On the Absence of Thiolhistidine in Insulin

Abstract

Considerable evidence has pointed to the possibility of the presence in insulin of sulfur other than that attributable to cystine, in which form the major portion of the sulfur is present. That thiolhistidine might account for part of this sulfur was a possibility to be considered. We therefore undertook studies to ascertain whether or not this amino acid is a constituent of the insulin molecule. On the basis of reports in the literature that the sulfur of thiolimidazoles could be split off as sulfate by boiling with ferric chloride and that the sulfur of cysteine was stable, we attempted to test for the presence of thiolhistidine. However, in testing this reaction with cystine, we found that ferric chloride will oxidize the sulfur of cystine to sulfate. 1 This reaction could not, therefore, be utilized to detect thiolhistidine in the presence of cystine in an insulin hydrolysate and some other method of approach was necessary.

In Barger and Ewin's early paper on the betaine of thiolhistidine, ergothioneine it was shown that the sulfur of this compound was oxidized by bromine to inorganic sulfate. 2 In addition, it is a well recognized fact that cystine upon treatment with bromine is converted to cysteic acid and that no inorganic sulfate is formed. 3 It occurred to us that these 2 observations might form the basis of the desired test but before applying this reaction to insulin for the detection of thiolhistidine we felt that it was necessary to test the reaction on the amino acid itself rather than depend on the known behavior of its betaine. This amino acid was, therefore, synthesized by Harington's method, 4 using aspartic acid as starting material. We were able to check Harington's procedure in all essential details and obtained approximately the same yields reported in the various steps recorded by him.