INTERPRETATION OF INHIBITIONS BY SH REAGENTS 649 



disappearance of a free SH group. These new groupings have varying sizes 

 and frequently electrical fields. It is quite possible that, all else being equal, 

 a smaller reagent of a particular type may exert less inhibition, due simply 

 to the fact that it exerts less steric hindrance to the catalytic process. Thus 

 the inhibition by methylmercuric chloride may be different from that by 

 p-chloromercuribenzoate for this reason. The groups introduced by iodo- 

 acetate and p-chloromercuribenzoate are negatively charged, whereas those 

 from iodoacetamide and phenylmercuric acetate are uncharged, and this 

 could well be responsible for some of the differences observed between these 

 inhibitors. This is one reason why many studies with SH reagents would 

 profit from a quantitative comparison of the effects of a large number of 

 inhibitors of different types. 



Another factor of some importance may be the influence of the reaction 

 of certain SH groups on the reactions of other SH groups. Further reaction 

 apparently may be either depressed or accelerated. In phosphorylase a the 

 reaction of the first SH group seems to facilitate the combination of the 

 remaining 18 groups with p-chloromercuribenzoate (Madsen and Gurd, 1956). 

 On the other hand, reaction of one SH group on hemoglobin prevents the 

 further reaction of one or two other groups with Ag+, implying that the SH 

 groups here occur in clusters (Ingram, 1955). The number of molecules of 

 SH reagent bound to the protein may thus not be equivalent to the number 

 of SH groups. 



Boyer (1959) has emphasized that insufficient consideration has usually 

 been given to the possible secondary structural changes induced in enzymes 

 by reaction with SH reagents. If some SH groups are unreactive because 

 of steric blocking or chemical combination, these hindrances must be over- 

 come in order to react these groups, and this could imply a modification 

 in the protein structure that in itself might be inhibitory. It has been ob- 

 served frequently that the poorly reacting SH groups are more important 

 in the enzyme structure than the free readily reactive ones. One of the first 

 statements of the importance of structure in the inhibition by SH reagents 

 resulted from work on urease by Desnuelle and Rovery (1949). Phenyliso- 

 cyanate reacts with certain SH groups rapidly but this does not inhibit; 

 inhibition begins when the unreactive SH groups are attacked, and this 

 was attributed to a reversible change in the enzyme structure. Similarly, 

 various properties of aldolase change as the SH groups are progressively 

 reacted with p-chloromercuribenzoate: after 3-5 are reacted, the enzyme 

 begins to be more readily attacked by trypsin; after 10 are reacted, the tur- 

 bidity increases, denoting marked structural changes, and inhibition is ob- 

 served (Szabolcsi and Biszku, 1961). There is a progressive labilization of 

 the tertiary structure, accompanied by appearance of previously masked 

 SH groups, with further reaction and eventual denaturation. In many cases, 

 the initial reactions must reduce the protein stability, perhaps only locally. 



