650 4. SULFHYDKYL REAGENTS 



and this spreads and progresses rapidly as further groups are attacked, just 

 as in other types of denaturation. The blocking of the SH groups of phos- 

 phoglyceraldehyde dehydrogenase changes the optical rotation and the in- 

 trinsic viscosity, the latter increasing linearly with the equivalents of p- 

 chloromercuribenzoate reacted (Elodi, 1960). Reversibility with cysteine 

 varies with the time of exposure to the mercurial, at first the effects being 

 completely reversible and eventually irreversible, again indicating a pro- 

 gressive breakdown of the protein structure. Elodi postulated three phases: 

 (1) a reversible reaction with certain SH groups, (2) an unfolding of the 

 polypeptide helices as a result of the alteration of the SH groups, and (3) 

 precipitation due to intermolecular bridges formed between the new groups 

 appearing on the protein surface. Ribonuclease is perhaps another example 

 of structural changes resulting from the scission of disulfide bonds, of which 

 there are 4 in the native enzyme: breaking 1 does not alter the activity, 

 breaking 2 inhibits about 20%, breaking 3 inhibits about 40%, and then 

 suddenly the activity drops to zero as the last disulfide is split (Resnick 

 et al., 1959). The — S — S — bonds were believed to be of importance in pro- 

 viding stability to the secondary structure of the enzyme, their breaking 

 leading to progressive unfolding. 



PROTECTION AND INHIBITION REVERSAL BY THIOLS 



Some of the problems involved in protection and reversal experiments 

 with SH reagents and thiols were discussed at some length in Volume I 

 (pages 622-626). A few of the conclusions reached there will be briefly sum- 

 marized. (1) Protection or reversal by a thiol depends on the relative affin- 

 ities of the SH reagent for the enzyme SH groups and the thiol, and the 

 relative concentrations of the components, and hence every degree of re- 

 versibility of SH-inhibited enzymes may be observed. (2) Protection or 

 reversal by a thiol does not provide conclusive information on the mecha- 

 nism of the inhibition or the enzyme groups attacked. (3) Irreversibility is 

 brought about not only by very tightly bound reagents, but by progressive 

 structural changes in the enzyme, as discussed above. This type of irrever- 

 sibility thus increases with the concentration of the SH reagent and the 

 time of exposure. (4) The amount of useful information relative to the 

 mechanism of inhibition obtained from such studies is much less then 

 commonly believed. 



The stability of the product formed by reaction of an enzyme SH group 

 with an SH reagent varies with many factors, most of which have been 

 mentioned in connection with the differential reactivity of these groups. 

 Sometimes the product is completely stable for all experimental purposes 

 and the reacted enzyme is permanently altered; such would be the case 

 with most of the alkylating agents. Then the mercaptide complexes vary 



