Metabolism and mode of action 



Measurements of sulphydryl disappearance 



The reactivity of sulphydryl with a-^ unsaturated ketones was first 

 described by Posner (1902). Dickens (1933) established the nature of the 

 reaction of iodoacetic acid with sulphydryls, namely that the iodine was 

 displaced by the sulphur resulting in the formation of a thio-ether. Geiger and 

 Conn (1945) published some preliminary evidence that the activity of some 

 antibiotics might be destroyed by cysteine, and in the same year Cavallito 

 and Haskell (1945) published additional evidence for the reaction of some 

 other lactones with cysteine. In no case, however, has the disappearance of 

 the sulphydryl groups been measured quantitatively. 



A simple system was used to follow sulphydryl disappearance. One- 

 millilitre aliquots containing various concentrations of growth substances and 



2 3 V- 5 



Concn. of iodoacefate 



exIO ^M 



Figure 4. Cysteine disappearance with iodoacetate. 

 Volume ; 1 • 1 ml. 

 Incubation time : 6 hrs. 

 Reactants adjusted to pH 6-5 ±0-3. 

 Free sulphydryl estimated by nitroprusside assay following dilution with 3-0 ml sat. NaCl. 



including the sulphydryl compound cysteine were incubated in vacuo at 

 approximately 28°C for 6 hours and the amount of free sulphydryl remaining 

 was then measured by the nitroprusside test of Grunnert and Phillips (1951). 



We proposed to begin following the disappearance of free sulphydryls in the 

 presence of a model sulphydryl inactivating agent. We selected iodoacetic 

 acid as the model substance and cysteine as the sulphydryl. The disappearance 

 of sulphydryls with iodoacetate is presented in Figure 4. It can be seen that 

 for each mole of iodoacetic acid there was a disappearance of approximately 

 one-tenth of a mole of sulphydryl in 6 hours. 



Turning to another halogenated acid, Figure 5 shows the results obtained 

 with TIBA. It is evident at once that this compound is at least as active as 

 iodoacetate. For each mole of TIBA added approximately one-half of 

 a mole of sulphydryl disappeared. The apparent reaction between TIBA and 

 cysteine was followed in time as shown in Figure 6. The slow decrease of the 

 sulphydryls in the control was presumably due to auto-oxidation. 



If TIBA forms aryl-thio-ethers analogous to those formed by iodoacetate, 

 it is interesting to compare the relative — SH activity of halogenated acetic 



274 



