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 publication used to be proposed, it was once expected that the dialogue of the total box of protein se quencing can be lined in one quantity - from purification and characterization of the protein via fragmentation by way of chemical or enzymic ability and, eventually, to reassembly of the pointed out person peptides into the reconstructed overall series. It quickly grew to become obtrusive that this could no longer be attainable. whereas the rationale was once to limit the reveal of methods in simple terms to that info interested in "hands on" rainy chemistry, it turned obvious thorough presentation will require, additionally, a dialogue of sure instrumental and extra theoretical ways no longer integrated within the first quantity. moreover, the full knowing of the sector of protein sequencing has complicated radically because the inception of this publication. the aim of the 1st quantity was once to supply sensible details in adequate element to allow the researcher to adopt the particular sequencing methods in his personal laboratory.
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Additional info for Advanced Methods in Protein Sequence Determination
Sequence and cyanogen bromide peptides of lobster glyceraldehyde-3 phosphat dehydrogenase Na - and NE-amino groups, the other by Na _ only. On treatment of the resin with acid, the arginine peptide will be released (minus its Nterminal) and the lysine peptide alone will be sequenced. Variations of this strategy include sequencing of lysine peptides contaminated 37 X A. NH2 I , 1 NH 2------------Arg------------Lys-COOH diisothiocyanate, resin x NHResin , , NH 2------------Arg------------Lys-COOH , NH2 B.
With aminopropyl or S-aminoethylaminopropyl siloxanes to give aminoglass supports, which can be used directly or further activated with p-phenylene diisothiocyanate (Fig. 11). is]:j- 0 / SiCH2CH2CH2NH-C-NHo-N=C=S "'-0 DITC-glass .. Protein-(NH 2) n B-aminoethylaminopropyl glass (B-APG) Fig. 11. Amino-glass supports for immobilization of proteins virtue of being rigid, so that many of the mechanical problems associated with polystyrene resins are avoided. They also have an enormous surface area, so that the reactive groups, which are all on the surfac are very accessible.
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. GIn. -------Lys,0,-------Rse121 ~ Trypsin ~ soluble pep tides II. -------Lys,01-------Rse121 ~BIOCk Lys ~ Trypsin ~ Rse coupling x III. GIn. -------L~S101-------Hse121-Resin Fig. 17. Sequencing strategy for azurin peptides 57-121. ~ the intact peptide is coupled and partially sequenced through about 20 residues.