The document discusses proteins, detailing their structure, classification, and functions, highlighting that they are essential nitrogenous organic compounds made up of amino acids. Key functions include biocatalysis, transport, muscle contraction, and supporting structures, with proteins classified based on solubility into fibrous and globular types, and further into simple, conjugated, and derived proteins. It also outlines protein structure levels from primary to quaternary and describes various protein assay methods for estimating protein concentration in research applications.

1. PROTEIN: STRUCTURE, FUNCTION CLASSIFICATION
AND ASSAY METHODS.
[Document subtitle]
JANUARY 1, 2018
SARDAR HUSSAIN
Gsc/cta

2. Lecture notes proteins/SH/BT/GSc
1Protein: structure function classification and assay methods
Proteins are nitrogenous organic compounds of high molecular weight which play a vital or prime role in
living organisms. They are made up of 20 standard a-amino acids.
Functions of proteins :
Proteins carry out most diverse and possibly the largest volumes of cellular functions. Some of the key
functions are summarized as below :
• Biocatalysis- Almost all the biological reactions are catalyzed by the enzymes. These are substrate
specific and carry out reactions at very high rates under mild physiological conditions. Several thousand
enzymes have been identified to date.
• Membrane are constitute of lipoprotein and some proteins are integral part of membrane. Receptors
found on the membrane are also protein in nature.
• Transport and storage proteins - small molecules are often carried by proteins in the physiological
setting e.g. haemoglobin is responsible for the transport of oxygen to tissues.
• Muscle are made up of proteins and their contraction is done by actin and myosin protein.
• Mechanical support - skin and bone are strengthened by the protein collagen.
• Antibodies of immune system are protein structures.
Classification of Proteins:
Proteins are classified based upon:
(1) Their solubility and (2) Their structural complexity.

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2Protein: structure function classification and assay methods
A. Classification Based upon Solubility:
On the basis of their solubility in water, proteins are classified into:
1. Fibrous proteins:
These are insoluble in water. They include the structural proteins. They have supportive function (e.g.,
collagen) and/or protective function (e.g., hair keratin and fibrin).
2. Globular proteins:
They are soluble in water. They include the functional proteins, e.g., enzymes, hemoglobin, etc.
B. Classification Based upon Structural Complexity:
On the basis of their structural complexity they are further divided into:
(1) Simple
(2) Conjugated and
(3) Derived proteins.
1. Simple proteins:
Proteins which are made up of amino acids only are known as simple proteins.
They are further sub-divided into:
(a) Albumins:
They are water soluble, heat coagulable and are precipitated on full saturation with ammonium sulphate,
e.g., serum albumin, lactalbumin and ovalbumin.
(b) Globulins:
They are insoluble in water, but soluble in dilute salt solutions. They are heat coagulable and precipitate on
half-saturation with ammonium sulphate, e.g., serum globulin and ovo-globulin.
(c) Glutelins:
They are insoluble in water and neutral solvents. Soluble in dilute acids and alkalies. They are coagulated by
heat, e.g., glutelin of wheat.
(d) Prolamines:
Water insoluble but soluble in 70% alcohol, e.g., gliadin of wheat, proteins of corn, barley, etc.
(e) Histones:
Water soluble, basic in nature due to the presence of arginine and lysine, found in nucleus. They help in
DNA packaging in the cell. They form the protein moiety of nucleoprotein.
(f) Protamine’s:
Water soluble, basic in nature, not-heat coagulable. Found in sperm cells, hence component of sperm
nucleoprotein.
(g) Globin’s:
They are water soluble, non-heat coagulable. e.g., globin of haemoglobin.

