Biomedical Engineering Reference
In-Depth Information
to protein production must be thoroughly validated. Apart from this, only limited
routes of delivery are available for proteins and peptides due to their lability and
susceptibility to degrade in an acidic environment and in the presence of proteolytic
enzymes. Two major aspects associated with protein formulations are their safety
and efficacy. It should always be borne in mind that bioavailability should not be
increased at the risk of raising safety concerns.
�.5.2.1.5 Levels of Protein Structure
8.5.2.1.5.1
Primary to Quaternary Structure
The arrangement of amino acids or
their sequential arrangement is referred to as the primary structure and is determined
genetically by ribonucleic acid (RNA) during the process of transcription, and ultimately
on the sequence of nucleotides in DNA. These 20 amino acids will be present in triplet
codons in various combinations within a biological system. The secondary structure of a
protein delineates the arrangement of individual amino acids along the polypeptide back-
bone of a protein, resulting in -helices, -sheets, reverse turns, and random coils.
Tertiary structure is the three-dimensional arrangement of a protein molecule and
depicts the mode of interaction of secondary structural elements.
Quaternary structure is the conformation of a protein when it exists in a solid
state or in solution and mainly based on noncovalent interaction or stereochemical
arrangement of individual protein subunits (monomers).
8.6 Protein and Peptide Synthesis
A wide literature is available for protein and peptide synthesis. We have given an
overview of various methods used for protein synthesis. They may be synthesized by
solution or using the solid phase method.
8.6.1 Classical Solution Phase Synthesis
8.6.1.1 Racemization
The underlying principle for all types of peptide synthesis is based on the reaction of
an amine component with an activated carboxyl component to form a peptide bond.
Racemization occurs due to proton abstraction followed by base-catalyzed enoliza-
tion of the parent peptide or formation of the 5(4
H
)-oxazolone (azalactone) interme-
diate from the activated peptide.
8.6.1.2 Carbodiimide Method
Sheehan and Hess introduced the use of dicyclohexylcarbodiimide (DCC) as a cou-
pling reagent for the preparation of amide bonds in 1955. DCC has been used since
then for peptide synthesis. The mechanism involves formation and propagation of
an amide or peptide bond via an
O
-acylisourea active intermediate. The intermedi-
ate is violently reactive and difficult to isolate. It reacts vigorously with nucleophiles
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