1/ Reaction involving residues in proteins

The chemical modification of protein is of importance for a number of reasons. It

provides derivatives suitable for sequence analysis, identifies the reactive groups in
catalytically active sites of an enzyme, enables the binding of protein to a carrier
(protein immobilization) and provides changes in protein properties which are important
in food processing. In contrast to free amino acids and except for the relatively small
number of functional groups on the terminal amino acids, only the functional groups on
protein side chains are available for chemical reactions.

Tryptophan residue

Tryptophan contains an α-amino group, an α-carboxylic acid group, and a side chain
indole, making it a non-polar aromatic amino acid. It is essential in humans, meaning
that the body cannot synthesize it and it must be obtained from the diet.

Since tryptophans are usually located within the interior of native proteins and since
their indole groups are intrinsically less reactive than thiol or amino groups, tryptophans
are less apt to be modified by most reagents. Nevertheless, under the right conditions,
they are susceptible to substitutions, oxidative cleavage, and reductions.

N-Bromosuccinimide oxidizes the tryptophan side chain

The reaction is used for the selective cleavage of peptide chains and the
spectrophotometric determination of tryptophan.

2/ Hydrolysis of proteins

Proteins are polypeptides, polymers made up of a large number of amino acid units
joined together by peptide bonds.

To analyze the amino acids contained within these biomolecules, it is critical that the
bonds be hydrolyzed to form free amino acids.

Protein hydrolysis is carried out by chemical and enzymatic methods

By acidic agents

Acid hydrolysis refers to a hydrolysis reaction occuring under acidic conditions

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Acid hydrolysis is the most common method for hydrolyzing a protein sample. Although
a range of different acids can be used for this reaction, the most common is 6 M HCl

However, not all amino acids are completely recovered under hydrolysis by HCl. Some
amino acids are hydrolyzed to their acid forms, such as asparagine and glutamine,
which form aspartic acid and glutamic acid, respectively

We can break the peptide bonds (amide links) joining the amino acid units together by
using acidified water in an acid hydrolysis reactions

Given the structure of a protein molecule, the structures of the amino acids used to form
it can be shown by simply breaking every peptide link to leave amine and carboxyl
groups.

Gly Ala Gly

This disrupts the primary structure of the protein and the protein breaks down into
smaller pieces, eventually resulting in many amino acids

During acid hydrolysis, water (H20) adds across the peptide bond (-CO-NH-), resulting
in the formation of carboxyl functional groups (-COOH) and amine functional groups (-
NH2)

Alkaline hydrolysis: alkaline or base hydrolysis is often used to measure tryptophan.

Because tryptophan is stable under basic conditions, this technique gives accurate
quantitation of tryptophan and is widely used for a variety of samples, from foods and

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feeds to peptides and proteins. Alkaline hydrolysis typically uses NaOH or KOH as the
reagent

However, alkaline hydrolysis cannot replace acid hydrolysis for the quantitation of all
amino acids

By proteolytic enzymes

When humans eat food containing proteins, like meat, fish and eggs, the proteins are
broken down into amino acids using various enzymes collectively called peptidases or
proteases

Different enzymes catalyse the hydrolysis of the peptide bond (amide link) between
different amino acid residues. Proteolytic enzymes hydrolyze proteins at the optimum
temperature and pH and usually target specific peptide cleavage bonds, resulting in
digestion consisting of amino acids and peptides of varying size.

