As noted earlier, the mean A/G ratio of 1.36 decreases in various disease processes, for example in nephrotic syn-drome, where albumin levels are low and/or globulin levels are high. Abnormal electrophoretic pattern, with decreased albumin and a prominent ␣2-globulins are seen.
Some other important conditions where A/G ratio is reversed are malnutrition and liver diseases (albumin decreases), and chronic infections and multiple myeloma (globulin rises). Abnormal electrophoretic patterns are also seen; for example, in multiple myeloma a sharp dis-tinct M band appears in ␥-globulin region, in infections the ␣1 and ␣2 bands are increased.
Advanced liver diseases: The serum globulin levels are typically elevated, often between 5–7 g/dL (normal ⫽ 1.8–
3.6 g/dL). Since the albumin concentration is usually in the lower part of the normal range, or reduced (because of decreased synthesis), the A:G ratio is low.
Multiple myeloma: It is a group of diseases characterized by proliferation of a single plasma cell clone to cause extraor-dinary accumulation of immunoglobulin molecules in the patient’s serum. Being derived from a single ancestral B cell, the proliferating plasma cells all produce the same (mono-clonal) immunoglobin. Such immunoglobulins are termed paraproteins, and they produce a sharp peak in the
␥-globulin fraction. The paraproteins may be of the IgG, IgA, IgM, IgD or IgE type. The malignant plasma cells grow in the bone marrow, where they cause bone pain, patho-logical fractures and radiopatho-logical abnormalities. In many cases, a multiple myeloma patient excretes protein in the urine. This protein called Bence-Jones protein, is a dimer of or -chains that are formed in paraprotein.
Diagnosis: Demonstration of paraprotein upon serum electrophoresis is a reliable test for diagnosis of multiple myeloma. In addition, the classical heat test is performed.
It involves the precipitation of Bence-Jones proteins when slightly acidifi ed urine is heated to 40º⫺50ºC and
Hb released from RBC
Heme binds to haemopexin
Removed by liver Haemopexin
Dissociation into ab dimers
Haptoglobin
Formation of Hb-Hpt complex
Removed by reticulo-endothelial cells RBC
Fig. 5.13. Iron conservation by haemoglobin and haemopexin.
many plasma cells, each producing a distinct antibody, that gives rise to the multiple antibody specifi cities. Spe-cifi c affi nity of an antibody is not for the entire macro-molecular antigen but for a particular site on it called antigenic determinant (or epitope).
This property is widely used in purifi cation of proteins and in affi nity chromatography (Chapter 4). The affi nity of antibody for an epitope is due to the complementary structure of the two. If there is a cleft on the surface of the epitope, a corresponding elevation is envisioned on the antibody. If an antigen is injected, the body generates an antibody specifi c to each of the epitopes on the antigen.
Immunoglobulins or antibodies, a group of plasma pro-teins that are secreted by plasma cells, play an impor-tant role in body’s defenses by specifi cally recognizing foreign molecules (antigens) and facilitating their selec-tive elimination.
A. Structure
The basic structural unit of an immunoglobulin is a hetero-tetramer, made up of two light chains (L) and two heavy chains (H), joined through non-covalent interaction and disulphide bridges. Thus, the simplest description of an antibody molecule is H2L2 (Fig. 5.14). The light chains are simple proteins of 212 amino acids (MW 23,000 each) and the heavy chains are glycoproteins comprising approx-imately 440 amino acids (MW 53,000–75,000) each.
The light chains are of two types: or . A given immu-noglobulin contains 2 or 2 chains, but never a mixture of the two. In humans, 60% light chains are of type.
There are fi ve types of heavy chains: ␥, , ␣, and ␦. Depending on the heavy chain make up, the immu-noglobulins are differentiated into fi ve classes: IgA (␣-chain), IgD (␦-chain), IgE (-chain), IgG (␥-chain), and IgM (-chain). All classes of immunoglobulins are present in every human being. (Within a class are found subclasses due to subtle differences in the amino acid sequences of heavy chains of the same class.)
Thus, structure of IgG can be represented as 2␥2 or as
2␥2; the IgA molecule as 2␣2 or as 2␣2 and so on. The heavy chains are hinged (fl exible) in the vicinity of two inter-chain disulphide bridges. At these places, several prolyl residues are present, which confer a degree of fl ex-ibility on the molecule.
Human immunoglobulins exist as IgA, IgD, IgE, IgG and IgM classes, which contain ␣, ␦, , ␥ and heavy chains respectively.
C. Fibrinogen
Fibrinogen, the precursor of fi brin clot, appears to be formed in the liver. It is a soluble glycoprotein that con-sists of three pairs of identical polypeptide chains, called the ␣-, - and ␥-chains (MW 63,500, 56,000 and 47,000, respectively). Blood coagulation is initiated by the action of thrombin on fi brinogen, whereby two small peptides are removed from the fi brinogen. This results in forma-tion of a fi brin monomer. Several monomers then aggre-gate via physical interactions to form a polymer that has properties of a soft gel. Further polymerization to form tough insoluble clots is accomplished by a specifi c enzyme called factor XIII: its action serves to join a number of linear fi brin polymers to form cross-linked fi brin lattices.
