The covalent insulin-protamine product molecules formed by heat stress in Neutral Protamine Hagedorn formulations of insulin and the insulin analogue [LysB28,ProB29] were examined by mass spectrometry. The results demonstrated that the covalent cross-link between insulin and protamine was not caused by linkage through the protamine N-terminal amino group, as had been previously thought. Our results indicate that the linkage was formed between the side chain of a protamine arginine and a histidine in the insulin B chain, resulting in a net mass change of -5 Da, compared to the sum of the protamine and insulin molecular masses. A mechanism for this new type of covalent cross-linking reaction is proposed.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.