生物化学英文版biochemistry-chapter3aaandprimarystructureofproteinpart3.ppt

3.9 Determining the Sequence  of Amino Acid residues Proteins can be sequenced in two ways: 1. Real amino acid sequencing 2. Sequencing the corresponding DNA in the gene Protein Sequencing Procedure in real amino acid sequencing 1. Determination of polypeptide chain number; 2. If more than one polypeptide chain, separate them; 3. Cleave (reduce) disulfide bridges; 4. Determine amino acid composition of each chain; 5. Determine N- and C-terminal residues; 6. Cleave each chain into smaller fragments by site-specific proteases or chemicals; 7. Determine the sequence of each chain by Edman degradation or MS; 8. Reconstruct the sequence of the protein from the sequences of overlapping fragments; Determination position of disulfide bridges. §1. Determine N- terminal residues 1. FDNB： §2. Determine C- terminal residues 1. Carboxypeptidase： (1)Carboxypeptidase A cleaves all except for P, R, K (It can not done if second AA is Pro); (2)Carboxypeptidase B cleaves only R and K (It can not done if second AA is Pro); (3)Carboxypeptidase C cleaves the C terminal AA that only second AA is Pro; (4)Carboxypeptidase Y cleaves all; 2. Hydrazinolysis： 3. Reduction： Peptide-COOH + sodium borohydride Peptide-CH2OH Hydrolysis AAs + AA-CH2OH （1）Proteases cleavage： 1．Trypsin：C-terminal of Arg, Lys. High specificity 2．Chymotrypsin： C-terminal of Phe, Trp, Tyr. 3．Thermolysin：some hydrophobic amino acids, Low specificity; 4．Pepsin：some hydrophobic amino acids, pH2, Low specificity; 5．Papain： c-terminal of Arg, Lys. Low specificity; 6．Staphylococcal protease（Glu protease）： Phosphate Buffer(pH7.8): c-terminal of Glu,Asp NH4HCO3 Buffer(pH7.8)： c-terminal of Glu NH4Ac Buffer(pH4.0)： c-terminal of Glu 7. Clostripain（Arg protease ）： c-terminal of Arg. Edman degradation uses Edman reagent to determine the order of amino acids from the N-terminal end of a prote