Chemical modification of immunoglobulins and the effects on antigen binding site affinity

(Uncorrected OCR) Abstract of thesis entitled CHEMICAL MODIFICATION OF IMMUNOGLOBULINS AND THE EFFECTS ON ANTIGEN BINDING SITE AFFINITY submitted by CHAN Lui Sek for the degree of Doctor of Philosophy at the University of Hong Kong in September 1993 Modified antibodies are in demand both for their intrinsic scientific interest and as articles of commerce, chiefly for use in analytical laboratories. Direct chemical modification has been relatively neglected and the objective of present study is to determine whether such modification of amino acid residues accessible on the surface of the molecule has any effects on hapten-binding of rabbit anti-DNS IgGs ( the hapten used was a fluorescent dye, DNS-lysine ). The principle was to induce covalent chemical modifications of different types of antibody amino acid residues ?? tryptophan by 2-hydroxy-5-nitrobenzylbromide, tyrosine by tetranitromethane, lysine by acetic anhydride and glutamate by 1-ethyl-3-(2-dimethylaminopropyl)-carbodiimide and glycine ethyl ester ?? in such a way that largely only the surface amino acid residues, outside the active site of the antibody molecule, would have been modified. The modified antibodies were then subjected to hapten-binding experiments designed to detect subtle changes in intrinsic affinity constant. The method of fluorescence enhancement was employed for this particular purpose because the method is highly sensitive for affinity measurements whilst measurement errors arising from non-specific binding or non-specific protein impurities are very low, and because results could be i obtained within a short period of time. The results suggested that surface-located hydrophobic residues rather than surface-located charged residues of the rabbit anti-DNS IgGs have more critical influence on the intrinsic affinity of the antibody. For example, just one tyrosine/IgG or just 1.5 tryptophan/IgG being chemically modified caused a degree of reduction in the antibody intrinsic affinity similar to 8 glutamate or aspartate/IgG or 40 lysine/IgG being modified, i.e. from the normal value of 1.2 x 108 M-1 down to 0.6 x 108 M-1 after modification. This is probably because surface hydrophobic residues are more important in maintaining the proper conformation of the Fab arms ( and therefore the conformation of the active sites ) of the anti-DNS IgG molecule, because they play an important role in the antibody conformation in solution. They might make certain non-covalent contacts between the Fab arm and the Fc part of the antibody molecule or with residues of the complementarity-determining regions in the active site of the antibody molecule and thus alter the functional conformation of the anti-DNS active site. ii

並列關鍵字

Immunoglobulins. ； Antigen-antibody reactions. ； Binding sites (Biochemistry)

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