A novel protein cross-linking reaction in stressed Neutral Protamine Hagedorn formulations of insulin.
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.
Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, DC 3811, Indianapolis, Indiana 46285, USA.
R C
RC
Beavis
M D
MD
Kneirman
D
D
Sharknas
M A
MA
Heady
B H
BH
Frank
M R
MR
DeFelippis
D002626:Chemistry, Pharmaceutical; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003692:Delayed-Action Preparations; D007328:Insulin / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000737:chemistry; D013058:Mass Spectrometry; D011479:Protamines / Q000008:administration & dosage* / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003432:Cross-Linking Reagents; D003692:Delayed-Action Preparations; D007328:Insulin; D011479:Protamines; C110077:neutral protamine lispro insulin