ovalbumin and ethylthiol-trifluoroacetate

A vapor of S-ethyltrifluorothioacetate was found to specifically cleave the amino side of serine and threonine peptide bonds. The cleavage reactions were carried out at 50 degrees C for 6 h-24 h or at 30 degrees C for 24 h. When vapors were generated in a solution containing several conventional organic solvents, the cleavage reactions were reduced or stopped, or modification took place. When the reagent vapor was made in an aqueous solution, the cleavage reaction at glycine residues was enhanced. This reagent did not oxidize any amino acid residues, such as methionine, cysteine and tryptophan. The cleavage was also effective on proteins on membranes blotted or electroblotted from polyacrylamide gels. This method therefore may be used for the peptide mass fingerprinting [Patterson, S. D. (1995) Electrophoresis 16, 1104-1114] after two-dimensional electrophoresis.

Topics: Alcohol Dehydrogenase; alpha-Endorphin; Amino Acid Sequence; Capsid; Fluoroacetates; Gases; Glucagon; Molecular Sequence Data; Mosaic Viruses; Motilin; Myoglobin; Ovalbumin; Peptide Fragments; Peptide Mapping; Peptides; Proteins; Sequence Analysis; Serine; Spectrometry, Mass, Fast Atom Bombardment; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Threonine; Trifluoroacetic Acid

A 19F-labeled derivative of hen egg-white lysozyme, in which the six epsilon-amino groups are trifluoroacetylated (LF6), was prepared by reaction of lysozyme with S-ethyltrifluorothioacetate. The reaction mixture was fractionated by cation-exchange chromatography at pH 7.3. A comparison of the circular dichroic spectra and the activity towards Micrococcus lysodeikticus of both LF6 and native lysozyme reveals that the labeling causes no major conformational changes of the polypeptide backbone. Assignment of the six resonances present in the 19F-NMR spectrum of LF6 was accomplished by using a variety of techniques: specific chemical modifications, the effect of the inhibitor (GlcNAc)3, 19F-shift/pH information and relaxation parameters.

Topics: Acetylglucosamine; Animals; Chemical Phenomena; Chemistry; Chickens; Chromatography, Ion Exchange; Circular Dichroism; Disulfides; Fluoroacetates; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Micrococcus; Muramidase; Ovalbumin; Protein Conformation; Trisaccharides