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 while the manuscript for the 1st a part of this publication was once proposed, it was once expected that the dialogue of the whole box of protein se quencing may be coated in one quantity - from purification and characterization of the protein via fragmentation via chemical or enzymic ability and, eventually, to reassembly of the pointed out person peptides into the reconstructed overall series. It quickly grew to become obvious that this may now not be attainable. whereas the reason was once to limit the divulge of approaches simply to that info inquisitive about "hands on" rainy chemistry, it turned obvious thorough presentation will require, furthermore, a dialogue of definite instrumental and extra theoretical methods no longer incorporated within the first quantity. in addition, the complete knowing of the sector of protein sequencing has complex extensively because the inception of this ebook. the aim of the 1st quantity used to be to supply useful info in adequate aspect to allow the researcher to adopt the particular sequencing systems in his personal laboratory.
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Additional info for Advanced Methods in Protein Sequence Determination
The enzyme was first isolated from a selected strain of Pseudomonas fZuorescens (120), but it can be prepared from a variety of bacterial and other sources (20, 121, 422, 423). , Strain No. 25289), but most of the comments also seem to apply to the enzyme isolated from other sources (119). 1. Assay Method PCA-peptidase is not a particularly stable enzyme, and as such it is important that the investigator trying to cleave a terminal PCA from some valuable peptide or protein be certain that an active enzyme preparation is actually being employed.
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. 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.
3. MethoYlOlysis The fact that many peptides and proteins can be successfully esterified with methanolic-HCI without rupture of peptide bonds led Kawasaki and Itano (229) to undertake an investigation of the effects of this treatment on the pyrrolidone ring of a variety of small PCA-peptides. They found that 1N HCl/methanol at 25-40° for 1-3 days was quite effective in converting the pyrrolidone residues to the y-methyl ester of glutamic acid. Although carboxyl groups are speedily esterified under these conditions, most peptide bonds are unaffected, and step-wise degradation procedures on the products ought to be possible.