4. Reaction of AA with Nitrous Acid:- AAs react with nitrous acid (NaNo3+HCl) to form hydroxy acids and nitrogen

5. Reaction of AA with Sanger’s reagent:- AAs react with 2,4,-dinitroflurobenzene also known as Sanger’s reagent to produce yellow colored dinitrophenylamino acids or DNP-amino acids. This reaction is very important in the determination of structure of peptides and proteins and N-terminal amino acid

Glycine

CH2 COOH +H2N HONO

Nitrous acid

CH2 COOH +N2 +H2O

HO

Glycolic acid

H+

α-amino acid

CH N-H +R

2,4, DNFBCOOH

H

F NO2

DNPAA (Yellow)

NO2 +HFCH NR

COOH

H

6. Reaction of AA with Ninhydrin:- All α-AAs react with ninhydrin to produce the purple complex. This reaction is commonly used to test the presence of α-AAs

C

C

COH

OH

Ninhydrin

CH COOHR

NH2

α-amino acid

+

O

O-H2O

C

C

C

Purple complex

O

O

N

C

C

C

O-

O

+ RCOOH

6. Reaction of AA with Edman reagent:- AAs react with phenyl isothiocyanate to produce phenyl thiocarbamoyl which on treatment with mild acidic conditions form a cyclic compound phenyl thiohydantoin. This reaction is used for the detection of N-terminal amino acid.

Phenyl thiocarbamoyl

CH COOHR

NH2

α-amino acid

+ SC=N CH COOHR

NH-SC-NH

H+

CH COR

NH-SCN

+ H2O

Phenyl Iso-thiocyanate

Phenyl thiohydantoin

ProteinProteins are organic compounds made of amino acids

arranged in a linear chain and folds into globular form. The polymer of amino acid are joined together by the peptide bonds between the carboxyl groups of adjacent amino acid residues.

Like other biological macromolecules such as polysaccharide and nucleic acids, protein are essential parts of organisms and participate in virtually every process within cells.

Many proteins are enzyme that catalyze biochemical reactions and are vital to metabolism. Proteins are important in cell signaling, immune responses, cell adhesion and in the cell cycle. Proteins are also necessary in animals diet, since animal cannot synthesis all amino acids they need and must obtain essential amino acids from food.

Protein Biochemistry1. Most proteins are linear polymers built from series of upto

20 different α-amino acids.2. All amino acids possess common structural features,

including a α-carbon to which an amino group, a carbonyl group and a variable side chain are bonded.

3. The side chains of the AAs have a great variety of chemical structures and properties.

4. The AAs in a polypeptide chain are linked by peptide bonds.5. In a protein chain, an individual amino acid is called a

residue and the linked series of carbon, nitrogen and oxygen atoms are known as the protein backbone.

6. The end of protein with a free carboxyl group is known as the C-terminal or carboxy terminal whereas the end with a free amino group is known as the N-terminal or amino terminal

7. The words protein, polypeptide and peptide have a little difference

Protein is generally used to refer the complete biological molecule in a stable conformation

Peptide is generally used for short amino acid oligomer lacking a stable three dimensional structure

Polypeptide can refer to any single linear chain of amino acids usually regardless of length and often an absence of a defined conformation

Level of Protein Structure

There are 4 types of protein structure, such as

Primary structureThe primary structure refers to the sequence of the different

amino acids of the peptide or protein. The primary structure is held together by covalent or peptide bonds, which are made during the process of protein biosynthesis or translation. The two ends of the polypeptide chain are referred to as the carboxyl terminus (C-terminus) and the amino terminus (N-terminus) based on the nature of the free group on each extremity. Counting of residues always starts at the N-terminal end (NH2-group), which is the end where the amino group is not involved in a peptide bond.

The primary structure of a protein is determined by the gene corresponding to the protein. A specific sequence of nucleotides in DNA is transcribed into mRNA, which is read by the ribosome in a process called translation. The sequence of a protein is unique to that protein, and defines the structure and function of the protein. The sequence of a protein can be determined by methods such as Edman degradation or tandem mass spectrometry.

Secondary structureAn alpha-helix with hydrogen bonds

Secondary structure refers to highly regular local sub-structures. Two main types of secondary structure, the alpha helix and the beta strand, were suggested in 1951 by Linus Pauling and coworkers. These secondary structures are defined by patterns of hydrogen bonds between the main-chain peptide groups. They have a regular geometry, being constrained to specific values of the dihedral angles ψ and φ on the Ramachandran plot. Both the alpha helix and the beta-sheet represent a way of saturating all the hydrogen bond donors and acceptors in the peptide backbone. Some parts of the protein are ordered but do not form any regular structures. They should not be confused with random coil, an unfolded polypeptide chain lacking any fixed three-dimensional structure. Several sequential secondary structures may form a "supersecondary unit".

Tertiary structure

Tertiary structure refers to three-dimensional structure of a single protein molecule. The alpha-helices and beta-sheets are folded into a compact globule. The folding is driven by the non-specific hydrophobic interactions (the burial of hydrophobic residues from water), but the structure is stable only when the parts of a protein domain are locked into place by specific tertiary interactions, such as salt bridges, hydrogen bonds, and the tight packing of side chains and disulfide bonds. The disulfide bonds are extremely rare in cytosolic proteins, since the cytosol is generally a reducing environment.

