8 DAVID PRESSMAN 



400 



/ICO 



^°o ^00 eoo 



600 



1250 



I05O 



750 



Fig. 5. Effect of chlorosubstilucnt in indicated position on the free energy of 



combination of (/>'-hydroxyphenylazo)benzoates with antibody. (Reproduced from 



the Journal of the .\merican Chemical Society, 76, 6339 (1954) with permission of 

 the Editor.) 



benzene ring so that the carboxylate group no longer fits the antibody site as 

 it did in the original hapten. 



In correlation with this tilt effect, we find that antibodies against the oiilio- 

 azobenzoate ion show very low interference with substituents in the orlho posi- 

 tion. This is presumably due to the fact that the carboxylate in the hapten 

 against which the antibody was formed is already tilted out of the plane of 

 the benzene ring, so that the antibody formed against this nonplanar carboxyl- 

 ate can accommodate a chlor-substituent in the ortho position. 



So far, I have discussed substituents which exhibit a steric interference on 

 the combination of hapten with antibody. What happens if a substituent is 

 placed on the hapten in the position occupied by the azo group of the injected 

 hapten (position of attachment of hapten with antigen)? Since the antibody 

 was formed against the azo group, it can accommodate other substituents in 

 this position. Indeed, a substituent in this position almost always increases 

 the combining power of the hapten. 



In the case where no hydrogen bonds are formed, one might well expect 

 the greatest interaction to take place with those radicals which have the great- 

 est van der Waals interaction, and this is actually the case. 



Fig. 6 shows the increase of combining power with van der Waals attraction 

 in several systems for the case where the substituent is in the position of attach- 

 ment of the hapten group to the antigen. It can be seen that for each system, 

 the order for strength of combination is methyl < chlorine < bromine < io- 

 dine, which is in the order of the polarizabilities of the groups and indicates 

 an attraction of these groups for the part of the antibody directed toward the 

 azo group. 



The order holds throughout except in the o?-///o-azobenzoate system, where 

 there is the problem of tilt. As the substituent becomes larger, there is a tend- 

 ency toward decreased combination due to increased tilt of the carboxyl on 

 the one hand and a tendency toward increased combination due to polariza- 



