lec 7-8 - Sequence Determination An eight step strategy 1....

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An eight step strategy 1. Separate, if more than one polypeptide chains 2. Cleave (reduce) disulfide bridges 3. Determine composition of each chain Sequence Determination 4. Determine N- and C-terminal residues 5. Cleave each chain into smaller fragments and determine the sequence of each chain 6. Repeat step 5, using a different cleavage procedure to generate a different set of fragments 7. Reconstruct the sequence of the protein from the sequences of overlapping fragments 8. Determine the positions of the disulfide cross-links { 1/26/2007 Step 1: separation of peptide chains Peptide-chain separation is based on weak force interactions 1/26/2007 Step 2: Cleavage of Disulfide bridges Performic acid oxidation • Sulfhydryl reducing agents BME - b-mercaptoethanol DTT - dithiothreitol or dithioerythritol • to prevent recombination, followed with an alkylating agent like iodo-acetate 1/26/2007 Step 3 Determine amino acid composition how many of each types of amino acids are there in the protein •Separation is achieved with: - extreme pH - 8M urea - 6M guanidine HCl - high salt concentration (usually ammonium sulfate) Step 4.1 Identify N-terminal residues • N-terminal analysis: – Use Edman's reagent: phenyl-iso-thio- cyanate – derivatives are phenylthiohydantoins (PTHs)
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lec 7-8 - Sequence Determination An eight step strategy 1....

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