PROTEIN DENATURATION BY ADDITION AND REMOVAL OF ACETONITRILE - APPLICATION TO TRYPTIC DIGESTION OF ACETYLCHOLINESTERASE

Bovine erythrocyte acetylcholinesterase was prepared for tryptic diges tion by radiomethylating with [C-14]HCHO and NaCNBH3, cleaving with pu rified bacterial phosphatidylinositol-specific phospholipase C to remo ve the lipid portion of the glycoinositol phospholipid anchor, and red ucing and alkylating the intersubunit disulfide bonds. Two alternative denaturation procedures were then compared prior to incubation with t rypsin. In the conventional procedure, acetylcholinesterase was treate d 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 evapora tion. The CH3CN concentration during evaporation could be estimated fr om the apparent pH of the solution (20 mM phosphate buffer), which var ied linearly over the range of 0-75% CH3CN. CH3CN was removed in a mix ture of constant composition (approximately 11% H2O-89% CH3CN), so tha t a final CH3CN content of 0-5% could be monitored by solution weight alone. The tryptic digests of the two denatured stocks yielded compara ble HPLC profiles for A(215) and radioactivity. This new denaturation protocol may be of general utility because of its convenience and gent le conditions. (C) 1995 Academic Press, Inc.