TETRATHIONATE 69? 



its effects in complex systems more difficult to interpret. The first use of 

 tetrathionate as a reagent for protein SH groups was by Anson (1941), who 

 demonstrated that it would titrate denatured ovalbumin, although the reac- 

 tion is slower than with ferricyanide, porphyrindin, or p-chloromercuriben- 

 zoate, not being complete in 3 min at neutrality. It has not been used ex- 

 tensively for this purpose, but it may well be a valuable reagent in certain 

 types of work; a detailed description of the method is given by Chinard 

 and HeUerman (1954). 



Chemistry and Reaction with SH Groups 



Sodium tetrathionate is prepared from the thiosulfate by oxidation with 

 iodine in 90% ethanol. The precipitate is purified by redissolving it in an 

 equal weight of water and filtering it into absolute ethanol, in which it re- 

 precipitates. It is washed with ethanol and dried in vacuo. Sodium tetra- 

 thionate crystallizes with 2 waters of hydration. When it is kept at 0° in 

 the dark, both the solid and 0.1 M solutions are stable for many weeks 

 (Pollock and Knox, 1943), but it is unstable when kept under ordinary 

 conditions. For all accurate work it is necessary to be certain that it is free 

 of appreciable thiosulfate and other impurities, and it should be recrystal- 

 lized as above. 



Tetrathionate rapidly oxidizes simple thiols, such as cysteine, homocys- 

 teine, and glutathione, according to the reaction: 



2 R— SH + S^Og" ^ R— S— S— R + 2 S^Og" + 2 H+ 



In titrations of SH groups the thiosulfate is determined iodometricaUy. 

 According to Baernstein (1936), it is specific for SH groups and does not 

 react with other amino acid groups. It is not a strong oxidant, since the 

 standard oxidation-reduction potential for the reaction 



2 §203= ±? 8406= + 2 e- 



is + 0.08 V. Although it has always been assumed that tetrathionate oxi- 

 dizes SH groups to disulfide, Pihl and Lange (1962) have obtained evidence 

 that sulfenyl thiosulfate groups may be formed: 



R— SH + 8406= -> R— 8— 8^03= + 8^03= + H+ 



Incubation of phosphoglyceraldehyde dehydrogenase with tetrathionate-S^^ 

 leads to the appearance of S^^ bound to the protein, and the binding of each 

 S^^ is associated with the disappearance of one SH group. 



One of the few thorough kinetic studies of SH group oxidants was made 

 by Goffart and Fischer (1948). It was shown that tetrathionate oxidizes 

 protein SH groups more slowly than cysteine or glutathione (Fig. 5-1). 



