GENERAL CONSIDERATIONS OF THE USES OF SH REAGENTS 651 



greatly in stability, so that in some instances they can be split at a rate 

 too rapid to be technically measurable, while in others the rate is too slow 

 to measure. It is not necessary that this stability be correlated with struc- 

 tural changes in the enzyme or irreversible inactivation. The inability to 

 reactivate an enzyme inhibited by an SH reagent can be attributed to a 

 variety of factors, some of which are listed below. 



(A) The binding of the SH reagent to the enzyme is stronger than to the 

 reversor; one must use the proper reversor and concentration (e.g., dimer- 

 caprol will reverse some inhibitions untouched by cysteine). 



(B) The enzyme is chemically altered by the SH reagent so that it is not 

 a question of a tightness of binding; reversal can occur only by a chemical 

 transfer of the attached group to another radical. 



(C) The enzyme is structurally altered irreversibly (denatured) by the 

 blocking of the SH groups. 



(D) The SH reagent has caused a splitting off of some coenzyme or co- 

 factor, which must be added back following restoration of the SH group 

 for activity to be evident. 



(E) The reversor may in some manner inhibit the enzyme, even though 

 restoring the free SH groups initially, as when the SH groups are oxidized 

 by disulfides formed from the oxidation of the added thiols. 



In order that irreversibility be correctly attributed to protein denaturation, 

 these other possibilities must be ruled out. Complete reversibility is more 

 easily interpretable, and one can quite confidently say that at least no per- 

 manent derangements in the enzyme structure have been induced by the 

 SH reagent. 



Protection experiments, in which some thiol is added previous to, or with, 

 the SH reagent, are, as has been emphasized earlier, of little value, since all 

 one is doing is reducing the concentration of the free SH reagent (assuming 

 that it reacts with the thiol). Actually, it is a little difficult to speak of this 

 as protection, inasmuch as one usually would not call a reduction in inhib- 

 itor concentration a type of protection. It is very unlikely that worthwhile 

 information can be obtained from such experiments, aside from the practical 

 determination of the ability of substances to reduce the toxic effects of SH 

 reagents. 



GENERAL CONSIDERATIONS OFTHE USES OF SH REAGENTS 



Numerous types of reagent are available for satisfactorily specific reaction 

 with SH groups but no single one is adequate for all purposes. The most 

 useful information on the nature of enzyme SH groups, their locations and 



