure of a polypeptide chain. There are two general classes
of proteins based on tertiary structure: fibrous and globular.
 Fibrous proteins serve mainly structural roles, have simple repeating elements of secondary structure.
 Globular proteins have more complicated tertiary structures, often containing several types of secondary structure in
the same polypeptide chain. The first globular protein structure to be determined, using x-ray diffraction methods,
was that of myoglobin.
 Complex structures of globular proteins can be analyzed by examining stable substructures called supersecondary
structures, motifs, or folds. The thousands of known protein structures are generally assembled from a repertoire of
only a few hundred motifs.
 Regions of a polypeptide chain that can fold stably and independently are called domains.
 Quaternary structure results from interactions between the subunits of multisubunit (multimeric) proteins or large
protein assemblies.
 Some multimeric proteins have a repeated unit consisting of a single subunit or a group of subunits referred to as a
protomer. Protomers are usually related by rotational or helical symmetry.
Protein tertiary and quarternary structure

29.  For proteins that consist of a single polypeptide chain, monomeric proteins, tertiary structure is the highest level of
organization.
 Multimeric proteins contain two or more polypeptide chains, or subunits, held together by noncovalent bonds. Quaternary
structure describes the number (stoichiometry) and relative positions of the subunits in a multimeric protein. Hemagglutinin
is a trimer of three identical subunits; other multimeric proteins can be composed of any number of identical or different
subunits.
 Many proteins contain one or more motifs built from particular combinations of secondary structures. A motif is defined by
a specific combination of secondary structures that has a particular topology and is organized into a characteristic three-
dimensional structure.
 The tertiary structure of large proteins is often subdivided into distinct globular or fibrous regions called domains.
Structurally, a domain is a compactly folded region of polypeptide. For large proteins, domains can be recognized in
structures determined by x-ray crystallography or in images captured by electron microscopy. These discrete regions are
well distinguished or physically separated from other parts of the protein, but connected by the polypeptide chain.
Hemagglutinin, for example, contains a globular domain and a fibrous domain
…Protein tertiary and quarternary structure

30. Concept Note
What is the main difference between protein
domains and protein subunits?
 Each subunit is a separate polypeptide chain,
while domains usually constitute a part of a
polypeptide chain
 Protein domains do not have secondary structure,
whereas protein subunits always have secondary
structures
 The tertiary structure of domains is stabilized by
hydrogen bonds, while the structure of subunits is
stabilized by disulfide bonds
 Domains are parts of membrane proteins, while
subunits are parts of water-soluble proteins

31. Biuret Test
 Biuret is a compound formed by heating urea at 1800
 It is the result of the condensation of 2 molecules of urea. The peptide bonds in Biuret give a positive result for the
test hence the reagent is named so.
 Biuret test is a general test for compounds (proteins and peptides) having two or more peptide (CO-NH) bonds.
 Histidine is the only amino acid that gives Biuret test positive

32. Principle
 When biuret is treated with dilute copper sulfate in alkaline medium, a purple colored compound is formed. It is
believed that the color is due to the formation of a copper co-ordinated complex or chelate complex.
 Cupric ions or Cu (II) ions form a violet-colored chelate complex with unshared electron pairs of peptide nitrogen
and oxygen of water.
 The chelate complex absorbs light at 540 nm and hence appears violet. The color change from blue to violet
indicates the presence of proteins.
 The greater the number of peptide bonds in a protein, the greater the color intensity. Hence, the color change is from
blue to pink if the concentration of peptide bonds is low like in short-chain peptides.
 The principle of biuret test is conveniently used to detect the presence of proteins in biological fluids.
Biuret reagent preparation
 It is prepared by adding 3 g of CuSO4•5H2O and 9 g of sodium potassium citrate to 500 mL of 0.2 N NaOH solution,
followed by the addition of 5 g of KI.
 The resulting solution was then brought to a total volume of 1 L with 0.2 N NaOH.
…Biuret Test

33. Protein Denaturation
NB: Primary structure of protein is
not affected by denaturation process

34. References for Protein
1. Voet and Voet
2. Lehninger
3. U. Satyanarayana
4. https://www.ncbi.nlm.nih.gov/books/NBK26830/
5. https://www.ncbi.nlm.nih.gov/books/NBK21581/