Binding Properties of Immunoglobulins

For reasons discussed in detail by Singer (1971) the current concept of membrane structure is that of a lipid bilayer intercalated with proteins, referred to as the fluid mosaic model (Fig. 11). It is based primarily on thermodynamic arguments (Tanford, 1973) concerned with the fact that it is energetically favorable for the ionic portion of an amphipathic substance to be in direct contact with water while its hydrophobic tail is sequestered from water and interacts with other nonpolar molecules. The role of hydrophobic interactions in protein conformation was first emphasized by Kauzman (1959). It is not our purpose here to review the area of membrane structure (see articles by Singer, 1971 Tanford, 1973), but certain points are important to the discussion of effector functions. 

The average amino acid composition of membrane proteins does not show a clear difference from that of soluble proteins (Woodward and Munkres, 1966 Engelman and Morowitz, 1968), and yet membrane pro- 

The lipid layer exhibits a fluidity allowing molecules to diffuse laterally in the plane of the membrane but not to rotate from one surface of the membrane to the other. This fluidity is temperature dependent, diminishing at lower temperatures. Membranes vary in their protein, lipid, and carbohydrate content, and it appears that the protein content in particular increases as the specialization of the membranes increases (Guidotti, 1972). In general, the percentage of protein varies from 18% 

There are two populations of lymphocytes differing in function and mode of development bursal derived, or B cells, and thymic dependent, or T cells. In general, B cells are responsible for humoral immunity, and the T cells mediate cellular immunity (delayed hypersensitivity), although T cells also cooperate with B cells in antibody formation (Katz and Benacerraf, 1972). 

Several hypotheses have been suggested to explain the occurrence of IgM and IgD as both secreted (aqueous-soluble) and membrane-bound proteins. Any explanation must account for the fact that membrane immunoglobulins which have been extracted with detergent are insoluble in physiological buffers in the absence of detergents (or some other dena-turant) i.e., membrane IgM and IgD are to a degree hydrophobic (Melcher et aL, 1975) while the secreted form is not. Possible explanations which have been suggested are that (1) the two forms differ in sequence, (2) a separate insertional piece is added to the membrane form, or (3) the conformation of the membrane-associated position of the molecule differs from that of its soluble counterpart. The membrane-favored conformation would have to be maintained in a polar medium to 

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SpA-derived proteins containing different number and composition of domains have been produced by recombinant methods. The binding properties of intact SpA and some SpA-derived protein fragments to IgG, IgA, IgM, and F(ab )2 have been analyzed. The results suggest that the binding is affected both by the number of immunoglobulin binding domains and by the composition of the domains. 

Size exclusion chromatography (or gel filtration ) has a unique set of attributes which sets it apart from other methods. Its applications are limited because it has low resolution, low capacity, and is slow. However, it is the only practical separation method based on size, whidi is a relatively invariant property of immunoglobulins in contrast to charge, hydrophobidty, and other specific binding interactions. The resolution achieved by SEC may not be spectacular however separations are predictable and nearly independent of buffer conditions. so the need for method development is minimal. 

Akerstrom, B. and Bjorck, L., A physiochemical study of protein G, a molecule with unique immunoglobulin G-binding properties, J. Biol. Chem., 261, 10240-10247, 1986. 

In this context, Greene and coworkers [159, 160] have reported the first low-molecular-mass immunoglobulin mimetic 207, Scheme 62, developed on the basis of an X-ray structure analysis of the antigen-antibody complex. Compound 207 is resistant toward proteases and imitates the binding and functional properties of the native antibody. 

There is no one procedure or combination of procedures that is applicable to all cases. The class of antibody to be purified has to be considered. Conventional procedures for the production of polyclonal antisera will generally result in immunoglobulin G, and procedures for the generation of monoclonal antisera may result in any of the immunoglobulin classes. Immunoglobulins from different species have broadly similar properties, but protein A, for example, will not bind all of the subclasses of human IgG, nor is it particularly effective in the purification of IgG from rat, sheep, or goat (2). Thus a certain amount of trial and error may be required to reach the best protocol for a particularly demanding application, but the methods described here should provide an adequate purity for most purposes. 

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