北大生物化学完整Chapter3 2课件

1、 Amino Acids, Peptides and Proteins 氨基酸、肽及蛋白质Nelson, D. L., and Cox, M. M. (2005) Lehninger Principles of Biochemistry, 4th edition.Protein sequences and Evolution！Introduction to Bioinformatics:Sequence alignment; Homologs;Paralogs; orthologs;Blosum (blocks substitution matrix) ; Signature sequence

2、s; Current phylogeny tree of life (by Carl Woese) Characteristic titration curves of amino acids The principal components a spectrophotometerProteins And Prosthetic groups (辅基)Analytical versus Preparative;Sources of proteins: Blood (serum); Tissue cells and microbial cells;Extraction, fractionation

3、, separation and purification. Working with ProteinsMethods for separating proteins take advantage of the physical properties such as charge, size, and solubility, which vary from one protein to the next. Because many proteins bind to other biomolecules, proteins can also be separated on the basis o

4、f their binding properties. Proteins can be separated and purified Centrifugation; Electrophoresis; Liquid Chromatography;Edman degradation; Mass spectrometry; Other Physical means: X-ray, NMR, Electron Microscopy; light scattering methods; different spectrophotometries; Thermo measurements; etc Man

5、y ways to work with Proteins based on their physical and chemical properties Svedberg studied proteins with the methods of ultra-centrifugation (超速离心), and defined sedimentation coefficient (s).e.g. Hemoglobin:Column chromatography Ion-exchange chromatography Size-exclusion chromatography Hydrophobi

6、c interaction chromatography Isoelectric focusing chromatography Affinity chromatography:Antibodies, His-tags, Protein-A, Protein-G, GST-, MBP-fusion proteins etc, Ion-exchange Chromatography离子交换 分离氨基酸常用的是带有耐酸性非常强的磺酸根SO3Na（以盐的形式出现）的强阳离子交换树脂。首先将这种树脂填充到柱子中，然后注入含有样品的流动相，样品中含有阳离子成分X，通过静电吸引，与树脂中的带电基团相互作用

7、，结果X与Na交换，即发生阳离子交换后，形成SO3X。 Size-exclusion Chromatography 分子筛This method separates proteins according to size. The column contains a cross-linked polymer with pores of selected size. Larger proteins migrate faster than smaller ones, because they are too large to enter the pores in the beads and henc

8、e take a more direct route through the column. The smaller proteins enter the pores and are slowed by the more labyrinthian path they take through the column. Isoelectric focusing Isoelectric focusing is a procedure used to determine the isoelectric point (pI) of a protein. A pH gradient is establis

9、hed by allowing a mixture of low molecular weight organic acids and bases to distribute themselves in an electric field generated across the gel. When a protein mixture is applied, each protein migrates until it reaches the pH that matches its pI. Proteins with different isoelectric points are thus

10、distributed differently throughout the gel.AmershamBiosciencesAKTA purifierQuantification of protein, an Enzyme: Activity versus specific activity Proteins can be characterized by electrophoresis In addition to chromatography, another important set of methods is available for the separation of prote

11、ins, based on the migration of charged proteins in an electric field, a process called electrophoresis. Electrophoresis is especially useful as an analytical method. Its advantage is that proteins can be visualized as well as separated, permitting a researcher to estimate quickly the number of prote

12、ins in a mixture or the degree of purity of a particular protein preparation. Also, electrophoresis allows determination of crucial properties of a protein such as its isoelectric point and approximate molecular weight. Arne Wilhelm Kaurin TiseliusUppsala UnuversityBorn 1902Ph.D. 1930Prof. 1938Nobel

13、 price 1948Tiselius developed the methods of electrophoresis seprating and purifying proteins. 20vg 10vg 5vg 2vg M 1vg 500ng 200ng 100ng 50ng 20ngBSACoomassie blue stainingSensitivity of silver stain: 3ng on BSA 300ng 30ng 3ng 1ngBSAMark1vl 5vl66.2KDSDS(SDS-PolyAcrylamide Gel Electrophoresis)Isoelec

14、tric focusing Isoelectric focusing is a procedure used to determine the isoelectric point (pI) of a protein (Fig. 6-6). A pH gradient is established by allowing a mixture of low molecular weight organic acids and bases (ampholytes; see p. 118) to distribute themselves in an electric field generated

15、across the gel. When a protein mixture is applied, each protein migrates until it reaches the pH that matches its pI. Proteins with different isoelectric points are thus distributed differently throughout the gel.Frederick SangerCambridgeBorn 1912Nobel price 1958 and 1980Sanger developed methods for

16、 sequencing both proteins and nucleic acids, and got the price twice. Insulin1953， Frederick Sanger has Sequenced the first protein (peptide)Short polypeptides are sequenced using automated procedures Breaking disulfide bonds in proteins Large Proteins Must Be Sequenced in Smaller Segments Fragmenti

17、ng proteins prior to sequencing, and placing peptide fragments in their proper order with overlaps. 2002, Nobel Prize for Chemistry:John B. Fenn for developing (ESI: Electro-spray Ionization MS) and Koichi Tanaka for developing (MALDI: Matrix-assisted laser desorption/ ionization MS); Kurt Wuthrich:

18、 NMR ( structure analyses of biological macromolecules).John B. FennVirginia Commonwealth University Richmond, VA, USAKoichi TanakaShimadzu Corp. Kyoto, JapanThe Nobel Prize in Chemistry 2002for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules John B. FennKoichi Tanaka Investigating proteins with Mass Spectrometry (MS)Obtaining protein sequence information with tandem mass spectrometryR. Bruce MerrifieldRockefeller University New York, NY, USA The Nobel Prize in Chemistry 1984for his devel