Diseases and Disorders
Decreased fi brinogen level in plasma is observed in severe liver diseases (e.g. acute hepatic necrosis). Low fi brinogen levels observed in antepartum haemorrhage, quickly return to normal after delivery.
Increased fi brinogen level is found in acute infections such as pneumonia, rheumatic fever, and tuberculosis. Since erythrocyte sedimentation rate (ESR) is affected by fi brin-ogen, it is markedly increased in these conditions. Hence, measurement of ESR is used for assessing the severity and course of these disorders. In contrast to most other acute infections, fi brinogen level is reduced in typhoid fever.
V. Immunoglobulins
The immune system is remarkable for its ability to gener-ate a variety of specifi c proteins that include antibodies (immunoglobulins). The antibody reacts specifi cally with the antigen that triggered its production. Antigens are mostly proteins, but can be nucleic acids or polysac-charides. Thus, various bacterial and viral cell components may act as antigens. Antibodies recognize the antigen on the surface of the intruding organisms. The specifi c antigen-antibody interaction leads to activation of the complement system which destroys the foreign cell. Antibodies play important role in providing resistance against bacterial infections. Though it is mainly the ␥-fraction of plasma proteins that comprises immunoglobulins, sometimes they separate with - and ␣-globulin bands of electro-phoretogram. Plasma concentration of immunoglobu-lins is about 1.0–2.5 g/dL.
Immunoglobulins are synthesized by plasma cells.
Plasma cells develop from B lymphocytes that have con-tacted foreign antigen. A single plasma cell can make only one type of antibody molecule. It is the accumulation of
CH2 domain binds the complement.
CH3 domain helps adherence to the cell surface.
All immunoglobulins are glycoproteins, and the degree of glycosylation in the region of the domain CH3 is variable.
An antibody consists of two light chains and two heavy chains joined by disulphide bonds. There are two antigen binding sites, each formed by a light chain and a heavy chain.
B. Variability
The amino acid sequences at the N-termini of both light and heavy chains show considerable variation between individual immunoglobulin molecules. Half of the light chain and quarter of the heavy chain constitute the vari-able region. Rest of the molecule, known as constant region, shows little variation within one immunoglobulin class (Fig. 5.14b). The light chain contains one variable (VL) and one constant (CL) region, whereas the heavy chain contains one variable and three or more constant regions. The variations in the amino acid sequences of the variable region permit the body to construct a large variety of three dimensional structures.
Moreover, within these V regions, certain segments are hypervariable. The 29–32, 48–52, and 93–96 aminoacyl residues in light chain and 30–36, 50–53 and 93–98 positions in heavy chains are termed hypervariable regions. They defi ne the actual binding site for antigen.
They are also termed the complementarity-determining regions (CDRs) as they form antigen binding site com-plementary to the topology of the antigen structure. In the Each of the four chains consists of variable region and
a constant region, discussed later.
Both heavy and light chains have distinct globular regions called domains that have a highly organized tertiary structure. Each of these structural domains comprise about 110 aminoacyl residues. The light chain has two such domains, one in the variable region of the chain (VL) and the other in the constant (CL) region. The heavy chain, has four or fi ve domains. The latter are termed VH or CH depend-ing on whether they are present in the variable region or the constant region of heavy chain. Figure 5.15 shows domain arrangement of a typical immunoglobulin molecule.
Each domain is stabilized internally by a disulphide bond and generates a globular three-dimensional struc-ture. Thus, antibody molecule can be visualized as being made up of distinct domains, connected by relatively non-ordered polypeptide segments. These domains have well defi ned biological functions.
VL and VH are responsible for forming antigen-binding site.
Fig. 5.15. Structure of an immunoglobulin molecule showing the disulphide-bridge limited domains. Domains on light chain are VL and CL; and the domains on heavy chain are: VH, CH1, CH2, CH3.
Fig. 5.14. Structure of an antibody molecule. (a) Each antibody (immunoglobulin) molecule has two identical light chains (L) and two identical heavy chains (H) consisting of 212 and 440 amino acids respectively, (b) Each chain has a variable region, where amino acid sequences shows considerable variations between individual immunoglobulin molecules and constant region where little variation occurs within one immunoglobulin class. The generic terms for these regions in the light chain are VL and CL and for heavy chain are VH and CH.
The Fab (fraction antibody) fragment binds with the corresponding antigen. It is constituted by whole of the light chain and N-terminal half of the heavy chain. Fc (fraction crystallizable) portion, constitutes the remain-ing part of the immunoglobulin molecule. It is con-cerned with activation of complement cascade and interaction with the cellular elements of the immune response. It triggers immune response that leads to the lysis of the intruding organisms.
Immunoglobulins from any class can also exist in two types: This is due to presence of either kappa () or lambda () light chain, giving rise to 10 different permu-tations of heavy and light chains. The chains are more common in humans than the chains.
While IgD, IgE, and IgG occur as a single Y-shaped antibody unit, IgM occurs as a pentamer and IgA can exist as a monomer, dimer, or trimer. Characteristics of various classes of immunoglobulins are summarized in Table 5.4.
There are fi ve classes of heavy chains that defi ne the class of immunoglobulin. Different classes of immuno-globulins have different properties.