652 4. SULFHYDRYL REAGENTS 



relation to the catalysis, can be obtained by the proper use of several types 

 of SH reagent. It is also advisable to use different concentrations of the 

 reagents (it is not very informative to report that 1 mM of some SH reagent 

 inhibits 100%) and calculate a K, that characterizes the potency of the in- 

 hibition and the affinity of the enzyme for the reagent. In order to present 

 the kinetics properly, it is necessary to determine if the inhibitions under 

 the experimental conditions used are reversible, and for this purpose it is 

 best to perform the reversal study in nitrogen. It is also useful to determine 

 the degree of inhibition and the number and type of SH groups reacted 

 simultaneously in order to correlate reactivity and relationship to the active 

 center. Finally, at least some simple rate studies should be done to deter- 

 mine if the inhibitions observed are for equilibrium conditions. In many 

 reports one finds only that the enzyme was incubated with the SH reagent 

 for a certain period (even this information is frequently omitted) and it is 

 impossible to determine if the inhibition observed is maximal or not. SH 

 reagents have perhaps been used during the past several years more com- 

 monly than any other type of enzyme inhibitor and yet they have been 

 used with little concern for the many complexities involved in the interpre- 

 tation of the results, with a few notable exceptions. 



Despite the generally good specificity of these reagents for SH groups, 

 they are not specific inhibitors from the metabolic standpoint in most cases. 

 Since SH groups are present not only in many enzymes but in most other 

 proteins of the cell, one must expect that in complex systems there will be 

 many components reacted. Whether some of these reactions will be of im- 

 portance in what is measured will depend on the nature of the work. It is 

 probably justifiable to suggest that the use of most SH reagents be restrict- 

 ed at the present time to enzyme studies for the purpose of determining the 

 nature of the enzyme SH groups. As the complexity of the system increases, 

 the value of SH reagents diminishes, at least if one is trying to correlate 

 some enzymic or metabolic process with over-all cellular metabolism or 

 function. In this connection it is interesting to note that although the most 

 reactive SH reagents are generally best for pure enzyme work, this is not 

 necessarily true for more complex systems. What one usually requires in 

 metabolic or functional investigations is specificity with respect to a partic- 

 ular enzyme or metabolic pathway. Thus iodoacetate, although it is a rather 

 poor reagent for the detection of SH groups and has frequently been ma- 

 ligned for this purpose, is actually more valuable in cellular work than most 

 of the others since it has the ability, if used properly, of inhibiting the phos- 

 phoglyceraldehyde dehydrogenase and glycolysis without affecting other 

 systems significantly, whereas a more reactive inhibitor, such as p-chloro- 

 mercuribenzoate, is valueless for producing specific metabolic blockade. The 

 choice of SH reagent to be used should always be made on the basis of the 

 type of work to be done. Another factor to be considered in work with eel- 