Quaternary structure

Quaternary structure is a larger assembly of several protein molecules or polypeptide chains, usually called subunits in this context. The quaternary structure is stabilized by the same non-covalent interactions and disulfide bonds as the tertiary structure. Complexes of two or more polypeptides (i.e. multiple subunits) are called multimers. Specifically it would be called a dimer if it contains two subunits, a trimer if it contains three subunits, and a tetramer if it contains four subunits. The subunits are frequently related to one another by symmetry operations, such as a 2-fold axis in a dimer. Multimers made up of identical subunits are referred to with a prefix of "homo-" (e.g. a homotetramer) and those made up of different subunits are referred to with a prefix of "hetero-" (e.g. a heterotetramer, such as the two alpha and two beta chains of hemoglobin). Many proteins do not have the quaternary structure and function as monomers.

Protein has a large number of important functions in the human body. In fact, the human body is about 45% protein. It’s an essential macromolecule without which our bodies would be unable to repair, regulate, or protect themselves

Protein has a range of essential functions in the body, including the following:

Required for building and repair of body tissues (including muscle) Enzymes, hormones, and many immune molecules are proteins Essential body processes such as water balancing, nutrient transport, and muscle contractions require protein to function. Protein is a source of energy. Protein helps to keep skin, hair, and nails healthy. Protein, like most other essential nutrients, is absolutely crucial for overall good health.

Function of Protein in Human Body

Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest.

The starting material is usually a biological tissue or a microbial culture. The various steps in the purification process may free the protein from a matrix that confines it, separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Separation of one protein from all others is typically the most laborious aspect of protein purification.

Separation steps may exploit differences in (for example) protein size, physico-chemical properties, binding affinity and biological activity.

Protein Purification

Purification may be done by two way, such as1. Preparative 2. Analytical

1.Preparative purifications:- The aim of PPs is to produce a relatively large quantity of purified proteins for subsequent use. For example, the preparation of commercial products such as enzymes (e.g. lactase), nutritional proteins (e.g. soy protein isolate), and certain biopharmaceuticals (e.g. insulin).

2.Analytical purification:- It produces a relatively small amount of a protein for research or analytical purposes, such as identification, quantification, and studies of the protein's structure, post-translational modifications and function.

The first purified proteins were urease and Concanavalin A.

Methods of Protein Purification

Strategies of Protein Purification

Select a starting material is key to the design of a purification process. Usually a particular protein isn't distributed homogeneously throughout the body in a plant or animal. Use of only the tissues or organs with the highest concentration decreases the volumes needed to produce a given amount of purified protein.

If the protein is present in low abundance, or if it has a high value, scientists may use recombinant DNA technology to develop cells that will produce large quantities of the desired protein (this is known as an expression system).

Fig. Recombinant bacteria can be grown in a flask containing growth media

An analytical purification generally utilizes three properties to separate proteins.

1.First, proteins may be purified according to their isoelectric points by running them through a pH graded gel or an ion exchange column.

2.Second, proteins can be separated according to their size or molecular weight via size exclusion chromatography or by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis.

3.Thirdly, proteins may be separated by polarity/hydrophobicity via high performance liquid chromatography (HPLC) or reversed-phase chromatography (RPC).

Fig. Picture of an SDS-PAGE. The molecular marker is in the left lane

There are several methods for protein extraction, such as

1.Repeated freezing and thawing2.Sonication3.Homogenization by high pressure4.Filtration (either via cellulose-based depth filters or cross-flow filtration) or5.Permeabilization by organic solvents.

The method of choice depends on how fragile the protein is andhow study the cells are.

Extraction of Protein

Different methods are available for the concentration of purified protein which are mention below-

1. Lypophilic:- If the solution doesn't contain any other soluble component than the protein in question can be lyophilized (dried). This is commonly done after an HPLC run. This simply removes all volatile component leaving the proteins behind.

2. Ultrafiltration:- concentrates a protein solution using selective permeable membranes. The function of the membrane is to let the water and small molecules pass through while retaining the protein. The solution is forced against the membrane by mechanical pump or gas pressure or centrifugation.

Concentration of Protein

Figure:- A selectively permeable membrane can be mounted in a centrifuge tube. The buffer is forced through the membrane by centrifugation, leaving the protein in the upper chamber.

Extraction of Protein from Eggs

1. The eggs (100 g) were taken in a morter and crush uniformly with a pestle within a possible short time

2. Pre-cooled 1% acetic acid was added to this mixture and homogenize uniformly by homogenizer

3. Then mixture was transferred to a beaker and keep 24 hour at 40C with occasional stirring

4. The suspension was then filtered through a filter paper or by cotton at 40C

5. Then the filtrate was collected and further clarification by centrifugation at 1200 rpm for 6 min

6. The clear supernatant was then saturated by the addition of ammonium sulfate with gentle stirring at 40C. The protein was salting out and supernatant become turbid due to the precipitation of protein

7. Then the mixture was again centrifuged at 1000 rpm for 15 mins

8. The ppt was collected and dissolved in minimum volume of deionized water and dialyzed for 24 hours. It was again centrifuged at 800 rpm for 6 mins to remove any insoluble materials present

9. The clear supernatant was transferred carefully in a plastic container and preserved in a deep freeze for further work

Separation and Isolation of Extracted protein

There are several techniques for the separation and isolation of extracted protein. Some of the techniques are mentioned below-1.Precipitation and differential solubilization2.Dialysis3.Chromatographic techniques

Ion exchange chromatographyAffinity chromatographySize exclusive chromatographyHigh performance liquid chromatography