LEC 8 - Insulin consists of two polypeptide chains, A and...

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Insulin consists of two polypeptide chains, A and B, held together by two disulfide bonds. The A chain has 21 residues and the B chain has 30 residues. The sequence shown is that of bovine insulin. Frederick Sanger was the first sequenced the two chains of insulin - in 1953
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Determining the Sequence 1. Separate if more than one polypeptide chain. 2. Cleave (reduce) disulfide bridges 3. Determine composition of each chain 4. Determine N- and C-terminal residues 5. Cleave each chain into smaller fragments and determine the equence 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 crosslinks
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Step 1: Step 1: Separation of chains Subunit interactions depend on weak forces Separation is achieved with: - extreme pH - 8M urea - 6M guanidine HCl - high salt concentration (usually ammonium sulfate)
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Step 2: Step 2: Cleavage of Disulfide bridges Performic acid oxidation Sulfhydryl reducing agents - BME: mercaptoethanol - DDT: dithiothreitol or dithioerythritol - to prevent recombination, follow with an alkylating agent like iodoacetate
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Step 3:
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LEC 8 - Insulin consists of two polypeptide chains, A and...

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