EFRAIM RACKER 



as acetylations, oxidations, reductions. The workers in this field 

 have been fully aware of the diffculties of this approach and have 

 emphasized the importance of demonstrating the purity of the 

 new protein derivative and, if possible, the reversibility of the 

 induced modification. Extensive acetylation of pepsin (13) 

 resulted in loss of enzyme activity which was fully restored after 

 removal of the acetyl groups. Among other types of modifica- 

 tions, two of more recent data may be quoted. Diisopropyl 

 fluorophosphate (DFP) inhibits certain esterases as well as 

 proteolytic enzymes with esterase activity, whereas other 

 enzymes appear unaffected {cf. ref. lb). Cholinesterase and 

 chymotrypsin are completely inactivated after reacting with one 

 mole of inhibitor per mole of enzyme. From the inactive 

 proteins, 0-phosphoryl serine was isolated after hydrolysis (40). 

 Serine is therefore strongly implicated as part of the active site, 

 provided steric hindrance, which will be discussed below, can 

 be ruled out. Since an acyl shift from nitrogen to the hydroxyl 

 group of serine may have occurred secondarily during the pre- 

 parative procedure, the hydroxyl group may not be the primary 

 reactor {cf. ref. 6a), It is significant that no phosphothreonine 

 was found in the hydrolyzates. What determines the specificity 

 of the interaction between DFP and enzyme-serine is unknown. 

 Free serine or enzyme-threonine does not react with DFP. 

 Perhaps sequence analysis of peptides containing the radioactive 

 phosphate of DFP^^^ obtained after partial hydrolysis, may yield 

 information on this point. The biological reactivity of hydroxy 

 amino acids has been recently demonstrated by an entirely 

 different approach. A rapid incorporation of labeled inorganic 

 phosphate (P^^) was shown to take place into the phospho- 

 protein fractions from which phosphorylated hydroxy amino 

 acids were isolated (1,18). 



The susceptibility of TDH to iodoacetate (lAA) has been 

 known for many years. Since it has been shown (23) that TDH 

 contains firmly bound glutathione (GSH), which can be liberated 

 by proteolytic enzymes, the interaction with lAA was reinvesti- 

 gated (24). A very rapid reaction was found to take place 



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