生物化学（英文版）Biochemistry-chapter 3 AA and primary structure of proteinpart 3.ppt
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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 AAs＋peptides
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, λ=280nm;
Tyr, λ=275nm;
Phe, λ=257nm;
朗伯－比尔（Lambert-Beer）定律
在特定波长下，溶液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=εCl
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 Edman degradation
§1. Determine N- terminal residues
1. FDNB：
DNP
DNP
DNP
2. DNS：
3. PITC (Edman reagent):
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 that only second AA is Pro;
(4)Car
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