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 might be lined in one quantity - from purification and characterization of the protein via fragmentation via chemical or enzymic capability and, ultimately, to reassembly of the pointed out person peptides into the reconstructed overall series. It quickly grew to become glaring that this is able to now not be attainable. whereas the motive was once to limit the reveal of techniques basically to that details eager about "hands on" rainy chemistry, it grew to become obvious thorough presentation will require, furthermore, a dialogue of definite instrumental and extra theoretical ways no longer incorporated within the first quantity. moreover, the whole figuring out of the sphere of protein sequencing has complex greatly because the inception of this ebook. the aim of the 1st quantity used to be to supply useful info in enough aspect to allow the researcher to adopt the particular sequencing systems in his personal laboratory.
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Extra resources for Advanced Methods in Protein Sequence Determination
Activated side-chain carboxyls either react with amines to form amides or rearrange to the inactive N-acylurea. 23 o R-N=C=N-R II I ~ ~'_R' o W ~-NH-CHR-C-N-R II o N-Acylurea \ NHR II ~-NH-C-o-C ~-NH-CHR-COOH NH2-R' I O-Acylurea ft ~-NH-CHR-C-NR , II C=O 0 I NHR / '--------' Fig. 3. Reactions of carbodiimide-activated carboxyl groups In the case of Asp-Gly bonds, some cyclic imide may be formed as noted with carbonyldiimidazole. A variety of carbodiimide coupling procedures has been described. The simplest involves adding an excess of N-dimethylaminopropyl-N'-ethyl carbodiimide to a mixture of the unblocked peptide and resin in dimethylformamide-water (246, 389).
It must be dialyzed away before the enzyme is used for digestion purposes. Generally speaking, the purer the preparation of pyrrolidone carboxylyl peptidase, the more liable it is to decay during storage. Hence, "G-200 enzyme", frozen as pellets after precipitation with ammonium sulfate, is stable at -20 0 for several months, whereas "A-25 enzyme" stored the same way loses about half its activity in six weeks. Purer preparations decay even more rapidly. ::'1 these preparations is preserved considerably longer when storage is at -70 0 (119).
The material is then dialyzed against the same buffer to remove the residual ammonium sulfate and 2-pyrrolidone (the stabilizer is also an inhibitor). Usually three successive 1 h dialyses against 250 ml buffer each are sufficient. 0 x 10 cm Sephadex G-25 column to remove the ammonium sulfate and the 2-pyrrolidone. The optimum concentrations of enzyme and substrate will vary with the particular peptide under study, and it is useful to make a preliminary examination of the reaction progress on a small amount of the peptide before attempting to prepare a large amount for subsequent degradation or other follow-up characterization.
Advanced Methods in Protein Sequence Determination by Professor Saul B. Needleman (auth.)