Protein denaturation by addition and removal of acetonitrile: Application to tryptic digestion of acetylcholinesterase

Robert Haas, Terrone L. Rosenberry

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)425-427
Number of pages3
JournalAnalytical Biochemistry
Volume224
Issue number1
DOIs
StatePublished - 1995
Externally publishedYes

Fingerprint

Protein Denaturation
Denaturation
Acetylcholinesterase
Digestion
alkylate 215
Phosphoinositide Phospholipase C
Vacuum evaporation
Guanidine
Radioactivity
Anchors
Disulfides
Trypsin
Phospholipids
Buffers
Evaporation
Proteins
Temperature
Phosphates
Vacuum
Lipids

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Cell Biology

Cite this

Protein denaturation by addition and removal of acetonitrile : Application to tryptic digestion of acetylcholinesterase. / Haas, Robert; Rosenberry, Terrone L.

In: Analytical Biochemistry, Vol. 224, No. 1, 1995, p. 425-427.

Research output: Contribution to journalArticle

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