生物化学英文.pptxVIP

生物化学英文;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;1. Separation of amino acids by ion exchange chromatography;第5页/共57页;第6页/共57页;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）： ;;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;;第16页/共57页;§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.