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 Amino Acid Separation Unfortunately, amino acids are not colored as described in this overhead. Therefore, what methods would you use to first check if an amino a
3、cid is indeed present? 2. Separation of amino acids by HPLC 3. Separation of amino acids by partition chromatography （一一）分配层析的原理：分配层析的原理： 分配系数分配系数 ( (K Kd d ) ) K Kd d = = C CA A/C/CB B 有效分配系数有效分配系数 ( (KeffKeff) )： 某物质在某物质在A A相中的总量相中的总量 Keff Keff 某物质在某物质在B B相中的总量相中的总量 （二）分配柱层析：（二）分配柱层析： （三）纸层析：（三）纸层
4、析： R Rf f X XY Y （四）薄层层析：（四）薄层层析： （五）气相色谱（五）气相色谱（气液层析气液层析，属分配色谱）：，属分配色谱）： 薄层层析：薄层层析： 氨基酸的光学性质氨基酸的光学性质 1.1. 旋光性旋光性: : AAAA的旋光符号和的旋光符号和 大小取决大小取决于于R R基的基的 性质性质, ,且与且与pHpH有关有关. . 2. 2. 紫外吸收紫外吸收: : TrpTrp, , =280nm;=280nm; Tyr, Tyr, =275nm;=275nm; Phe, Phe, =257nm;=257nm; 朗伯比尔（朗伯比尔（Lambert-Beer）定律定律 在特定波
5、长下，溶液中物质的光吸收与其浓度在特定波长下，溶液中物质的光吸收与其浓度 C C（以（以mmolmmol- -1 1为单位）和溶液中光径长为单位）和溶液中光径长l l（以（以cmcm 为单位）成正比。为单位）成正比。 A=ClA=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 Seque
6、ncing 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 int
7、o 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; 9. Determination position of disulfide bridges. Edman degradation 1. Determine N-
8、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 i
9、f 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 与与苯甲醛苯甲醛反应沉淀反应沉淀 3. Cleave each chain into smaller fragments
10、by site- specific proteases or chemicals Requirement：1. Less cleave sites; 2. High 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
11、：some hydrophobic amino acids, pH2, Low specificity; 5Papain： c-terminal of Arg, 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
12、）：）： c-terminal of Arg. （2）Chemical cleavage： 1CNBr： c-terminal of Met. CNBr is useful because proteins usually have only few Met residues. N H CH C CH2 H2C S H3C H N O O R Br C N N H CH C CH2 H2C S H3C H N O O R C N displacement of Br N H C H C H2C H2 C S H3C N O C N + + Methyl thiocyanate N H C H
13、C H2C H2 C O O H2O C-terminal Peptide+ Homoserine lactone 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） PI
14、TC with fluorescent or luminescence groups； （3）Solid phase sequencing; 5. Cleave of disulfide bond 1. Oxidation of a disulfide bond by performic acid: N H CHC CH2 O O S H NCHC CH2 O O S N H CHC CH2 O O SO3 H NCHC CH2 O O SO3 CO O HOH performic Acid 2. Reduction of S-S bridges by sulfhydryl compound:
15、 6. Reconstruct the sequence of the protein from the sequences of overlapping fragments 7. Determination of sites of disulfide bonds 1. Diagonal electrophoresis： 2. Partial reduction： MS TCEP（三羧乙基磷酸）（三羧乙基磷酸） CDAP（1-氰基氰基-4- （二甲氨基）吡啶四（二甲氨基）吡啶四 氟硼酸盐）氟硼酸盐） An example: Trypsin cleavage: A-E-F-S-G-I-T-P-K
16、 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 Enzymatic hydrolysis： aminopeptidase or carboxypeptidase, only short sequencing. Mass Spectrometer Sequencing the corresponding DNA in the gene： First PCR cycles Second PCR cycl
17、es Third PCR cycles 3.10 Comparisons of the Primary Structures of Proteins Reveal Evolutionary Relationships homogenous protein ? 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 3The End of Chapter 3 Thank You For Attention!Thank You For Attention!
