784 7. MERCURIALS 



strate, for example, does not necessarily imply that the substrate reacts 

 with an SH group nor that the SH group is involved in the catalysis, al- 

 though these may well be the case. A positive result is more valuable than 

 a negative one. The failure to achieve protection may be due to an inade- 

 quate concentration of the protector, too low a relative affinity of the en- 

 zyme for the protector, or a long incubation wherein equilibrium is reached, 

 and yet the substance examined may participate in the reaction and inter- 

 act with the enzyme in the same way as effective protectors. Definite pro- 

 tection allows one to make the reasonable assumption that the mercurial 

 binds somewhere in the region occupied by the protector. Potter and Du- 

 Bois (1943) postulated an SH group to be located between the two cationic 

 groups binding succinate to the dehydrogenase; protection against mercuri- 

 als by succinate simply implies that succinate is able to shield this SH 

 group from the mercurial, and not that the SH group is involved in the 

 succinate binding or participates in the oxidation-reduction reaction. For 

 steric reasons, the smaller the molecular sizes of the protector and the mer- 

 curial, the more certain can one be that a common SH group is involved 

 in the binding of both. 



Displacement of Coenzymes and Cofactors from Enzymes 



Closely allied to protection experiments are those in which a mercurial 

 is shown to dissociate an enzyme-coenzyme or enzyme-cofactor complex. 

 It is now believed that several coenzymes and metal ion activators may be 

 bound to apoenzymes through SH groups in part (Shifrin and Kaplan, 

 1960), and if this is so one would expect tightly bound SH reagents, such 

 as the mercurials, to displace the coenzymes or activators. Certain coen- 

 zymes or cofactors, such as NAD, have been shown to react with thiols 

 (van Eys and Kaplan, 1957 b), but in most cases the evidence for binding 

 to enzyme SH groups is circumstantial. Certainly such displacement of 

 necessary components of the enzyme reaction would be an important 

 mechanism in the inhibition produced by mercurials, especially in vivo where 

 the total binary or ternary complexes usually occur. One molecule of crys- 

 talline horse liver alcohol dehydrogenase binds 2 molecules of NADH at 

 physiological pH and this is accompanied by a shift in the absorption 

 spectrum of the NADH. Addition of p-MB was found by Theorell and 

 Bonnichsen (1951), to reverse the spectral shift, and it was concluded 

 that the bond between an enzyme SH group and the NADH pyridine 

 ring is broken by the mercurial. However, some doubts have recently been 

 cast on this simple interpretation. The liver alcohol dehydrogenase mol- 

 ecule has 28 SH groups as determined by p-MB titration; the presence 

 of NADH does not reduce this number, although NADH protects the en- 

 zyme moderately (Witter, 1960). On a rather tenuous basis, Witter postulat- 

 ed that the function of the SH groups is to maintain the stable enzyme 



