3] CONFIGURATION OF GLOBULAR PROTEINS 47 



The effect of ionic strength on interaction energies, for impenetrable 

 models such as these, is quite small. This is shown by the calculation of this 

 effect for model A. It should be noted, incidentally, that the choice of im- 

 penetrable models is not an arbitrary one. As already mentioned, it is forced 

 on us by the small experimentally observed effect of ionic strength on titra- 

 tion curves of compact proteins.^' 



Table 2 indicates that, whatever the forces stabilizing a compact con- 

 figuration near the isoelectric point, electrostatic interaction is the principal 

 force which makes such a configuration unstable as one moves away from 

 the isoelectric region. For the strength of 'hydrophobic' bonding, of peptide 

 hydrogen bonds and of disulfide bonds is not affected at all by pH changes. 

 Hydrogen bonds between side chain groups and intramolecular ion pairs 

 are affected, since titration of the side chain groups involved in such bonds 

 would lead to their weakening or rupture. However, all the energy contri- 

 buted in this way to a shift in the free energy balance between a compact 

 and an extended configuration must be reflected by an exactly equal anoma- 

 lous free energy of ionization of the groups concerned. Whether such 

 anomahes in fact occur, is debatable. If one assumes a uniform density of 

 charged sites on the surface of the protein molecule, then anomalies certainly 

 exist. ^2. 86 jf^ Qjj ^jje other hand, one allows for reasonable variation from 

 one protein to another in the relative locations of charged sites, then such 

 apparent anomahes can be explained on the basis of electrostatic interaction 

 of these sites with the acidic or basic groups under consideration.^" In any 

 event, any instabihty contributed by hydrogen bond rupture would be an 

 addition to and not a substitute for the instabihty due to electrostatic forces. 



It should be noted again, however, that, though Coulombic forces prob- 

 ably provide the principal basis for the thermodynamic instability of a com- 

 pact configuration, hydrogen bonds may block the kinetic pathway to a 

 configurational change. It is not impossible to visualize mechanisms by 

 which a change in configuration is acid- or base-catalysed so that a change 

 in pH per se may affect the rate at which a change in configuration can occur. 



Before considering the effect of pH on globular proteins, it is of interest 

 to examine its effect on a simple synthetic polyelectrolyte, such as poly- 

 methacryhc acid. This polymer is isoelectric when all of its carboxyl groups 

 are un-ionized. It then has no charges at all, and is randomly coiled. When 

 it becomes charged, by ionization of the carboxyl groups, Coulombic repul- 

 sion sets in. Relatively compact configurations, such as that represented by 

 Fig. \b, acquire a much higher electrostatic interaction energy than relatively 

 extended configurations, such as that represented by Fig. \d. The average 

 configuration therefore becomes much more extended, and this is reflected 

 by corresponding changes in viscosity, sedimentation coefficient, etc. (A 

 theoretical treatment of this effect has recently been presented by Harris 

 and Rice.^*'^' The same authors have also considered polyampholytes with 

 equal numbers of acidic and basic groups. ^^) Experimental verification of 



