生物化学（英文版）biochemistry-chapter 3 aa and primary structure of proteinpart 3教学材料.pptVIP

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;;;2. Separation of amino acids by HPLC;3. Separation of amino acids by partition chromatography （一）分配层析的原理： 分配系数 (Kd ) Kd = CA/CB 有效分配系数 (Keff)： 某物质在A相中的总量 Keff ＝ 某物质在B相中的总量 （二）分配柱层析： （三）纸层析： Rf ＝ X／Y （四）薄层层析： （五）气相色谱（气液层析，属分配色谱）： ;薄层层析：;氨基酸的光学性质 ; 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;Protein Sequencing Procedure in real amino acid sequencing;;;§1. Determine N- terminal residues 1. FDNB：; ;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)Carboxypeptidase Y cleaves all;;;2. Hydrazinolysis： 3. Reduction： Peptide-COOH + sodium borohydride Peptide-CH2OH Hydrolysis AAs + AA-CH2OH; §3. Cleave each chain into smaller fragments by site- specific proteases or chemicals Requirement：1. Less cleave sites; 2. High specificity； 3. More production.;（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