北大生物化学完整课件Chapter3 2经典案例.pptx

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 sequences; Current phylogeny tree of life (by Carl Woese) Characteristic titration curves of amino acids The principal components a spectrophotometerLevels of structure in proteinsProteins And Prosthetic groups (辅基) Working with ProteinsAnalytical versus Preparative;Sources of proteins: Blood (serum); Tissue cells and microbial cells;Extraction, fractionation, separation and purification. Proteins can be separated and purified Methods 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 of their binding properties. Many ways to work with Proteins based on their physical and chemical properties Centrifugation; Electrophoresis; Liquid Chromatography;Edman degradation; Mass spectrometry; Other Physical means: X-ray, NMR, Electron Microscopy; light scattering methods; different spectrophotometries; Thermo measurements; etc…Svedberg studied proteins with the methods of ultra-centrifugation (超速离心), and defined sedimentation coefficient (s).e.g. Hemoglobin:The(odor) Svedberg Uppsala UniversityBorn 1884Ph.D. 1908Professor 1912Nobelprice 1926蛋白质可以通过各种生物化学技术纯化利用蛋白质的溶解度、净电荷、大小以及与配体结合特异性上的微小差异。有透析、凝胶过滤、离子交换层析、亲和层析、电泳（垂直板电泳、等电聚焦电泳、双向电泳）等分离纯化方法。透析Salting out and dialysis(透析)Column chromatography Ion-exchange chromatography Size-exclusion chromatography Hydrophobic 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SO3－Na＋（以盐的形式出现）的强阳离子交换树