For example, In the small intestine, trypsin breaks down proteins, continuing the
process of digestion that began in the stomach, it helps break the peptide bond when
the amino acid residues arginine or lysin are contributing the –CO- functional group to
the peptide bond

Note: Under relatively mild conditions of temperature and acidity, certain proteolytic
enzymes like pepsin and trypsin hydrolyse the proteins. Enzyme hydrolysis is used for
the isolation of certain amino acids like tryptophan. Two important drawbacks with this
type of hydrolysis are:

It requires prolonged incubation and Hydrolysis may be incomplete

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2/ Colour reaction

A color test for a protein tests for a particular functional group or structural component
present in a protein or in a particular amino acid found in proteins

Biuret test: It is a general test used for detecting the presence of proteins and peptides

It is based on the biuret reaction in which a peptide structure containing at least two
peptide links produces a violet color when treated with alkaline copper sulfate

The Biuret reagent is a solution composed of sodium hydroxide (NaOH) or potassium

hydroxide (KOH), hydrated copper (II) sulfate, and potassium sodium tartrate. Sodium
hydroxide and Potassium hydroxide provide the alkaline medium and potassium sodium
tartrate is added to chelate and thus stabilize the cupric ions in the solution or to
maintain their solubility in alkaline solution.

In presence of an alkaline solution, blue-colored copper II ion can form a complex with
the peptide bonds since the peptide has unshared electron pairs in nitrogen and oxygen
of water. The colored coordination complex is formed between Cu2+ ion and carbonyl
oxygen (>C=O) and amide nitrogen (=NH) of the peptide bond. Once this complex has
been formed, the solution turns from blue to purple. The deeper the purple color, the
higher is the number of peptide-copper complexes.

The reaction occurs in any compound containing at least two H2N-C, H2N-CH2-, H2N-
CS- or similar groups joined together directly or through a carbon or nitrogen atom. One
copper ion is probably linked to 6 nearby peptide linkages by co-ordinate bonds. The
intensity of the color is directly proportional to the number of the peptide bonds present
in the protein molecule that is reacting and also the number of the protein molecules
present in the reaction system.

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Uses: Biuret reaction with protein is applicable to the quantitative determination of total
protein by spectrophotometric analysis. The use of bovine or human serum albumin to
standardize the biuret method is well established

3/Reactions Involved in Food Processing

Maillard reaction (carbonyl - amine browning)

This reaction was described for the first time by Louis Maillard in 1912.

is also known as nonenzymatic browning. It is an extremely complex process and is the

reaction between reducing sugars and proteins by the impact of heat./ The Maillard
reaction occurs between reducing sugars and principally free amino acids and peptides
(usually from proteins) when heated

The Maillard reaction also takes place at room temperature but at a much slower rate
and occurs at its slowest at low temperatures, low pH and low Aw levels.

Excellent examples of the Maillard reaction are the crust of roast pork or baked bread.
The Maillard reaction also creates, besides colour, countless complex flavours at the
same time.

Explain 1: The Maillard reaction is divided into three stages: the early Maillard reaction,
the advanced Maillard reaction, and final Maillard reaction. The early Maillard reaction

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consists of condensation of the reducing sugar with the amino groups and leads, via
formation of Schiff’s base and the Amadori rearrangement, to the so-called Amadori
product. The advanced Maillard reaction consists of the breakdown of the Amadori
product into numerous fission products of the sugar–amino compound. The final
Maillard reaction consists of the condensation of amino compounds and sugar
fragments into polymerized protein and brown pigments, called melanoidins (brown
compound).

The formation of the melanoidins during the advanced stages of the Maillard reaction
lead to the color changes toward brown

Melanoidin is a general term for colored, polymeric structures that form as a result of the
Maillard reaction in thermally processed foods. A common characteristic feature of
melanoidins is strong absorption in the UV–blue range, which provides the familiar
browning color to many baked and roasted foods

The sulphur-containing amino acids methionine and cysteine play a primary role within
the formation of the flavour-intensive components gained during the Maillard reaction.

The flavour of such roasted or grilled meats also contains heterocyclic compounds
derived from amino acids, nucleotides and sugars from the Maillard reaction such as
oxopropanol and hydroxymethylfurfural. Unsaturated fatty acids, as well as aldehyde–
fatty–acid components, also contribute to the formation of odorous heterocyclic flavour
compounds during the Maillard reaction.

Maillard reaction form products that are desirable or undesirable

Desirable: caramel aromas and golden brown colors,

undesirable foods: darkness and off-flavor development, loss of nutrition component

(Lysine)