Oxidation of Cystine Sulfur to Sulfate by Ferric Chloride

Abstract

The oxidation of thiol-imidazole sulfur to sulfate by FeCl₃ is frequently used in the conversion of thiol-imidazole compounds to imidazoles. One would assume from the literature that cystine sulfur is not similarly oxidized by this reagent. 1 This suggested the utilization of this reaction as a means of testing for the possible presence of a thiol-imidazole compound in insulin. So far only about 70% of the sulfur of insulin has been accounted for. The highly specific Sullivan reaction indicates the presence of about 8.4% cystine, whereas if all the sulfur of insulin (3.2%) were present as cystine, 12.0% should be present.

To test the feasibility of this approach experiments with cystine were first undertaken. These experiments showed contrary to our expectations that cystine sulfur was quite readily oxidized to sulfate by FeCl₃. Twenty mg. of cystine heated with 2.5 cc. of 6.5% FeCl₃ for 1 hour in the boiling water bath gave a positive test for sulfate. Under the same conditions no sulfate was formed from cysteic acid.

Other experiments on cystine were conducted in which the compound was heated in FeCl₃ (6.5%) with HCl varying in concentration from N/10 to 10%. All samples showed appreciable amounts of sulfate after being heated for an hour. The amount of oxidation decreased in a roughly proportional manner with the increase in acid content, yet we found no conditions under which no sulfate was obtained.

To determine the amount of sulfate formed a 5 gm. sample of l-cystine was suspended in 625 cc. of a 6.5% FeCl₃ solution and was heated on a boiling water bath for a total of 21 hours. Samples were removed at intervals for sulfate determination. The oxidation followed roughly an exponential curve of low coefficient. At 21 hours approximately 60% of the sulfur was accounted for as sulfate.