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3Protein: structure function classification and assay methods
(h) scleroproteins:
Insoluble in all neutral solvents, dilute acids or alkalies, e.g., keratin of hair and proteins of bone and
cartilage.
2. Conjugated proteins:
Proteins which are made up of amino acids and a non-amino acid/protein substance called the prosthetic
group are known as conjugated proteins.
The various types of conjugated proteins are:
(a) Chromo proteins:
Here the non-protein part is a coloured compound in addition to the protein part. Ex. Haemoglobin has
heme as the prosthetic group and cytochromes also have heme.
(b) Nucleoproteins:
These proteins are bound to nucleic acids, e.g., chromatin (histones + nucleic acids).
(c) Glycoproteins:
When a small amount of carbohydrate is attached to a protein it is known as glycoproteins, e.g., mucin of
saliva. (Note: Glycoproteins have major amounts of protein and some amount of carbohydrates and
proteoglycans contain major amounts of carbohydrates and little amount of proteins).
(d) Phosphoprotein:
Phosphoric acid is present with the protein. Ex. Milk casein and egg yolk (vitellin).
(e) Lipoproteins:
Proteins in combination with lipids, e.g., LDL, HDL.
(f) Metalloproteins:
They contain metal ion in addition to the amino acids, e.g., hemoglobin (iron), ceruloplasmin (copper).
3. Derived proteins:
They are the proteins of low molecular weight produced from large molecular weight proteins by the
action of heat, enzymes or chemical agents.
Proteins → Proteans → Proteoses → Peptones → Peptides → Amino acids
Protein conformation/ structure
• The spatial arrangement of atoms in a protein is called its conformation.
• The possible conformations of a protein include any structural state that can be achieved without
breaking covalent bonds. A change in conformation could occur, for example, by rotation about single
bonds. Of the numerous conformations that are theoretically possible in a protein containing hundreds of
peptide bonds, one or (more commonly) a few generally predominate under biological conditions.
• The need for multiple stable conformations reflects the changes that must occur in most proteins as
they bind to other molecules or catalyze reactions.

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4Protein: structure function classification and assay methods
• The conformations existing under a given set of conditions are usually the ones that are
thermodynamically the most stable, having the lowest Gibbs free energy (G). Proteins in any of their
functional, folded conformations are called native proteins.
Structure :
• Proteins have a total of four levels of structures.
• Primary structure.
• Secondary structure.
• Tertiary structure.
• Quaternary structure.
• Primary structure :-
• The simple amino acid sequence of a protein is called as its primary structure.
• Since the possible way of arrangement of the chain will depend on the sequence of amino acid residues
leading to proper protein folding, the primary structure dictate three dimensional structure of the
proteins.

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5Protein: structure function classification and assay methods
Secondary structure :-
• This defines the interaction of closely located amino acids in a chain. Two main types of secondary
structures observed in the proteins are helices (α helices) and pleated sheets (β pleated sheets).
• Alpha helix is a helical structure around an axis.
• This is coiled in clockwise (right handed) manner. It has an average of 3.6 amino acids per turn. Please
write other dimensions too.
• There are 3.6 residue per turn with in a fix pitch of 5.40A
• Thus the rise per residue come out to be 1.50A. In a typical α - helix φ value ranges from 113 to 1320 and ψ
from 123 to 1360.

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6Protein: structure function classification and assay methods
The reason as to why alpha helices form more readily in proteins than any other possible conformations is
that these arrangements make optimal use of internal hydrogen bonds to attain stability. The helix is
stabilized by hydrogen bonding between the carbonyl of each first amino acid of the chain to the NH of the
amino acid four residues away. All main chain amino and carboxyl groups are thus hydrogen bonded, and
the R groups stick out from the structure in a spiral arrangement.
Beta pleated sheet is composed of two or more straight chains that are hydrogen bonded side by side. If
the amino termini are on the same end of each chain, the sheet is termed parallel, and if the chains run in
the opposite direction (amino terminal on opposite ends), the sheet is termed antiparallel.
Pleated sheets may be formed from a single chain if it contains a beta turn, which forms a hairpin loop
structure. Often a proline can be found in a beta turn, since it places a "kink" in the chain.
Glycine and Ala are predominant amino acids in beta sheet.
• Tertiary structure :- refers to the arrangement of amino acids in the space i.e. in three dimensional form.
• Distinct amino acid are brought closer in chain are further linked by :
- polar-polar interaction,
- hydrophobic interaction,
- ionic interaction,
- disulfide bonds,
- Van der Waals forces.
- hydrogen bonds.
• Hydrophobic amino acids :- are buried inside the core of protein and charged and polar group are located
on the surface which tend to cluster and exclude water. This allows a protein to have greater water
solubility.
• Quaternary structure :- If protein consists of more than one polypeptide chains, their association with

