BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

PART V. MOLECULAR PHYSIOLOGY

CHAPTER 33. IMMUNOGLOBULINS

33.2. Synthesis of Specific Antibodies in Response to an Antigen

Animals are capable of synthesizing specific Antibodies against virtually any chemical group. Dinitrochlorobenzene (DNP) is particularly effective at stimulating antibody production and is therefore widely used as a hapten determinant. Antibodies against DNP are obtained as follows.

1. DNP groups are covalently attached to a carrier protein, such as bovine serum albumin (BSA), via the reaction of fluorodinitrobenzene with the side chains of Lysine or other nucleophilic residues of the protein (Fig. 33.4).

Class="center">Fig. 33.4. Dinitrophenyl derivative of bovine serum albumin (DNP-BSA), an effective antigen

2. DNP-BSA is administered to a rabbit as an immunogen (antigen). Within a few days, the level of anti-DNP antibodies begins to rise (Fig. 33.5). These first-appearing antibodies belong to the class of IMMUNOGLOBULINS M (IgM) with a Molecular Weight of about 1000 kDa.

Fig. 33.5. Kinetics of the appearance of immunoglobulins M and G (IgM and IgG) in serum following immunization

3. Approximately 10 days after antigen administration, the level of immunoglobulin M begins to decline, while simultaneously the concentration of another class of antibodies—specifically immunoglobulins G (IgG) with a molecular weight of about 150 kDa—increases.

4. About 3 weeks after antigen injection, the level of anti-DNP antibodies of the immunoglobulin G class reaches a plateau. Administering another dose of DNP-BSA during this period triggers a further increase in the concentration of anti-DNP antibodies in the rabbit's serum.

5. Blood is collected from the immunized rabbit, and the serum is isolated (referred to as antiserum, since it is obtained after immunization). The concentration of anti-DNP antibodies in this serum can reach 1 mg of antibody per 1 mL of serum. DNP is exceptionally effective as an agent stimulating The production of large amounts of specific antibodies. Almost all of these antibodies belong to the class of immunoglobulins G, the major serum immunoglobulin class.

6. The next stage involves separating anti-DNP antibodies from antibodies of other specificities and from other Serum Proteins. Anti-DNP antibodies differ from the remaining antiserum proteins by their exceptionally high affinity for DNP. Consequently, they can be isolated by Affinity Chromatography. To accomplish this, a Column is prepared containing dinitrophenyl groups covalently linked to an insoluble carbohydrate matrix. The antiserum is applied to the column and then washed with a buffer solution. Most of the antiserum proteins pass straight through the column because their affinity for both the DNP groups and the carbohydrate matrix is negligible or entirely absent. At the same time, anti-DNP antibodies bind tightly to the column. Next, these antibodies are eluted from the column by applying a high concentration of DNP: the added DNP binds to the antibodies, displacing them from their attachment to the dinitrophenyl groups of the insoluble carbohydrate matrix. The soluble complex consisting of DNP and anti-DNP antibodies emerges from the column. Subsequently, DNP is removed via dialysis or Ion-exchange chromatography, yielding a purified antibody preparation.

33.3. Antibody Antigen-Binding Sites Resemble Enzyme Active Sites

The antigen-binding sites in antibody molecules are in many respects similar to the active sites of Enzymes.

1. Binding constants for haptens have been determined using equilibrium dialysis and spectroscopy Methods. For instance, the binding of a colored hapten such as a dinitrophenyl derivative quenches the fluorescence of Tryptophan residues in the antibody protein. The degree of quenching serves as a measure of the saturation of binding sites in the protein molecule. For most haptens, binding constants range from 10^-4 to 10¹⁰ M. Consequently, the Standard Free energy of binding ranges from -6 to -15 kcal/mol, falling within the typical range for enzyme-substrate and enzyme-coenzyme complexes. Furthermore, hapten-antibody complexes are formed by the same forces that drive enzyme-substrate interactions. A combination of weak non-covalent bonds, such as electrostatic, hydrogen, and Structure/103.html">Van der Waals interactions, ensures tight and specific binding.

2. The ability of dextran antibodies (Polysaccharides consisting of glucose residues) to bind oligomers was tested. It was found that full binding affinity is exhibited toward an oligomer composed of six glucose residues. This naturally invites a comparison with Lysozyme, whose active-site cleft also accommodates six carbohydrate residues. This comparison suggests that the length of the binding site in anti-dextran antibody molecules is 25 A.

3. Spectroscopic properties of a series of haptens can provide insight into the polarity of binding sites within antibody molecules. For example, certain naphthalenes exhibit weak yellow fluorescence in a highly polar environment (such as Water), but intense blue fluorescence in a markedly nonpolar environment (such as hexane). When such a naphthalene hapten binds to a specific antibody, an intense blue fluorescence appears, indicating the expulsion of water from the binding site. Typically, binding sites are nonpolar clefts within the antibody molecule; this enhances antigen-binding strength for reasons already discussed in relation to enzyme-substrate interactions (Section 6.8).

4. The hapten fits the binding site quite precisely in terms of structure. Binding Specificity is undoubtedly very high, though not absolute. The hapten is held firmly within the binding site and thus possesses limited rotational freedom. The association rate constant for many haptens is approximately 10⁸ M^-1 • s^-1.

Such a high constant indicates that The rate of the process is diffusion-controlled. Hapten binding does not appear to be accompanied by major structural rearrangements.

33.4. Antibody Preparations of Defined Specificity Are Generally Heterogeneous

Antibodies differ fundamentally from enzymes: most normal antibodies of a given specificity, such as anti-DNP antibodies, are heterogeneous in their molecular composition. Analysis of the binding of dinitrophenyl haptens to an anti-DNP antibody preparation revealed a whole spectrum of affinity values. Some antibody molecules bind DNP with K = 10^-6 M, whereas others bind with K = 10^-10 M. In contrast, enzymes are typically characterized by a single binding constant for a given substrate or coenzyme. Moreover, Electrophoresis of an anti-DNP antibody preparation or other specific antibodies reveals multiple protein bands. By contrast, enzymes subjected to electrophoresis yield a single band or a small number of discrete bands (for instance, Lactate dehydrogenase Isoenzymes).

The heterogeneity of antibodies with a given specificity is determined by the very Nature of the Immune Response. What is the reason for this heterogeneity? It turned out that antibodies produced by a single Cell are homogeneous. However, different Cells produce antibodies that vary in structure. Antibodies against DNP are synthesized by A large number of different cells, which accounts for their heterogeneity.