丙酮沉淀与盐析.PPT

Effect of pH on protein structures Negative charged at higher pH pH changing : alters the charge on proteins alters their solubility 溶解度 Isoelectric point (pI)*** --等电点 pI is the pH at which protein molecular becomes electrically neutral, which has no net electric charge. Positive charged: pH pI Negative charged: pHpI The pI value of most proteins in our body is around 5.0 Protein solubility in water is low at its pI ? Macromolecules -- MW: 10,000 ～ million -- diameter: 1～ 100nm Stabilizing elements of protein solution: -- protein bears the same charge -- protein is surrounded by hydration shell (水化层) The colloid property of proteins 蛋白质的胶体性质 Protein spatial structure is sensitive to denaturing agents (high T, urea (尿素), strong acids or bases, organic solvents, detergents(去污剂), heavy metal ions) Denaturation of proteins*** 蛋白质变性 Chloroform 氯仿, 三氯甲烷 Denaturation of proteins Denaturing agents result in unfolding and disorganization of protein spatial structure without change in AA sequences of primary structure, and associate with loss of biological activity Renaturation of the protein After softly denaturation, remove the denaturation factor, the conformation and activity of denatured protein are returned. Denatured proteins: no peptide bond broken biological activity is partially or completely lost, sensitive to protease effect physical and chemical properties changed usually the solubility is reduced The polypeptide chain of denatured protein is aggregated by hydrophobic interaction, and separated out from solution Denatured protein is easily to precipitate Precipitated protein may not be denatured Precipitation of protein（蛋白质沉淀 denatured protein→ flocculating(絮状) insoluble →solid coagulation clot Coagulation of protein（蛋白质凝固） UV absorption of protein--紫外吸收 Aromatic AAs：Trp, Tyr, Phe absorb ultraviolet light Proteins: λmax =280 nm A280 (OD280) = A280 : detect and quantify amount of proteins