A Mechanism of the Irreversible Inactivation of Bovine Pancreatic Ribonuclease by Diethylpyrocarbonate

The irreversible inactivation of ribonuclease with diethylpyrocarbonate was studied. The products of the reaction were electrophoresed on polyacrylamide gels in sodium dodecyl sulfate and found to contain polymers of ribonuclease. Other proteins reacted similarly with diethylpyrocarbonate. Ribonuclease treated with maleic anhydride was unable to polymerize when treated with diethylpyrocarbonate, but when the N‐maleyl group was removed by incubation at low pH, the capacity of the ribonuclease to polymerize was restored. It was concluded that the diethylpyrocarbonate reaction forms amide bonds between ɛ‐amino groups of lysine and carboxylic groups of aspartic or glutamic both intermolecularly or intramolecularly. In either case the enzyme was inactivated. A mechanism based on the known chemical reactivities of diethylpyrocarbonate is proposed. The conclusions are further substantiated by studies of tryptic peptides and microbiuret positive products obtained from a reaction of diethylpyrocarbonate with assorted amino acids.