Covalent attachment of peptides to cytochrome C for automated sequence determination. 1977

M D Garrick, and R L Sloan

Because small peptides are lost into the organic solvents used, it is virtually impossible to obtain the complete amino acid sequence of a small peptide using only an automated peptide sequencer of the spinning cup type. To overcome this problem we have extended peptides at the carboxy terminus by attachment to equine cytochrome c by a water soluble carbodiimide, relying on the acetylated N-terminus of the cytochrome to minimize its direct contribution to recovery of PTH-amino acids. The Model Peptide H-Leu-Trp-Met-Arg-phe-Ala-OH was used for most experiments. After reaction of 3H-peptide with cytochrome c, about one-third of the tritium counts migrated with cytochrome c during gel filtration. After attachment, the amino acid sequence of the hexapeptide was readily determined with a single cleavage Quadrol program in a Beckman 890B sequencer, whereas only the N-terminal residue was recovered without attachment. The repetitive yield after attachment was 95-96%, with 21-27+ overlap and an initial yield of 18-20%. Sequence data with other peptides illustrate applications and present limitations of our approach.

UI MeSH Term Description Entries
D010455 Peptides Members of the class of compounds composed of AMINO ACIDS joined together by peptide bonds between adjacent amino acids into linear, branched or cyclical structures. OLIGOPEPTIDES are composed of approximately 2-12 amino acids. Polypeptides are composed of approximately 13 or more amino acids. PROTEINS are considered to be larger versions of peptides that can form into complex structures such as ENZYMES and RECEPTORS. Peptide,Polypeptide,Polypeptides
D002850 Chromatography, Gel Chromatography on non-ionic gels without regard to the mechanism of solute discrimination. Chromatography, Exclusion,Chromatography, Gel Permeation,Chromatography, Molecular Sieve,Gel Filtration,Gel Filtration Chromatography,Chromatography, Size Exclusion,Exclusion Chromatography,Gel Chromatography,Gel Permeation Chromatography,Molecular Sieve Chromatography,Chromatography, Gel Filtration,Exclusion Chromatography, Size,Filtration Chromatography, Gel,Filtration, Gel,Sieve Chromatography, Molecular,Size Exclusion Chromatography
D003574 Cytochrome c Group A group of cytochromes with covalent thioether linkages between either or both of the vinyl side chains of protoheme and the protein. (Enzyme Nomenclature, 1992, p539) Cytochromes Type c,Group, Cytochrome c,Type c, Cytochromes
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001322 Autoanalysis Method of analyzing chemicals using automation. Autoanalyses
D012997 Solvents Liquids that dissolve other substances (solutes), generally solids, without any change in chemical composition, as, water containing sugar. (Grant & Hackh's Chemical Dictionary, 5th ed) Solvent

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