SALT COXCEXTRATION AND IONIC STRENGTH 817 



at 37°, where y is the ionic activity coefficient, z is the charge number for 

 the ions (usually unity for most enzyme work), s is the ionic strength, /„ 

 is the closest distance of approach of two oppositely charged ions, and K^^ 

 is the salting-out constant (pertaining to the nonionic portions of the mole- 

 cules and related to the change in the dielectric constant). The acid disso- 

 ciation constant for a group in a salt solution can then be written as: 



pX. . pi... - ^^^^^ ,15-69, 



" 1 + 0.327ro V s 



where p^^ is the dissociation constant when the ionic strength is zero. If 

 we assume unit charges on the ions and )\ = 4 A, the change in the \>K^ 

 upon changing the ionic strength from s^ to s^ would be given by: 



zlpZa = 0.509 



Vs, V" 



1 + 1.31 Vsi 1+1.31 \/s2 . 



;i5-70) 



Addition of KCl or NaCl to increase the ionic strength from a physiological 

 value of 0.16 to 0.5 would decrease the p^^ by 0.053 on this theoretical basis. 

 The piC^ of acetic acid in NaCl solutions at 25° falls from 4.527 to 4.479 

 when the ionic strength is increased from 0.16 to 0.5 and thus in this case 

 Zlp^a = 0.048. The piC^'s of phosphoric acid have been found experimen- 

 tally to obey an equation of the form, p-K", = A — B y s , where the con- 

 stants A and B have different values for the three ionizations: A^ = 2.12 

 and 5i = 0.467, A^ = 6.944 and B.^ = 0.562, and A^ = 12.185 and B^ = 

 1.494 (Elliot et al., 1958), and it may be expected that most acidic ioniza- 

 tions will take either this form or that of Eq. 15-69. It is seen that JvK^ 

 is never very large in the range of ionic strength usually used but if the piC^ 

 is close to the experimental pH of the enzyme medium, an appreciable al- 

 teration of the equilibrium may be induced. Not only will the local charge 

 pattern on an enzyme be changed but the total protein charge will usually 

 be shifted by a modification of the ionic strength, and this can have signi- 

 ficant effects on the association of substances with the enzyme as discussed 

 in Chapter 6. It is well known that the electrophoretic behavior of proteins 

 is modified by the salt concentration, reflecting changes in the over-all 

 protein charge. This is an expression of the fact that the isoionic point is 

 a function of the ionic strength. In addition, increase in the ionic strength 

 reduces the mobility by the effect of the ionic atmosphere on the potential. 

 It is quite possible in certain instances that a change in salt concen- 

 tration could alter the protein structure by affecting the normal forces 

 between groups on the polypeptide chains or by changing the hydration 

 of the proteins. In those proteins or enzymes where association between 

 units occurs, this may also be modified by salt concentration, and if the 

 enzyme activity or the susceptibility to inhibition varies with this associa- 



