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 booklet was once proposed, it was once expected that the dialogue of the total box of protein se quencing may be coated in one quantity - from purification and characterization of the protein via fragmentation through chemical or enzymic potential and, ultimately, to reassembly of the pointed out person peptides into the reconstructed overall series. It quickly turned glaring that this could no longer be attainable. whereas the reason used to be to limit the divulge of techniques merely to that info serious about "hands on" rainy chemistry, it grew to become obvious thorough presentation will require, moreover, a dialogue of convinced instrumental and extra theoretical techniques no longer incorporated within the first quantity. additionally, the whole realizing of the sphere of protein sequencing has complex substantially because the inception of this publication. the aim of the 1st quantity used to be to supply sensible details in adequate aspect to allow the researcher to adopt the particular sequencing systems in his personal laboratory.
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Extra info for Advanced Methods in Protein Sequence Determination
I the peptide is coupled and the N-terminal segment sequenced. II a sample of peptide is cleaved with trypsin and the C-terminal segment is coupled and sequenced ment, the cyanogen bromide-digested protein can be further digested with trypsin, and the resulting peptide mixture coupled again using the homoserine lactone method and sequenced. In this way, residues 1944 can be determined and this information combined with previous data to give the sequence of the first 44 residues. It has been shown (198) that the homoserine coupling method can be used to obtain nearly quantitative removal of C-terminal homoserine peptides from peptide mixtures.
Although the enzyme is active towards this substrate, the poor solubility of the material in water at neutral pH (ca. 1 mg/ml) limits its usefulness. We still find the ninhydrin assay and PCA-ALA to be the most sensitive and convenient. Several PCA-dipeptides, including PCA-ALA, are available commercially (Cyclo Chern. ). In addition, we have published a complet description of the synthesis and properties of several others (443). 2. Preparation of PCA-Peptides from the Original Strain of Pseudomonas Fluoreseens The enzyme can be prepared either from bacterial pellets frozen directly after harvesting, or from freeze-dried suspensions.
III a sample of the peptides is cleaved at arginine and is selectively attached and sequenced in length from 10 to 30 residues, can be sequenced either in a single run or by using the strategy for residues 57-121. The examples above also illustrate one of the pitfalls encountered in sequencing. Some of the largest peptides have N-terminal glutamine, which can be expected to cyclize to pyroglutamic acid, thus making Edman degradation impossible. Nevertheless some C-terminal sequence data can be obtained using the strategy outlined.
Advanced Methods in Protein Sequence Determination by Professor Saul B. Needleman (auth.)