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7Protein: structure function classification and assay methods
each other – implies the Quaternary structure.
• Accordingly protein are termed as dimeric ( wherein one chain is referred as monomeric unit), trimeric or
oligomeric. If the chains are similar i.e. have same amino acid sequence these are called homomeric or
heteromeric if chains are different.
• Proteins have also been put into two major groups :
(a) Fibrous proteins, having polypeptide chains arranged in long strands or sheets.
(b) Globular proteins, having polypeptide chains folded into a spherical or globular shape.
• The two groups are structurally distinct : fibrous proteins usually consist largely of a single type of
secondary structure; globular proteins often contain several types of secondary structure e.g a- keratins
are predominantly alpha helix whereas silk proteins are beta –sheets.
• The two groups differ functionally in that the structures that provide structure, support, shape, and
external protection to vertebrates are made of fibrous proteins, whereas most enzymes and regulatory
proteins are globular proteins.
Protein Assay methods
Introduction
Protein assays are one of the most widely used methods in life science research. Estimation of protein
concentration is necessary in protein purification, electrophoresis, cell biology, molecular biology and
other research applications. Although there are a wide variety of protein assays available, none of the
assays can be used without first considering their suitability for the application. Each assay has its own
advantages and limitations and often it is necessary to obtain more than one type of protein assay for
research applications.
Dye Binding Assays (Bradford) the dye binding protein assay is based on the binding of protein molecules
to Coomassie dye under acidic conditions. The binding of protein to the dye results in spectral shift, the

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8Protein: structure function classification and assay methods
color shifts from brown (Amax= 465nm) to blue (Amax= 610nm). The change in color density is read at
595nm and is proportional to protein concentration. The basic amino acids, arginine, lysine and histidine
play a role in the formation of dye-protein complexes color. Small proteins less than 3kDa and amino acids
generally do not produce color changes. CB™ and CB-X™ protein assays are dye binding protein assays.
SPN™ and SPN™-htp protein assays are spin column format dye binding assays
Copper Ion Based Assays (Lowry & BCA)
In the copper ion based protein assays, the protein solution is mixed with an alkaline solution of copper
salt. Under alkaline conditions, cupric ions (Cu2+) chelate with the peptide bonds resulting in reduction of
cupric (Cu2+) to cuprous ions (Cu+). If the alkaline copper is in excess over the amount of peptide bonds,
some of the cupric ions (Cu2+) will remain unbound to the peptide bonds and are available for detection
(Figure 1). Protein assays based on copper ions can be divided into two groups, assays that detect reduced
cuprous ions (Cu+) and assays that detect the unbound cupric (Cu2+) ions. The cuprous ions are detected
either with bicinchoninic acid (BCA) or Folin Reagent (phosphomolybdic/ phosphotungstic acid) as in the
protein assays based on Lowry method. Cuprous ions (Cu+) reduction of Folin Reagent produces a blue
color that can be read at 650-750nm. The amount of color produced is proportional to the amount of
peptide bonds, i.e. size as well as the amount of protein/peptide.
The presence of tyrosine, tryptophan, cysteine, histidine and asparginine in protein contributes to
additional reducing potential and enhances the amount of color produced. Hence, the amount of blue
color produced is dependent on the composition of protein molecules. The reaction of cuprous ions (Cu+)
with the bicinchoninic acid and color production is similar to that of Folin Reagent. In the assays based on
the detection of unbound cupric ions, the protein solution is mixed with an amount of alkaline copper that
is in excess over the amount of peptide bond. The unchelated cupric ions are detected with a color-
producing reagent that reacts with cupric ions. The amount of color produced is inversely proportional to
the amount of peptide bond