The Labile Sulfur of Insulin.

All observers agree that insulin material contains sulfur and Abel 1 has shown that sulfur is present in a very labile form, being split off as a sulfide by boiling with weak alkali. Abel believes that the sulfur is roughly proportional to the hypoglycemic potency. Brand and Sandberg 2 have shown that although the sulfur of cystine is relatively stable, the linkage of other amino acids with cystine so affects the sulfur that it is easily split out by boiling with weak alkali. Cystine can be determined quantitatively by the uric acid reagent of Folin and Denis, as shown by Folin and Looney. 3 The cystine is first reduced by sodium sulfite in saturated sodium carbonate to cysteine which then gives a deep blue color with the phosphotungstic acid reagent. Shonle and Waldo 4 found that insulin preparations respond to this test for cystine.

I have observed that insulin preparations do not give the reaction directly with the uric acid reagent but do so after reduction with sulfite. The insulin material upon boiling for thirty minutes with 0.1 N Na₂CO₃ gives a positive reaction without previous reduction, due to the formation of sulfide ion. Acid hydrolysates of the insulin material give the reaction only after reduction with sulfite. They do not, however, give the reaction directly with the uric acid reagent after boiling with 0.1 N Na₂C0₃, showing that the sulfur has somehow become more stable in the fragment split off by acid hydrolysis. It is necessary to reduce first with sodium sulfite to obtain the reaction as in the case of the unboiled acid hydrolysate.

This difference in the stability of the sulfur in the original and in the acid hydrolysed material and the behavior towards the uric acid reagent can be explained on the assumption that a S-S linkage exists in the insulin material.