768 7. MERCURIALS 



since the amount bound to bovine seralbumin increases with the dye con- 

 centration until the molar ratio of dye to protein is 0.66, following which 

 no more is bound although the dye concentration is increased 50-fold. This 

 ratio corresponds quite closely to the known SH content of the protein, 

 lodination of the seralbumin prevents the reaction with the mercurial. 

 Ovalbumin reacts readily with two of its SH groups, slowly with a third, 

 and more slowly with the fourth, the dye perhaps differentiating the rel- 

 ative reactivities more closely than does p-MB. This method has a high 

 sensitivity and can be used for very low concentrations of protein. 



A more recently examined mercurial dye, 4-(p-mercuriphenylazo)-l-naph- 

 thylamine-7-sulfonate, must also be dissolved in glyine buffer (Nosoh, 1961). 

 Absorption at 470 m// is determined and the titration of glutathione and 

 proteins appears to be quite satisfactory. 



INHIBITION OF ENZYMES 



The early concept of the mercurials as nonspecific denaturing and coagu- 

 lating agents for enzymes has gradually been abandoned in favor of a pic- 

 ture in which definite and often isolatable mercurial complexes are formed 

 under the proper experimental conditions. A selective reaction with SH 

 groups on enzymes is now generally assumed and the mercurials are exten- 

 sively used for the detection of these groups. The possibility of reaction with 

 other than SH groups has been discussed (pages 737 and 753) and should 

 never be ignored. We shall note instances in which a selective action on SH 

 groups is well established, and a few examples of inhibition not involving 

 SH group. We shall also see that mercurial inhibition does not necessarily 

 imply an SH group within the active center or the participation of an SH 

 group in the catalysis. In this connection, it is well to bear in mind the dif- 

 ferent groups which are introduced on the surfaces of enzymes when the 

 different mercurials are used (Fig. 7-10), inasmuch as the steric and elec- 

 trostatic effects of these side chains may be critical in producing inhibition. 



Crystalline Mercuri-enzymes 



The crystallization of the mercuric derivative of mercaptalbumin was 

 not the first instance of such a procedure. Warburg and Christian (1941, 

 1942) introduced this technique for the isolation of fermentation enzymes 

 and obtained the crystalline Hg-enolase complex from yeast, whereas the 

 normally active Mg-enolase could not be crystallized. Kubowitz and Ott 

 (1941) in Warburg's laboratory also crystallized the Hg++ complexes of 

 lactate dehydrogenases from Jensen sarcomata and rat muscle. The Hg++ 

 complexes in all cases are enzymically inactive, but dialysis against cyanide 

 solution removes the Hg++ and restores the activity. There is no better 

 evidence for the homogeneous, stoichiometric, and reversible Hg++ deriva- 



