INTERACTIONS OF HAPTENS WITH ANTIBODIES 



283 



chlorine atom (0.6 A) results in 1.22 kcal/mole less binding energy in the 

 o-position and the larger groups, such as — I or — NO.,, produce an even 

 greater effect. The origin of this effect may be sought in three possible 

 contributions: (1) the substituent group may prevent the adjacent parts 

 of the hapten molecule from approaching as closely to the antibody as 

 with the unsubstituted compound, (2) the substituent group may tilt the 

 plane of the benzene ring so that it and the planar carboxylate group are 

 not oriented for maximal interaction, and (3) the substituent group may 

 dilate that region of the enclosing antibody, a process requiring energy. 

 The important conclusion is that binding will be increased by a substituent 

 group only when there is space for it to occupy. Such large steric energies 



Table 6-21 

 The Effect of Substituents on Ben zo ate Interaction with the Antibody 



TO 29-(2>'-AzOPHENyLAZO)-BENZOATE '^ 



" From NisonofE and Pressman (1957). 



indicate that hapten-antibody interactions must generally occur within 

 cavities or trenches. The effects of substitution on interactions with a re- 

 latively flat protein surface would be expected to be of lesser magnitude. 

 With respect to position effects, it is worthwhile mentioning that separa- 

 tion of the benzene ring from the charged group by one — CH.,— group 

 results in loss of binding. Thus cp — CH2— AsOgH" is not bound to the an- 

 tibody for p-azophenylarsonate, whereas cp— AsOgH" is readily bound 

 (Pressman, 1957). Likewise, cp — CH^— COO" is not bound to antibody 

 for p-(p'-azophenylazo)-benzoate, while cp— COO" is bound (Nisonoff and 

 Pressman, 1957). Such increased separation would result in displacement 

 of the benzene ring or the charged group or both from the closely fitting 

 antibody region. The benzene ring is necessary for hapten binding, smce 

 Ko' is zero for both CHg-AsOgH" and CHg-COO", indicating that the 

 ring contributes at least 2.5 kcal/mole to the total binding energy. The 

 antibody to the p-azophenyltrimethylammonium group reacts well with 



