Protein Denaturation by Addition and Removal of Acetonitrile: Application to Tryptic Digestion of Acetylcholinesterase

Bovine erythrocyte acetylcholinesterase was prepared for tryptic digestion by radiomethylating with [¹⁴C]HCHO and NaCNBH₃, cleaving with purified bacterial phosphatidylinositol-specific phospholipase C to remove the lipid portion of the glycoinositol phospholipid anchor, and reducing and alkylating the intersubunit disulfide bonds. Two alternative denaturation procedures were then compared prior to incubation with trypsin. In the conventional procedure, acetylcholinesterase was treated with 6 M guanidine hydrochloride for 40 min at room temperature and dialyzed. In a new procedure, acetonitrile (CH₃CN) was added to 30% v/v for 10-15 min at room temperature and then removed by vacuum evaporation. The CH₃CN concentration during evaporation could be estimated from the apparent pH of the solution (20 mM phosphate buffer), which varied linearly over the range of 0-75% CH₃CN. CH₃CN was removed in a mixture of constant composition (approximately 11% H₂O-89% CH₃CN), so that a final CH₃CN content of 0-5% could be monitored by solution weight alone. The tryptic digests of the two denatured stocks yielded comparable HPLC profiles for A₂₁₅ and radioactivity. This new denaturation protocol may be of general utility because of its convenience and gentle conditions.