760 7. MERCURIALS 



may be better, appreciated. Third, it presents one clear instance in which 

 mercurials react specifically with protein SH groups, since there is no evi- 

 dence that other groups are even involved in the mercuration of mercaptal- 

 bumin. Last, it is significant in the use of mercurials in whole animals that 

 the serum contains sufiicient mercaptalbumin to bind most, if not all, the 

 mercurial present, a factor that must be considered in the penetration, dis- 

 tribution, and actions of the mercurials in animals. 



Effect of pH 



The competition between H+ and the mercurial for the S" group and the 

 effect this has on the over-all equilibrium have been discussed. On the basis 

 of only the ionization of the SH group, one would predict that mercurials 

 would react more rapidly and more completely at higher pH's (particularly 

 above p^^'s of the SH groups). However, there are many other factors 

 which may be important. Actually, it has generally been observed that the 

 rate of reaction of p-MB with proteins is decreased with a rise in the pH. 

 Both the extent and rate of reaction of p-MB with ovalbumin are affected 

 by pH: At pH 4.6, 4 moles of p-MB react rapidly with 1 mole of protein, 

 whereas at pH 7, only 3.2 moles of p-MB react in 24 hr (Boyer, 1954). 

 Reduction of the rate with increasing pH has also been reported for /5- 

 lactoglobulin and 3-PGrDH (Boyer and Segal, 1954), it being much faster 

 at 4.6 than at 7. The work of Huisman (1959) with hemoglobin illustrates 

 an important point; the rate and extent of reaction may be influenced dif- 

 ferently by pH, inasmuch as the rate of mercaptide formation is faster be- 

 tween 7 and 11.2 than at 4.6, but more SH groups are reactive at 4.6. One 

 also recalls that p-MB is dissociated more rapidly from ovalbumin at pH 

 7.9 than in the acid pH range (MacDonnell et al., 1951). Another factor 

 often overlooked is the effect of pH on the secondary denaturation of the 

 protein following mercuration. The rate of thermal denaturation of seral- 

 bumin is increased 13.2-fold at pH 3.6, while at pH 7 there is no denatura- 

 tion by Hg++ at 1.85 mM, while denaturation of /?-lactoglobulin is increased 

 89-fold at pH 3.6 and not at all at pH 7, this indicating that an acid pH 

 favors secondary configurational changes resulting from binding of the Hg++ 

 (Stauff and Uhlein, 1958). The reaction of mercurials with non-SH groups 

 is also pH-dependent, since the complexing of the azomercurial studied by 

 Horowitz and Klotz (1956) with glycine is maximal between pH 6.5 and 

 9.5; at low pH's the +H3N — CH2 — C00~ form of glycine dominates and 

 is less reactive, while at high pH's there is competition by 0H~. 



R-Hg-00C-CH3 + H2N-CH2-COO- ±? R-Hg-OOC— CH2-NH2 + CH3-COO- 



The following factors, in addition to the ionization of SH groups, must 

 thus be considered. ( 1 ) The pH may vary the number of reactive SH groups 



