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The very basic structure of an immunoglobulin (antibody) molecule can be demonstrated under following points:
Polypeptide chains:
Antibody molecules have a common structure of four polypeptide chains, having two different sizes. These are a pair of identical high molecular weight chains called Heavy chains (H-chains) and a pair of identical low molecular weight chains called Light chains (L-chains). Each light and heavy chain may be subdivided into homologous regions termed domains.
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H-chains:
H-chains have a molecules weight of 50-55 kd. Each H-chain has disulfide linkages (-S-S-) and also contains carbohydrate molecule attached to its asparagine residue. Each H-chain has N-terminal (NH3+) and C-terminal (Coo–) respectively.
L-chains:
L-chains have a molecular weight of 20-25kd. L-chains are covalently linked with H-chains by disulfide bridge (-S-S-). Each L-chain also has N-terminal and C-terminal respectively.
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Hinge region:
Electron microscopy of purified immunoglobulin (IgG) molecules, after negative staining reveals the Y-shape of the molecules indicating a flexible “hinge region” at about the middle of the H-chains where two H-chains are connected by disulfide bridge (-S-S-).
Antigen combinding site (ACS):
One variable region of a heavy chain (VH) and one variable domain of light chain (VL) together constitute an antigen combinding site (ACS) or in other words determine the antibody specificity.
Structure of Immunoglobulin Molecule (Ab) based on Amino Acid sequencing Studies:
Polypeptide chains:
Amino acid sequencing study reveals that each heavy and light chain in an immunoglobulin molecule contains an amino terminal variable (V) regions (VH → variable region of H-chain; VL → → variable region of L-chain) that consist of 100-110 amino acids and differ from one antibody to the next.
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The remainder of the molecule—the constant region (CH → constant region of Heavy-chain; CL → constant region of Light-chain) exhibits limited variation that defines two Light chain subtypes (k & λ) and five Heavy chain subtypes (γ, α, μ, δ, or ε).
Heavy and Light chains are folded into domain, each containing about 110 amino acid residues and an inter-chain disulfide bonds that forms a 60 amino acids loop.
Hinge region:
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Amino acid analysis of the hinge region has indicated an unusual feature—a large no. of proline residues present.
Because of its structure, proline prevents the peptide chains from assuming an α-helix conformation and thus the hinge region remains extended.
And the peptide bonds of the hinge region are accessible to the proteolytic enzymes shown below:
Antigen binding site:
Within the variable region of both Light and Heavy chain (VL and VH), amino acids at reversal positions are often substituted and at certain points, the substitution is at a notably higher frequency. These high frequency-regions are termed as “hyper-variable regions” or “hot spots”. Three regions on the Light (VL) chain and three on the Heavy (VH) chain lie relatively close to each other to form the antigen binding site.
Each hyper-variable region consists of five to ten amino acid residues. As the hyper-variable regions determine the combinding sites for antigenic determinant, that’s why they are also termed as complementarity-determining regions (CDRs). The variable sequences on either side of the hyper-variable regions termed as framework regions (FRs). Thus each variable (V) region consists of three CDRs and four FRs.
Proteolytic digestion of Immunoglobulin:
Immunoglobulin fragments produced by proteolytic digestion have proven very useful in elucidating structure /function relationships in immunoglobulin. Poster (1962) demonstrated that Pepsin digestion of IgG molecules produced three fragments, of which two are identical (each with a MW. 45,000 dt/45kd), each having a specific binding site for Ag and these two fragments were termed as Fab (fragment antigen binding) and the third segment (Mw. 50,000 dt/50kd) did not have combining site for Ag and also was found to crystallize during cold storage. This fragment was termed as Fc (fragment crystalliable).
But when pepsin proeolytic enzyme was administered, interestingly it was found that pepsin-digestion generated a single 100,000 dt. MW. fragment composed of two Fab-like segments and designated as F(ab’)2, able to precipitate Ag. But Fc fragment was not recovered from pepsin digestion because it had been digested into multiple Fc-fragments (F/Fc‘).
