Advanced Methods in Protein Sequence Determination by Professor Saul B. Needleman (auth.) PDF

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 full box of protein se­ quencing should be lined 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 obtrusive that this is able to no longer be attainable. whereas the purpose used to be to limit the divulge of tactics in simple terms to that info interested in "hands on" rainy chemistry, it grew to become obvious thorough presentation will require, additionally, a dialogue of convinced instrumental and extra theoretical techniques now not integrated within the first quantity. in addition, the full knowing of the sector of protein sequencing has complicated notably because the inception of this e-book. the aim of the 1st quantity used to be to supply useful info in enough element to allow the researcher to adopt the particular sequencing systems in his personal laboratory.

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Activation occurs by way of the imidazolide (Fig. 1). Carbonyldiimidazole reacts rapidly with nucleophiles such as amines and water. /\ I N Peptide~COOH ----------~~.. R~N Peptide~C-N Fig. 1. '" COOH I' ~C-NH-CH-C-NH~ ~ Carbonyldiimidazole o II 110 CH--C,-N_N .. --C CH. I \ \1 N'\/\/vv 'vvvvC-NH-CH __ C/ II o Fig. 2. Cyclic imide formation been distilled from P20S to remove traces of water and dimethylamine. The peptide amino groups must be protected, for example, with the tbutyloxycarbonyl group (249), which is easily removed under the conditions of the Edman degradation.

Care must be taken to maintain the temperature of the suspension during sonication in the range 0-10 0 . After sonication the material is centrifuged at 39,000 g at 00 for 3060 min. The supernatant liquid containing the enzyme activity is carefully decanted, and if necessary, it should be centrifuged a second time. The A280 of the supernatant should be at least 50; if it is higher, it can be diluted with the phosphate buffer described above. Nucleic acids may be precipitated by adding one volume of 1% protamine sulfate to six volumes of cold supernatant containing the enzyme.

After digestion of the protein with cyanogen bromide, the peptide mixture can by subjected to coupling via the homoserine lactone procedure. Only the N-terminal fragment contain· ing homoserine will become attached to the resin; sequencing will give the first 25-30 residues of the protein (Fig. 14). In another experi- 33 .. I. --------------Lys,s------------Hse44-Restl t Trypsin ~ Hse coupling II. ------------------------Hse44-Resin Fig. 14. Hypothetical strategy for sequencing the N-terminal BrCN fragment of acyl carrier protein.

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