鸡卵类粘蛋白分离提纯、活性测定与相对分子质量测定.docVIP

鸡卵类黏蛋白的分离纯化及性质测定 巩海雪，崔晓东，张岩，王晶晶 （河北大学生命科学学院，河北保定071002） 摘 要： 通过本次实验的学习，主要目的是掌握蛋白质的提取、分离、纯化及性质测定方法，了解其应用。掌握离子交换柱层析和聚丙烯酰胺凝胶电泳的原理与技术以及离心机、分光光度计、核酸蛋白检测仪、电泳仪等实验仪器的使用。 鸡卵类黏蛋白是从鸡卵清中分离的糖蛋白，在pH7.8~8.0的碱性条件下，具有很强的结合胰蛋白酶的活性，而且这种结合是可逆的，因此，可以制成亲和吸附剂，用以纯化胰蛋白酶，其抑制胰蛋白酶的摩尔比为1：1，1μg高纯度的卵类黏蛋白能抑制相当于0.86 μg的胰蛋白酶（比活力为12000BAEE U/mg）。本实验主要参照Kassell所述的方法，先将鸡蛋清经三氯乙酸-丙酮溶液和离心处理，除去沉淀物，然后经丙酮分级沉淀获得粗品，再经过DEAE纤维素柱层析纯化而得合格产品。运用考马斯亮蓝法测定蛋白质浓度；用BAEE法对鸡卵类粘蛋白活性进行测定，通过SDS聚丙烯酰胺凝胶电泳检测类粘蛋白的纯度及相对分子质量。 通过本实验的学习得出类粘蛋白是一种专一性很强的胰蛋白酶抑制剂，胰蛋白酶能水解酯键,酰胺键和肽键,利用这一性质用人工合成的底物或天然的蛋白质可以测定胰蛋白酶的活力,由此可计算类粘蛋白的活力。 关键词： 鸡卵类粘蛋白 纯化 胰蛋白酶 相对分子质量 抑制 活性 The purification and properties determination of CHOM GONG Haixue, CUI Xiaodong, ZHANG Yan, WANG Jingjing Abstract:Through the experimental，main purpose is to master the technology of protein extraction, isolation, purification and properties mensuration and understand the use.Master the principle and technique of ion exchange column chromatography and polyacrylamide gel electrophoresis and the usage of centrifuge, spectrophotometer, nucleic acidprotein detector, electrophoresis apparatus and so on. CHOM is a glycoprotein separated from egg white. In alkaline condition of pH7.8~8.0,it has a strong combination with trypsin and it is reversible. Thus it can be used to made the affinity adsorbent which is used for trypsin purification. Molar rattio of trypsin inhibion is 1:1. 1ug high purity ovomucoid can inhibits 0.86ug trypsin( specific activity: 12000BAEE U/mg).This experiment mainly refers to the method of Kassell said. First, remove the miscellaneous protein in egg white by TCA – acetone and centrifugal. Then, get the crude protein by acetone fractional precipitation. Then, get the qualified protein by the purification of DEAE cellulose column chromatography. Using coomassie brilliant blue method determines the concentration of protein; Using BAEE method determines the activity of CHOM. Through SDSpolyacrylamide gel electrophoresis to determine the purity and relative molecula