第三节蛋白质一级结构教程方案.ppt

第三节 蛋白质的共价结构 （一级结构）;蛋白质多肽链的氨基酸排列顺序和连接方式。;一、肽和肽键的结构;? 蛋白质分子中氨基酸连接的基本方式是肽键（peptide bond）。;Fig.1-9 The peptide bond is shown in its usual trans configuration.;Peptide bond;;? 肽键中的C-N键具有部分双键性质，不能自由旋转。;H;二、肽的物理和化学性质;例：试判断Gly-Glu-Lys-Ala 在pH6.0时的带电状态。;（2）化学反应;三、天然存在的活性肽;2. 脑啡肽;四、蛋白质的一级结构举例 ;Fig.1-11 The amino acid sequence of the two chains of bovine insulin, which are joined by disulfide cross-linkages. ; [美] Stanford Moore, 50s’末;3. 肌红蛋白(Mb) 血红蛋白(Hb);五、蛋白质分子氨基酸顺序的测定;（一）测定蛋白质分子中多肽链的数目;（二）N末端和C末端氨基酸残基的测定;苯异硫氰酸酯法（PITC法, Edman法）;Fig.1-14 Steps in sequencing a polypeptide. (a) Determination of amino acid composition and (b) identification of the amino-terminal residue are the first steps for many polypeptides. Sangers method for identifying the amino-terminal residue is shown here. The Edman degradation procedure (c) reveals the entire sequence of a peptide. For shorter peptides, this method alone readily yields the entire sequence, and steps (a) and (b) are often omitted. ;2. C末端氨基酸残基的测定;? 羧肽酶法;（三）多肽链的分离和二硫键的断裂;（四）氨基酸组成的鉴定;胰蛋白酶： R1= Lys, Arg R2?Pro;Fig. 1-15 Trypsin is a protease, that specifically cleaves only those peptide bonds in which arginine or lysine contributes the carbonyl function.;CNBr：;（六）肽段的aa顺序测定;（七）肽段在多肽链中次序的决定—重叠肽法;（八）二硫键位置的确定— 对角线电泳 （diagonal eletrophoresis）;Fig. 1-16 Illustration of Diagonal electrophoresis;例： 从下列信息推导多肽的氨基酸顺序。;Identification of the amino-terminal residue can be the first step in sequencing a polypeptide. Sanger’s method or Edman degradation can be used. 2. Breaking disulfide bonds Disulfide bonds(二硫键）, if exist, need to be broken first. The disulfide bonds can be reduced by dithiothreitol (DTT, 二硫苏糖醇) or b-mercaptoethanol(巯基乙醇). 3. Determine the amino acid composition;4. Polypeptide chains are cleaved into short fragments by chemical or enzymatic methods and then sequenced by Edman method. 3.1 Cyanogen bromide (CNBr) cleaves poly