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1、3.8 Amino Acid Composition of Proteins 3.9 Determining the Sequence of Amino Acid residues 3.10 Comparisons of the Primary Structures of Proteins Reveal Evolutionary Relationships,3.8 Amino Acid Composition of Proteins,Hydrolyzation of proteins： complete Hydrolyzation AAs incomplete Hydrolyzation AA
2、speptides acid Hydrolyzation Methods basic Hydrolyzation enzyme Hydrolyzation,1. Separation of amino acids by ion exchange chromatography,2. Separation of amino acids by HPLC,薄层层析：,氨基酸的光学性质,旋光性: AA的旋光符号和大小取决于R基的性质,且与pH有关. 2. 紫外吸收: Trp, =280nm; Tyr, =275nm; Phe, =257nm;,朗伯比尔（Lambert-Beer）定律 在特定波长下，溶液
3、中物质的光吸收与其浓度C（以mmol-1为单位）和溶液中光径长l（以cm为单位）成正比。 A=Cl,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 po
4、lypeptide 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. Dete
5、rmine 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., Edman degradation,1. Determine N- terminal residues 1. FDNB：,DNP,DNP,DNP,2. DNS：,3. PITC (Edman reagent):
6、,4. Amino peptidase：,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 th
7、at only second AA is Pro; (4)Carboxypeptidase Y cleaves all;,2. Hydrazinolysis： 3. Reduction： Peptide-COOH + sodium borohydride Peptide-CH2OH Hydrolysis AAs + AA-CH2OH,与苯甲醛反应沉淀,3. Cleave each chain into smaller fragments by site- specific proteases or chemicals Requirement：1. Less cleave sites; 2. H
8、igh specificity； 3. More production.,（1）Proteases cleavage： 1Trypsin：C-terminal of Arg, Lys. High specificity,2Chymotrypsin： C-terminal of Phe, Trp, Tyr.,3Thermolysin：some hydrophobic amino acids, Low specificity; 4Pepsin：some hydrophobic amino acids, pH2, Low specificity; 5Papain： c-terminal of Arg
9、, Lys. Low specificity;,6Staphylococcal 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.,（2）Chemical cleavage： 1CNBr： c-terminal of Met. CNBr is use
10、ful because proteins usually have only few Met residues.,2NH2OH：AsnGly，Asn Lue；AsnAla.,4. Determine the sequence of each chain 1. Edman degradation：,Edman degradation uses Edman reagent to determine the order of amino acids from the N-terminal end of a protein.,2. Other methods: （1）DNSEdman; （2） PIT
11、C with fluorescent or luminescence groups； （3）Solid phase sequencing;,5. Cleave of disulfide bond,Oxidation of a disulfide bond by performic acid:,2. Reduction of S-S bridges by sulfhydryl compound:,6. Reconstruct the sequence of the protein from the sequences of overlapping fragments,7. Determinati
12、on of sites of disulfide bonds,1. Diagonal electrophoresis：,2. Partial reduction：,MS,TCEP（三羧乙基磷酸） CDAP（1-氰基-4-（二甲氨基）吡啶四氟硼酸盐）,An example:,Trypsin cleavage: A-E-F-S-G-I-T-P-K L-V-G-K Chymotrypsin Cleavage: L-V-G-K-A-E-F S-G-I-T-P-K Edman degradation: L Correct sequence: L-V-G-K-A-E-F-S-G-I-T-P-K, Enzy
13、matic hydrolysis： aminopeptidase or carboxypeptidase, only short sequencing., Mass Spectrometer, Sequencing the corresponding DNA in the gene：,First PCR cycles,Second PCR cycles,Third PCR cycles,3.10 Comparisons of the Primary Structures of Proteins Reveal Evolutionary Relationships,homogenous prote
14、in ? Invariant residues Variant residues,Conservation and variation of cytochrome c sequences,27 invariant residues (yellow), conservative substitutions (blue) nonconservative or variable residues (unshaded),Phylogenetic tree for cytochrome c,Summary of chapter 3,Characterisation of amino acids Protein purification Protein sequencing,The End of Chapter 3 Thank You For Attention!,
