By Professor Saul B. Needleman (auth.)
Confusion now hath made his masterpiece Macbeth II iii seventy two Whence and what are these execrable form? Paradise misplaced Ib 1 681 Confusion worse confounded Paradise misplaced Ib 1 995 whilst the manuscript for the 1st a part of this ebook was once proposed, it was once expected that the dialogue of the whole box of protein se quencing should be coated in one quantity - from purification and characterization of the protein via fragmentation by way of chemical or enzymic capacity and, ultimately, to reassembly of the pointed out person peptides into the reconstructed overall series. It quickly turned obtrusive that this might now not be attainable. whereas the motive was once to limit the reveal of techniques basically to that details desirous about "hands on" rainy chemistry, it grew to become obvious thorough presentation will require, moreover, a dialogue of convinced instrumental and extra theoretical ways no longer incorporated within the first quantity. moreover, the complete realizing of the sector of protein sequencing has complicated greatly because the inception of this e-book. the aim of the 1st quantity used to be to supply sensible info in adequate element to allow the researcher to adopt the particular sequencing tactics in his personal laboratory.
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Additional info for Advanced Methods in Protein Sequence Determination
Wachter et al. (454) have recently summarized their experience with porous glass supports. For attachment of homoserine peptides, APG (Fig 11) is best for peptides containing 10-40 residues, while B-APG is better for larger peptides. The B-APG appears to be more reactive than APG because of the neighboring amine group which can participate in general base catalysis (Fig. 12) (69, 335). The same effect is seen with TETA polystyrene (197). DITC-glass couples large peptides and proteins in high yields, but gives poor results for small peptides.
Tryptic digestion of a second peptide sample, followed by attachment of the mixture via the homoserine lactone procedure will permit the sequencing of residue! 102-121 (Fig. 17). In a third experiment, the peptide lysines can then be blocked and the peptide cleaved at arginine. Homoserine coupling of the peptide mixture and sequencing of the single attached peptide shou: provide sufficient information to overlap the N- and C-terminal portiol of the peptide. The remaining cyanogen bromide peptides, which range 35 I.
The aliphatic amino groups (pKa ca. 6-10) are also better nucleophiles than aryl amino groups. For these reasons the TETA resin gives superior results in coupling carboxyl-activated peptides. For the combined coupling method, a support such as the EDAA-resin would probably give best results since it has both aryl and aliphatic amino groups. This, however, remains to be tested. After the peptide has attached to the support, excess resin amino groups are blocked, usually by reaction with methyl isothiocyanate.
Advanced Methods in Protein Sequence Determination by Professor Saul B. Needleman (auth.)